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Sample records for antibody conjugate dota-morab-003

  1. New Antibody Conjugates in Cancer Therapy

    Directory of Open Access Journals (Sweden)

    Serengulam V. Govindan

    2010-01-01

    Full Text Available Targeting of radiation, drugs, and protein toxins to cancers selectively with monoclonal antibodies (MAbs has been a topic of considerable interest and an area of continued development. Radioimmunotherapy (RAIT of lymphoma using directly labeled MAbs is of current interest after approval of two radiolabeled anti-CD20 MAbs, as illustrated with the near 100% overall response rate obtained in a recent clinical trial using an investigational radiolabeled anti-CD22 MAb, 90Y-epratuzumab. The advantage of pretargeted RAIT over directly labeled MAbs is continuing to be validated in preclinical models of lymphoma and solid tumors. Importantly, the advantages of combining RAIT with radiation sensitizers, with immunotherapy, or a drug conjugate targeting a different antigen are being studied clinically and preclinically. The area of drug-conjugated antibodies is progressing with encouraging data published for the trastuzumab-DM1 conjugate in a phase I clinical trial in HER2-positive breast cancer. The Dock-and-Lock platform technology has contributed to the design and the evaluation of complex antibody-cytokine and antibody-toxin conjugates. This review describes the advances made in these areas, with illustrations taken from advances made in the authors' institutions.

  2. Antibody-Conjugated Nanoparticles for Biomedical Applications

    Directory of Open Access Journals (Sweden)

    Manuel Arruebo

    2009-01-01

    Full Text Available Nanoscience and Nanotechnology have found their way into the fields of Biotechnology and Medicine. Nanoparticles by themselves offer specific physicochemical properties that they do not exhibit in bulk form, where materials show constant physical properties regardless of size. Antibodies are nanosize biological products that are part of the specific immune system. In addition to their own properties as pathogens or toxin neutralizers, as well as in the recruitment of immune elements (complement, improving phagocytosis, cytotoxicity antibody dependent by natural killer cells, etc., they could carry several elements (toxins, drugs, fluorochroms, or even nanoparticles, etc. and be used in several diagnostic procedures, or even in therapy to destroy a specific target. The conjugation of antibodies to nanoparticles can generate a product that combines the properties of both. For example, they can combine the small size of nanoparticles and their special thermal, imaging, drug carrier, or magnetic characteristics with the abilities of antibodies, such as specific and selective recognition. The hybrid product will show versatility and specificity. In this review, we analyse both antibodies and nanoparticles, focusing especially on the recent developments for antibody-conjugated nanoparticles, offering the researcher an overview of the different applications and possibilities of these hybrid carriers.

  3. Antibody Conjugates: From Heterogeneous Populations to Defined Reagents

    Directory of Open Access Journals (Sweden)

    Patrick Dennler

    2015-08-01

    Full Text Available Monoclonal antibodies (mAbs and their derivatives are currently the fastest growing class of therapeutics. Even if naked antibodies have proven their value as successful biopharmaceuticals, they suffer from some limitations. To overcome suboptimal therapeutic efficacy, immunoglobulins are conjugated with toxic payloads to form antibody drug conjugates (ADCs and with chelating systems bearing therapeutic radioisotopes to form radioimmunoconjugates (RICs. Besides their therapeutic applications, antibody conjugates are also extensively used for many in vitro assays. A broad variety of methods to functionalize antibodies with various payloads are currently available. The decision as to which conjugation method to use strongly depends on the final purpose of the antibody conjugate. Classical conjugation via amino acid residues is still the most common method to produce antibody conjugates and is suitable for most in vitro applications. In recent years, however, it has become evident that antibody conjugates, which are generated via site-specific conjugation techniques, possess distinct advantages with regard to in vivo properties. Here, we give a comprehensive overview on existing and emerging strategies for the production of covalent and non-covalent antibody conjugates.

  4. Goat serums for fluorescent antibody conjugates to chlamydial antigens.

    OpenAIRE

    Tessler, J.

    1984-01-01

    Serums from goats hyperimmunized with Chlamydia psittaci consistently produce antichlamydial fluorescent antibody conjugate of high titer. The titer of the fluorescent antibody conjugate prepared from a given serum correlated well with the titer obtained by agar gel precipitin, but not with the complement fixation. The agar gel precipitin test can be used to predict whether a given serum is satisfactory for use in production of a conjugate for direct fluorescent antibody tests. Serums with an...

  5. Antibody conjugate radioimmunotherapy of superficial bladder cancer

    Directory of Open Access Journals (Sweden)

    Alan Perkins

    2002-09-01

    Full Text Available The administration of antibody conjugates for cancer therapy is now proving to be of clinical value. We are currently undertaking a programme of clinical studies using the monoclonal antibody C595 (IgG3 which reacts with the MUC1 glycoprotein antigen that is aberrantly expressed in a high proportion of bladder tumours. Radioimmunoconjugates of the C595 antibody have been produced with high radiolabelling efficiency and immunoreactivity using Tc-99m and In-111 for diagnostic imaging, and disease staging and the cytotoxic radionuclides Cu-67 and Re-188 for therapy of superficial bladder cancer. A Phase I/II therapeutic trail involving the intravesical administration of antibody directly into the bladder has now begun.A administração de anticorpos conjugados para o tratamento do câncer está agora provando ser de valor clínico. Nós estamos atualmente realizando um programa de estudos clínicos usando o anticorpo monoclonal C595 (IgG3 que reage com a glicoproteína MUC1 que está aberrantemente expressa numa alta proporção de tumores de bexiga. Tem sido produzidos radioimunoconjugados do anticorpo C595, com alta eficiência de radiomarcação e a imunoreatividade, usando-se o Tc-99m e In-111, para o diagnóstico por imagem e estagiamento de doenças. Tem sido produzidos, também, radionuclídeos citotóxicos (Cu-67 e Re-188 para o tratamento de cânceres superficiais de bexiga. A fase terapêutica I/II já se iniciou, envolvendo a administração intravesical do anticorpo diretamente na bexiga.

  6. Biophysical characterization of a model antibody drug conjugate.

    Science.gov (United States)

    Arakawa, Tsutomu; Kurosawa, Yasunori; Storms, Michael; Maruyama, Toshiaki; Okumura, C J; Maluf, Nasib Karl

    2016-01-01

    Antibody drug conjugates (ADC) are important next-generation biopharmaceuticals and thus require stringent structure characterization as is the case for monoclonal antibodies. We have tested several biophysical techniques, i.e., circular dichroism, analytical ultracentrifugation, differential scanning calorimetry and fluorescence spectroscopy, to characterize a fluorescein-labeled monoclonal antibody as a model ADC. These techniques indicated possible small structure and stability changes by the conjugation, while largely retaining the tertiary structure of the antibody, consistent with unaltered biological activities. Thus, the above biophysical techniques are effective at detecting changes in the structural properties of ADC. PMID:27534450

  7. Recent advances in the construction of antibody-drug conjugates

    Science.gov (United States)

    Chudasama, Vijay; Maruani, Antoine; Caddick, Stephen

    2016-02-01

    Antibody-drug conjugates (ADCs) comprise antibodies covalently attached to highly potent drugs using a variety of conjugation technologies. As therapeutics, they combine the exquisite specificity of antibodies, enabling discrimination between healthy and diseased tissue, with the cell-killing ability of cytotoxic drugs. This powerful and exciting class of targeted therapy has shown considerable promise in the treatment of various cancers with two US Food and Drug Administration approved ADCs currently on the market (Adcetris and Kadcyla) and approximately 40 currently undergoing clinical evaluation. However, most of these ADCs exist as heterogeneous mixtures, which can result in a narrow therapeutic window and have major pharmacokinetic implications. In order for ADCs to deliver their full potential, sophisticated site-specific conjugation technologies to connect the drug to the antibody are vital. This Perspective discusses the strategies currently used for the site-specific construction of ADCs and appraises their merits and disadvantages.

  8. New type of antibody-enzyme conjugate which specifically kills Candida albicans.

    OpenAIRE

    Okuda, K; Ishiwara, K; Noguchi, Y.; Takahashi, T.; Tadokoro, I

    1980-01-01

    A new type of antibody-enzyme conjugate was made, and its possible application to Candida infection was studied. Both lactoperoxidase and xanthine oxidase were conjugated to specific antibody against Candida albicans. In vitro microbiocidal activity of the new antibody-enzyme conjugate, when incubated together with xanthine and minute amount of halides, showed a remarkable level of candidacidal ability. When the new antibody-enzyme conjugate was given to Candida-infected mice, followed by inj...

  9. Antibody conjugated graphene nanocomposites for pathogen detection

    Science.gov (United States)

    Sign, Chandan; Sumana, Gajjala

    2016-04-01

    Graphene oxide (GO), due to its excellent electrochemical properties and large surface area, known to be highly suitable material for biosensing application. Here, we report in situ synthesis of silver nanopaticles (AgNPs) onto the GO sheets for the electrochemical detection of Salmonella typhimurium (S.typhimurium). The GO-AgNPs composites have been deposited onto the indium tin oxide (ITO) coated glass substrate by the electrophoretic deposition technique. Carbodiimide coupling (EDC-NHS) has been used for the immobilization of antibodies of Salmonella typhimurium (anti-S.typhimurium) for detection of S.typhimurium. The electron microscopy and UV-visible studies reveal successful synthesis GO-AgNPs composites while FT-IR studies suggest the proper immobilization of anti-S.typhi. The cyclic voltammetry (CV) has been utilized for detection using anti-S.typhi/GOAgNPs/ITO based immunoelectrode as a function of S.typhimurium concentration. The fabricated immunosensor shows improved sensitivity of 33.04 μACFU-1mLcm-2 in a wide detection range of 101 to 106 CFUmL-1. This immunosensor may be utilized for the detection of other food borne pathogens like aflatoxin and E.coli also.

  10. Linkers Having a Crucial Role in Antibody-Drug Conjugates.

    Science.gov (United States)

    Lu, Jun; Jiang, Feng; Lu, Aiping; Zhang, Ge

    2016-01-01

    Antibody-drug conjugates (ADCs) comprised of a desirable monoclonal antibody, an active cytotoxic drug and an appropriate linker are considered to be an innovative therapeutic approach for targeted treatment of various types of tumors and cancers, enhancing the therapeutic parameter of the cytotoxic drug and reducing the possibility of systemic cytotoxicity. An appropriate linker between the antibody and the cytotoxic drug provides a specific bridge, and thus helps the antibody to selectively deliver the cytotoxic drug to tumor cells and accurately releases the cytotoxic drug at tumor sites. In addition to conjugation, the linkers maintain ADCs' stability during the preparation and storage stages of the ADCs and during the systemic circulation period. The design of linkers for ADCs is a challenge in terms of extracellular stability and intracellular release, and intracellular circumstances, such as the acid environment, the reducing environment and cathepsin, are considered as the catalysts to activate the triggers for initiating the cleavage of ADCs. This review discusses the linkers used in the clinical and marketing stages for ADCs and details the fracture modes of the linkers for the further development of ADCs. PMID:27089329

  11. DNA-templated antibody conjugation for targeted drug delivery to cancer cells

    DEFF Research Database (Denmark)

    Liu, Tianqiang

    2016-01-01

    Monoclonal antibodies have been used for the treatment of cancer because of their specificity to tumor markers. However, intact unmodified antibodies used as single agent therapeutics sometimes are not curative. To overcome this, there is a trend to empower them with bioactive payloads (toxins......, chemotherapeutics, radionuclides, cytokines) by means of antibody conjugation. Conventional chemical modification techniques for the conjugation of antibodies result in highly heterogeneous mixtures. This heterogeneity is far from optimal since for therapeutic purpose antibody conjugates are better in homogeneous...... conjugation strategy. Recently, a site-selective antibody conjugation method called “DNA-templated protein conjugation (DTPC)” was developed by our group. The site-selective covalently attachment of single-stranded DNA (ssDNA) to proteins was achieved by using a metal-affinity DNA probe and DNA...

  12. Platinum-conjugated antibodies for application in mass cytometry.

    Science.gov (United States)

    Mei, Henrik E; Leipold, Michael D; Maecker, Holden T

    2016-03-01

    Mass cytometry has overcome limitations of fluorescent single cell cytometry by allowing for the measurement of up to currently ∼40 different parameters on a single cell level. However, the cellular proteome comprises many more potential analytes, and current mass cytometry instrumentation allows for theoretically up to 121 different mass detection channels. The labeling of specific probes with appropriate metal ions is a significant hurdle for exploiting more of mass cytometry's analytical capacity. To this end, we here describe the labeling of antibody with natural abundance or isotopically purified platinum as formulated in cisplatin and circumventing the use of chelator-loaded polymers. We confirm the utility of cisplatin-antibody-conjugates for surface, intracellular, and phosphoepitope-specific immunophenotyping, as well as for application in cell surface CD45-based barcoding. Cisplatin-labeling of antibody increases the analytical capacity of the CyTOF(®) platform by two channels based on available reagents, and has the potential to add a total of six channels for detection of specific probes, thus helping to better extend the analytical mass range of mass cytometers. PMID:26355391

  13. Conference report: hot topics in antibody-drug conjugate development.

    Science.gov (United States)

    Thudium, Karen; Bilic, Sanela

    2013-12-01

    American Association of Pharmaceutical Scientists National Biotechnology Conference Sheraton San Diego Hotel and Marina, San Diego, CA, USA, 19-23 May 2013 The National Biotechnology Conference, is a premier meeting for biotechnology professionals covering a broad range of hot topics in the biotechnology industry. Attracting participants from academia, industry and regulatory, this meeting features sessions that aim to address emerging subjects of interest and allows for open exchange between scientists. The 2013 conference featured leading researchers in the fields of antibody-drug conjugates (ADCs) and immunogenicity. Herein, we present a summary of the ADC hot topics, including bioanalytical and PK considerations, quantitative evaluation of the impact of immunogenicity and ADME to understand ADC drug-drug interactions, and clinical considerations for ADC development. This article aims to summarize the recommendations that were made by the speakers during various sessions throughout the conference. PMID:24320125

  14. R-phycoerythrin-conjugated antibodies are inappropriate for intracellular staining of murine plasma cells.

    Science.gov (United States)

    Kim, Myun Soo; Kim, Tae Sung

    2013-05-01

    Phycoerythrin (PE) is a type of phycobiliproteins found in cyanobacteria and red algae. PE-conjugated antibodies are broadly used for flow cytometry and immunofluorescence microscopy. Because nonspecific binding of antibodies results in decreased analytic accuracy, numerous efforts have been made to unveil cases and mechanisms of nonspecific bindings. However, nonspecific binding of specific cell types by a fluorescent dye-conjugated form of antibody has been rarely reported. In the present study, we discovered that PE-conjugated antibodies, but not FITC- or APC-antibodies, selectively stained lamina propria plasma cells (LP-PCs) from the murine small intestine after membrane permeabilization. We demonstrated that LP-PC-selective staining with PE-antibodies was not due to interactions of antibody-epitope or antibody-Fc receptor. This unexpected staining by PE-antibody was not dependent on the mouse strain of LP-PCs, experimental methods, or origin species of the antibody, but dependent on PE itself. This phenomenon was also observed in plasma cells isolated from bone marrow, spleen, and mesenteric lymph nodes. Furthermore, in vitro activated B cells and in vivo generated LP-PCs were also selectively stained by PE-conjugated antibodies. Taken together, these results show that PE-conjugated antibodies are inappropriate for intracellular staining of murine plasma cells.

  15. Hacking into the granuloma: could antibody antibiotic conjugates be developed for TB?

    Science.gov (United States)

    Ekins, Sean

    2014-12-01

    Alternatives to small molecule or vaccine approaches to treating tuberculosis are rarely discussed. Attacking Mycobacterium tuberculosis in the granuloma represents a challenge. It is proposed that the conjugation of small molecules onto a monoclonal antibody that recognizes macrophage or lymphocytes cell surface receptors, might be a way to target the bacteria in the granuloma. This antibody drug conjugate approach is currently being used in 2 FDA approved targeted cancer therapies. The pros and cons of this proposal for further research are discussed.

  16. Overcoming Instability of Antibody-Nanomaterial Conjugates: Next Generation Targeted Nanomedicines Using Bispecific Antibodies.

    Science.gov (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

    2016-08-01

    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.

  17. High Turnover of Tissue Factor Enables Efficient Intracellular Delivery of Antibody-Drug Conjugates

    DEFF Research Database (Denmark)

    de Goeij, Bart E.C.G.; Satijn, D.; Freitag, C. M.;

    2015-01-01

    Antibody-drug conjugates (ADC) are emerging as powerful cancer treatments that combine antibody-mediated tumor targeting with the potent cytotoxic activity of toxins. We recently reported the development of a novel ADC that delivers the cytotoxic payload monomethyl auristatin E (MMAE) to tumor ce...

  18. Conjugation behaviours of CdTe quantum dots and antibody by a novel immunochromatographic method.

    Science.gov (United States)

    Wang, Y; Bai, Y; Wei, X

    2011-03-01

    Three water-soluble CdTe quantum dots (QDs) (green-emitting, yellow-emitting and red-emitting) were synthesised for different refluxing time with 3-mercaptopropionic acid (MPA) as stabiliser. Then the red-emitting CdTe QDs and mouse immunoglobulin G (IgG) were taken as the representative to study the conjugation behaviour of QDs and antibody by a novel immunochromatographic method. After comparing with several methods, that is, direct conjugation, 1-ethyl-3(3-dimethylaminopropyl) carbodiimides hydrochloride (EDC)-mediated conjugation, N-hydroxysuccinimide (NHS)-mediated conjugation, EDC/NHS-mediated conjugation by immunochromatographic strips, EDC and NHS were selected together as coupling agents to conjugate QDs with antibody efficiently. Finally, the K562 leukaemia cells were incubated with the EDC/NHS-mediated conjugates to evaluate the performance in practical application, and the result from fluorescence images showed that it was successfully applied to label cells. The immunochromatographic strip was a superior method to study the conjugation of the fluorophore and antibody. PMID:21241157

  19. Indocyanine green as effective antibody conjugate for intracellular molecular targeted photodynamic therapy

    Science.gov (United States)

    Wang, Sijia; Hüttmann, Gereon; Rudnitzki, Florian; Diddens-Tschoeke, Heyke; Zhang, Zhenxi; Rahmanzadeh, Ramtin

    2016-07-01

    The fluorescent dye indocyanine green (ICG) is clinically approved and has been applied for ophthalmic and intraoperative angiography, measurement of cardiac output and liver function, or as contrast agent in cancer surgery. Though ICG is known for its photochemical effects, it has played a minor role so far in photodynamic therapy or techniques for targeted protein-inactivation. Here, we investigated ICG as an antibody-conjugate for the selective inactivation of the protein Ki-67 in the nucleus of cells. Conjugates of the Ki-67 antibody TuBB-9 with different amounts of ICG were synthesized and delivered into HeLa and OVCAR-5 cells through conjugation to the nuclear localization sequence. Endosomal escape of the macromolecular antibodies into the cytoplasm was optically triggered by photochemical internalization with the photosensitizer BPD. The second light irradiation at 690 nm inactivated Ki-67 and subsequently caused cell death. Here, we show that ICG as an antibody-conjugate can be an effective photosensitizing agent. Best effects were achieved with 1.8 ICG molecules per antibody. Conjugated to antibodies, the ICG absorption peaks vary proportionally with concentration. The absorption of ICG above 650 nm within the optical window of tissue opens the possibility of selective Ki-67 inactivation deep inside of tissues.

  20. Indocyanine green as effective antibody conjugate for intracellular molecular targeted photodynamic therapy

    Science.gov (United States)

    Wang, Sijia; Hüttmann, Gereon; Rudnitzki, Florian; Diddens-Tschoeke, Heyke; Zhang, Zhenxi; Rahmanzadeh, Ramtin

    2016-07-01

    The fluorescent dye indocyanine green (ICG) is clinically approved and has been applied for ophthalmic and intraoperative angiography, measurement of cardiac output and liver function, or as contrast agent in cancer surgery. Though ICG is known for its photochemical effects, it has played a minor role so far in photodynamic therapy or techniques for targeted protein-inactivation. Here, we investigated ICG as an antibody-conjugate for the selective inactivation of the protein Ki-67 in the nucleus of cells. Conjugates of the Ki-67 antibody TuBB-9 with different amounts of ICG were synthesized and delivered into HeLa and OVCAR-5 cells through conjugation to the nuclear localization sequence. Endosomal escape of the macromolecular antibodies into the cytoplasm was optically triggered by photochemical internalization with the photosensitizer BPD. The second light irradiation at 690 nm inactivated Ki-67 and subsequently caused cell death. Here, we show that ICG as an antibody-conjugate can be an effective photosensitizing agent. Best effects were achieved with 1.8 ICG molecules per antibody. Conjugated to antibodies, the ICG absorption peaks vary proportionally with concentration. The absorption of ICG above 650 nm within the optical window of tissue opens the possibility of selective Ki-67 inactivation deep inside of tissues.

  1. Comparative assay of fluorescent antibody test results among twelve European National Reference Laboratories using various anti-rabies conjugates

    DEFF Research Database (Denmark)

    Robardet, E.; Andrieu, S.; Rasmussen, Thomas Bruun;

    2013-01-01

    Twelve National Reference Laboratories (NRLs) for rabies have undertaken a comparative assay to assess the comparison of fluorescent antibody test (FAT) results using five coded commercial anti-rabies conjugates (Biorad, Bioveta, Fujirebio, Millipore, and SIFIN conjugates). Homogenized positive...

  2. A rapid approach for characterization of thiol-conjugated antibody-drug conjugates and calculation of drug-antibody ratio by liquid chromatography mass spectrometry.

    Science.gov (United States)

    Firth, David; Bell, Leonard; Squires, Martin; Estdale, Sian; McKee, Colin

    2015-09-15

    We present the demonstration of a rapid "middle-up" liquid chromatography mass spectrometry (LC-MS)-based workflow for use in the characterization of thiol-conjugated maleimidocaproyl-monomethyl auristatin F (mcMMAF) and valine-citrulline-monomethyl auristatin E (vcMMAE) antibody-drug conjugates. Deconvoluted spectra were generated following a combination of deglycosylation, IdeS (immunoglobulin-degrading enzyme from Streptococcus pyogenes) digestion, and reduction steps that provide a visual representation of the product for rapid lot-to-lot comparison-a means to quickly assess the integrity of the antibody structure and the applied conjugation chemistry by mass. The relative abundance of the detected ions also offer information regarding differences in drug conjugation levels between samples, and the average drug-antibody ratio can be calculated. The approach requires little material (small-scale process development testing or as an early component of a complete characterization project facilitating informed decision making regarding which aspects of a molecule might need to be examined in more detail by orthogonal methodologies. PMID:26070852

  3. Photo-thermal therapy of bladder cancer with Anti-EGFR antibody conjugated gold nanoparticles.

    Science.gov (United States)

    Chen, Chieh Hsiao; Wu, Yi-Jhen; Chen, Jia-Jin

    2016-01-01

    The aim of this study was to enhance the effectiveness of photo thermal therapy (PTT) in the targeting of superficial bladder cancers using a green light laser in conjunction with gold nanoparticles (GNPs) conjugated to antibody fragments (anti-EGFR). GNPs conjugated with anti-EGFR-antibody fragments were used as probes in the targeting of tumor cells and then exposed to a green laser (532nm), resulting in the production of sufficient thermal energy to kill urothelial carcinomas both in vitro and in vivo. Nanoparticles conjugated with antibody fragments are capable of damaging cancer cells even at relatively very low energy levels, while non-conjugated nanoparticles would require an energy level of 3 times under the same conditions. The lower energy required by the nanoparticles allows this method to destroy cancerous cells while preserving normal cells when applied in vivo. Nanoparticles conjugated with antibody fragments (anti-EGFR) require less than half the energy of non-conjugated nanoparticles to kill cancer cells. In an orthotopic bladder cancer model, the group treated using PTT presented significant differences in tumor development. PMID:27100501

  4. Linker-free conjugation and specific cell targeting of antibody functionalized iron-oxide nanoparticles

    Science.gov (United States)

    Xu, Yaolin; Baiu, Dana C.; Sherwood, Jennifer A.; McElreath, Meghan R.; Qin, Ying; Lackey, Kimberly H.; Otto, Mario; Bao, Yuping

    2015-01-01

    Specific targeting is a key step to realize the full potential of iron oxide nanoparticles in biomedical applications, especially tumor-associated diagnosis and therapy. Here, we developed anti-GD2 antibody conjugated iron oxide nanoparticles for highly efficient neuroblastoma cell targeting. The antibody conjugation was achieved through an easy, linker-free method based on catechol reactions. The targeting efficiency and specificity of the antibody-conjugated nanoparticles to GD2-positive neuroblastoma cells were confirmed by flow cytometry, fluorescence microscopy, Prussian blue staining and transmission electron microscopy. These detailed studies indicated that the receptor-recognition capability of the antibody was fully retained after conjugation and the conjugated nanoparticles quickly attached to GD2-positive cells within four hours. Interestingly, longer treatment (12 h) led the cell membrane-bound nanoparticles to be internalized into cytosol, either by directly penetrating the cell membrane or escaping from the endosomes. Last but importantly, the uniquely designed functional surfaces of the nanoparticles allow easy conjugation of other bioactive molecules. PMID:26660881

  5. SPECIFIC UPTAKE OF MONOCLONAL ANTIBODY-CONJUGATED METHOTREXATE BY HUMAN LYMPHOCYTIC LEUKEMIC B CELLS

    Institute of Scientific and Technical Information of China (English)

    Zhu Zhenping; Yang Chunzheng; Tarunendu Ghose; Jaroslav Kralovec

    1998-01-01

    Objective: To analysis the uptake of free MTX and MTX conjugated to tumor specific monoclonal antibody by target and non-target cells. Methods: The folate antagonist methotrexate (MTX) was conjugated to two monoclonal antibodies (Mab) directed against human chronic lymphocytic leukemia (CLL), Dal B01 and Dal B02, by an active ester method. Both conjugates were more cytotoxic toward the target tumor cell line D10-1than to the non-target cell line MOLT-3, and Dal B02-MTX conjugate was more inhibitory to D10-1 cells than free MTX in a 6 h pulse exposure assay. Results: Drug uptake studies revealed that D10-1 cells took up much more Dal B01 and Dal B02-conjugated MTX than free MTX. The amounts of drug taken up by D10-1 cells incubated with Dal B01 and Dal B02-conjugated MTX were always 3 to 5-fold higher than that taken up by MOLT-3 cells, although the latter took up more drug when incubated with free MTX. Furthermore, tumor cells incubated with Dal B01 or Dal B02-conjugated MTX retained much larger amounts of drug for a prolonged period of time than those incubated with free MTX.Conclusion: The enhanced specific cytotoxicity of Dal B01 and Dal B02-MTX conjugates toward target tumor cells is therefore likely due to (Ⅰ) delivery of larger amounts of MTX to target cells when the drug is conjugated to Mab;(ii) longer retention of Mab-conjugated MTX by target cells; and (iii) slow, prolonged release of MTX from the surface-bound or endocytosed conjugates, rendering them into a sustained release dosage form.

  6. Quantum dot-antibody and amputator conjugates shift fluorescence upon binding bacteria

    International Nuclear Information System (INIS)

    CdSe/ZnS quantum dots (QDs) exhibited fluorescence emission blue shifts when conjugated to antibodies or DNA aptamers that are bound to bacteria. The intensity of the shifted emission peak increased with the number of bound bacteria. Curiously, the emission was consistently shifted to approximately 440-460 nm, which is distinctly different from the major component of the natural fluorescence spectrum of these QDs. This minor emission peak can grow upon conjugation to antibodies or aptamers and subsequent binding to bacterial cell surfaces. We hypothesize that the wavelength shift is due to changes in the chemical environment of the QD conjugates when they encounter the bacterial surface and may be due to physical deformation of the QD that changes the quantum confinement state. Regardless of the mechanism, these remarkable emission wavelength shifts of greater than 140 nm in some cases strongly suggest new applications for QD-receptor conjugates

  7. Salivary and Serum Antibody Response Against Neisseria meningitidis After Vaccination With Conjugate Polysaccharide Vaccines in Ethiopian Volunteers.

    Science.gov (United States)

    Bårnes, G K; Workalemahu, B; Kristiansen, P A; Beyene, D; Merdekios, B; Fissiha, P; Aseffa, A; Caugant, D A; Naess, L M

    2016-08-01

    Meningococcal conjugate vaccines induce serum antibodies crucial for protection against invasive disease. Salivary antibodies are believed to be important for hindering meningococcal acquisition and/or clearance of established carriage. In this study, we measured salivary IgA and IgG antibodies induced by vaccination with a monovalent serogroup A conjugate vaccine or a tetravalent A, C, W and Y conjugate vaccine, in comparison with antibody levels in serum. Saliva and serum samples from Ethiopian volunteers (1-29 years) collected before and eight times on a weekly basis after receiving the serogroup A conjugate vaccine, the tetravalent serogroup A, C, W and Y conjugate vaccine, or no vaccine (control group), were analysed using a multiplex microsphere immunoassay for antibody detection. Serogroup-specific IgG antibody levels in saliva increased significantly after vaccination with both vaccines. The monovalent serogroup A vaccine also induced an increase in salivary IgA antibodies. A strong correlation between serogroup-specific IgG antibodies in saliva and serum, and a somewhat lower correlation for IgA, was observed for all serogroups. There was also a strong correlation between specific secretory IgA and IgA antibodies in saliva for all serogroups. Meningococcal conjugate vaccines are able to elicit salivary antibodies against serogroup A, C, W and Y correlating with antibody levels in serum. The strong correlation between saliva and serum antibody levels indicates that saliva may be used as a surrogate of systemic antibody responses. PMID:27219622

  8. Gold Nanotheranostics: Photothermal Therapy and Imaging of Mucin 7 Conjugated Antibody Nanoparticles for Urothelial Cancer

    OpenAIRE

    Chieh Hsiao Chen; Yi-Jhen Wu; Jia-Jin Chen

    2015-01-01

    Objective. To kill urothelial cancer cells while preserving healthy cells, this study used photothermal therapy (PTT). PTT techniques target urothelial cancer cells using gold nanoparticles (GNPs) and a green light laser. Materials and Methods. The GNPs were conjugated with anti-Mucin 7 antibodies, which acted as a probe for targeting tumor cells. Conjugated GNPs were exposed to a green light laser (532 nm) with sufficient thermal energy to kill the transitional cell carcinomas (TCCs). Result...

  9. Study of conjugation and radiolabeling of monoclonal antibody rituximab for use in radionuclide therapy

    International Nuclear Information System (INIS)

    Lymphomas are tumors originated from the transformation of a lymphocyte in the lymphatic system. The most common lymphoma is the Non-Hodgkin Lymphoma (NHL). Advances in immunology and molecular biology have been improving NHL's detection and treatment strategies development, such as Radioimmunotherapy (RIT). Rituximab is an anti-CD20 monoclonal antibody used as immunotherapeutic to treat refractory or relapsed NHL. The goal of the present work was to conjugate this antibody to DOTA-NHS-ester bifunctional chelator and to radiolabel it with 177Lu radioisotope in order to develop a radio immunotherapeutic agent for NHL's treatment. Different rituximab to DOTA molar ratios (1:5, 1:10, 1:20, 1:50, 1:250, 1:500 and 1:1000) were evaluated in order to determine the best condition for obtaining the highest radiochemical purity of radio immunotherapeutic. The stability of the unlabeled immuno conjugated was evaluated by high performance liquid chromatography (HPLC) for up to 240 days in different storage conditions. The stability of the labeled preparations was evaluated either after storing at 2-8 degree C or incubation in human serum at 37 degree C. The binding to serum proteins was also determined. In vivo studies were performed in healthy Swiss mice, in order to characterize the biological properties of labeled conjugate. Finally, preliminary studies of radio immuno conjugated competitive binding to CD20 positive Raji cells were carried out in order to analyze if the process of conjugation and radiolabeling compromises the immunoreactivity of the antibody. The conjugation applying lower antibody to chelator molar ratios (1:5, 1:10 and 1:20) showed high stability when stored for up to 240 days in different conditions. The HPLC analysis showed that the monoclonal antibody conjugated in molar ratio 1:50 was labeled with higher radiochemical purity (> 95%) when purified in PD-10 column. This conjugate showed reasonable stability at 2-8 degree C. The analysis of the stability

  10. A monoclonal antibody-Pseudomonas toxin conjugate that specifically kills multidrug-resistant cells.

    OpenAIRE

    FitzGerald, D J; Willingham, M C; Cardarelli, C O; Hamada, H; Tsuruo, T.; Gottesman, M M; Pastan, I

    1987-01-01

    One form of multidrug resistance is due to the expression of a 170-kDa energy-dependent drug efflux pump called P-glycoprotein in the plasma membranes of human cancer cells. We have prepared conjugates of Pseudomonas toxin with the anti-P-glycoprotein monoclonal antibody MRK-16. These anti-P-glycoprotein-toxin conjugates specifically kill multidrug-resistant human KB cells. Similar conjugates could be useful in cancer therapy to reduce or eliminate multidrug-resistant tumor populations in tum...

  11. A rapid, single vessel method for preparation of clinical grade ligand conjugated monoclonal antibodies

    International Nuclear Information System (INIS)

    A rapid, single vessel method for the preparation of clinical grade chelate conjugated monoclonal antibodies has been developed. By use of an Amicon concentrator with reservoir, each of the steps necessary for the preparation of the conjugated drug may be performed in a single vessel. Advantages include reduced risk of metal, pyrogen and bacterial contamination; buffer exchanges are achieved rapidly and efficiently using a continuous dilution method. The radiolabeling efficiency, the radiochemical purity, the total immunoreactivity and the affinity of the final product have been evaluated in the production of CHXA-DTPA-chelate conjugated HuM195. The characteristics compare favorably to those achieved using our conventional synthetic methods

  12. Hacking into the granuloma: could antibody antibiotic conjugates be developed for TB?

    Science.gov (United States)

    Ekins, Sean

    2014-12-01

    Alternatives to small molecule or vaccine approaches to treating tuberculosis are rarely discussed. Attacking Mycobacterium tuberculosis in the granuloma represents a challenge. It is proposed that the conjugation of small molecules onto a monoclonal antibody that recognizes macrophage or lymphocytes cell surface receptors, might be a way to target the bacteria in the granuloma. This antibody drug conjugate approach is currently being used in 2 FDA approved targeted cancer therapies. The pros and cons of this proposal for further research are discussed. PMID:25287628

  13. FAST TRACK COMMUNICATION: Air plasma coupled with antibody-conjugated nanoparticles: a new weapon against cancer

    Science.gov (United States)

    Kim, G. C.; Kim, G. J.; Park, S. R.; Jeon, S. M.; Seo, H. J.; Iza, F.; Lee, J. K.

    2009-02-01

    Ambient air plasmas have been known to kill cancer cells. To enhance selectivity we have used antibody-conjugated nanoparticles. We achieved five times enhancement of melanoma cell death over the case of the plasma alone by using an air plasma with gold nanoparticles bound to anti-FAK antibodies. Our results show that this new interdisciplinary technique has enormous potential for use as a complement to conventional therapies.

  14. RECOMBINANT PRODUCTION OF HORSERADISH PEROXIDASE CONJUGATES WITH FAB ANTIBODIES IN PICHIA PASTORIS FOR ANALYTICAL APPLICATIONS

    OpenAIRE

    Koliasnikov, O.; Grigorenko, V.; Egorov, A.; S. Lange(Justus Liebig-Universität Gie\\ssen II Physikalisches Institut, Germany); Schmid, R

    2011-01-01

    Recombinant immunoconjugates of marker enzymes with antigens or antibodies present considerably more advantages than those obtained by conventional methods of chemical synthesis; i.e. they are homogeneous, have a strictly determined stoichiometry, and retain the functional activity of both a marker protein and an antigen/antibody. Based on the pPICZαB shuttle vector, we first managed to obtain a recombinant conjugate of key marker enzyme horseradish peroxidase (HRP) with Fab fragments of anti...

  15. Chemotherapy by Intravenous Administration of Conjugates of Daunomycin with Monoclonal and Conventional Anti-Rat α -fetoprotein Antibodies

    Science.gov (United States)

    Tsukada, Yutaka; Hurwitz, Esther; Kashi, Rina; Sela, Michael; Hibi, Nozomu; Hara, Akihiko; Hirai, Hidematsu

    1982-12-01

    Monoclonal antibodies to rat α -fetoprotein (AFP) were produced by hybridization of mouse myeloma cells with spleen cells from mice immunized with rat AFP. The monoclonal antibodies as well as horse anti-rat AFP were coupled via a dextran bridge to daunomycin. Both types of conjugates were tested in vitro and in vivo for their anti-tumor activity. They were equally cytotoxic to rat AH66 hepatoma cell line in culture. Rats challenged with hepatoma cells were treated with the conjugates either by intraperitoneal or intravenous injections. Daunomycin conjugates with horse anti-AFP and monoclonal mouse anti-AFP were capable of delaying the tumor development more efficiently than the controls of antibodies or free drug, mixtures of drug with antibodies, and a conjugate of drug and normal immunoglobulin. The specific conjugates were considerably more effective when the treatments were given intravenously. The specific conjugates produced 60% long-term survival, whereas the controls delayed only slightly tumor development.

  16. A sulfanyl-PEG derivative of relaxin-like peptide utilizable for the conjugation with KLH and the antibody production.

    Science.gov (United States)

    Katayama, Hidekazu; Mita, Masatoshi

    2016-08-15

    A small peptide-keyhole limpet hemocyanin (KLH) conjugate is generally used as an antigen for producing specific antibodies. However, preparation of a disulfide-rich heterodimeric peptide-KLH conjugates is difficult. In this study, we developed a novel method for preparation of the conjugate, and applied it to the production of specific antibodies against the relaxin-like gonad-stimulating peptide (RGP) from the starfish. In this method, a sulfanyl group necessary for the conjugation with KLH was site-specifically introduced to the peptide after regioselective disulfide bond formation reactions. Using the conjugate, we could obtain specific antibodies with a high antibody titer. This method might also be useful for the production of antibodies against other heterodimeric peptides with disulfide cross-linkages, such as vertebrate relaxins.

  17. 78 FR 18999 - Prospective Grant of Start-Up Exclusive License: Photosensitizing Antibody-Fluorophore Conjugates...

    Science.gov (United States)

    2013-03-28

    ...-fluorophore conjugates where the antibody is specific for cancer cells and the fluorophore is IR700 dye... for Photoimmunotherapy (PIT), or in combination with cancer therapeutic agents, to treat cancer or pre-cancerous hyperplasia'', and may be further limited to certain types of cancer and/or specific...

  18. Antibody response to Haemophilus influenzae type b capsular polysaccharide conjugated to tetanus toxoid in preterm infants

    DEFF Research Database (Denmark)

    Kristensen, Kim; Gyhrs, A; Lausen, B;

    1996-01-01

    OBJECTIVE: To evaluate the antibody response to a Haemophilus influenzae type b capsular polysaccharide (HibCP) tetanus toxoid (TT) conjugate vaccine (HibCP-TT) in preterm infants. SUBJECTS: Thirty-five healthy preterm infants with gestational ages (GA) from 27 to 36 weeks and birth weights from...

  19. An improved method for covalently conjugating morpholino oligomers to antitumor antibodies.

    Science.gov (United States)

    He, Jiang; Liu, Guozheng; Dou, Shuping; Gupta, Suresh; Rusckowski, Mary; Hnatowich, Donald

    2007-01-01

    Whether for conventional pretargeting, amplification pretargeting, or affinity enhancement pretargeting, it will be necessary to conjugate an antitumor antibody as the first injectate. This laboratory is investigating phosphorodiamidate morpholinos (MORFs) for pretargeting, and accordingly we are examining methods of attaching MORFs to antitumor antibodies that provide at least one group per molecule (gpm) without adversely influencing antibody properties. The aim of this investigation was to evaluate the commercial Hydralink for the conjugation of the anti-CEA MN14 antibody with an 18 mer amine-derivatized MORF. The conjugation was approached in both directions by first reacting MN14 with the NHS derivatives of 4-hydrozinonicotinate acetone hydrazone (SANH) or 4-formylbenzoate (SFB) and then combining with MORF that was previously reacted with SFB or SANH to yield MN14(SANH)-MORF and MN14(SFB)-MORF respectively. The storage stability, immunoreactive fraction, and the biodistribution in normal mice were compared for both conjugates. Thereafter, MN14(SANH)-MORF was used in a pretargeting study in tumored nude mice, and the results were compared to that obtained historically with MN14-MORF prepared by carbodiimide (EDC) coupling. Both new methods of conjugation provided between 1 and 2 gpm compared to 0.2 achieved previously by EDC. Furthermore, by repeat SE HPLC with and without CEA, both showed an unimpaired immunoreactive fraction. MN14(SANH)-MORF tolerated long-term storage best. More importantly, when labeled by hybridization with 99mTc-labeled complementary MORF (99mTc-cMORF), the biodistribution of MN14(SANH)-MORF was more favorable than that of MN14(SFB)-MORF in normal mice with lower liver (5.7 vs 9.4 %ID/g at 18 h) and spleen (3.5 vs 8.4 %ID/g) accumulations and higher blood levels (4.8 vs 3.4 %ID/g). Accordingly, only MN14(SANH)-MORF was used in a pretargeting study in tumored mice. When targeted with 99mTc-cMORF and at 2 days postinjection of antibody

  20. Comparison of the Photobleaching and Photostability Traits of Alexa Fluor 568- and Fluorescein Isothiocyanate- conjugated Antibody

    Directory of Open Access Journals (Sweden)

    Ahmad Reza Mahmoudi

    2011-01-01

    Full Text Available Objective: Synthetic fluorescent dyes that are conjugated to antibodies are useful tools toprobe molecules. Based on dye chemical structures, their photobleaching and photostabilityindices are quite diverse. It is generally believed that among different fluorescent dyes,Alexa Fluor family has greater photostability than traditional dyes like fluorescein isothiocyanate(FITC and Cy5. Alexa Fluor 568 is a member of Alexa Fluor family presumed tohave superior photostability and photobleahing profiles than FITC.Materials and Methods: In this experimental study, we conjugated Alexa Fluor 568 andFITC dyes to a mouse anti-human nestin monoclonal antibody (ANM to acquire their photobleachingprofiles and photostability indices. Then, the fluorophore/antibody ratios werecalculated using a spectrophotometer. The photobleaching profiles and photostability indicesof conjugated antibodies were subsequently studied by immunocytochemistry (ICC.Samples were continuously illuminated and digital images acquired under a fluorescentmicroscope. Data were processed by ImageJ software.Results: Alexa Fluor 568 has a brighter fluorescence and higher photostability thanFITC.Conclusion: Alexa Fluor 568 is a capable dye to use in photostaining techniques and ithas a longer photostability when compared to FITC.

  1. Quantification of residual solvents in antibody drug conjugates using gas chromatography

    Energy Technology Data Exchange (ETDEWEB)

    Medley, Colin D., E-mail: medley.colin@gene.com [Genentech Inc., Small Molecule Pharmaceutical Sciences, 1 DNA Way, South San Francisco, CA 94080 (United States); Kay, Jacob [Research Pharmaceutical Services, 520 Virginia Dr. Fort, Washington, PA (United States); Li, Yi; Gruenhagen, Jason; Yehl, Peter; Chetwyn, Nik P. [Genentech Inc., Small Molecule Pharmaceutical Sciences, 1 DNA Way, South San Francisco, CA 94080 (United States)

    2014-11-19

    Highlights: • Sensitive residual solvents detection in ADCs. • 125 ppm QL for common conjugation solvents. • Generic and validatable method. - Abstract: The detection and quantification of residual solvents present in clinical and commercial pharmaceutical products is necessary from both patient safety and regulatory perspectives. Head-space gas chromatography is routinely used for quantitation of residual solvents for small molecule APIs produced through synthetic processes; however residual solvent analysis is generally not needed for protein based pharmaceuticals produced through cultured cell lines where solvents are not introduced. In contrast, antibody drug conjugates and other protein conjugates where a drug or other molecule is covalently bound to a protein typically use solvents such as N,N-dimethylacetamide (DMA), N,N‑dimethylformamide (DMF), dimethyl sulfoxide (DMSO), or propylene glycol (PG) to dissolve the hydrophobic small molecule drug for conjugation to the protein. The levels of the solvent remaining following the conjugation step are therefore important to patient safety as these parental drug products are introduced directly into the patients bloodstream. We have developed a rapid sample preparation followed by a gas chromatography separation for the detection and quantification of several solvents typically used in these conjugation reactions. This generic method has been validated and can be easily implemented for use in quality control testing for clinical or commercial bioconjugated products.

  2. Quantification of residual solvents in antibody drug conjugates using gas chromatography

    International Nuclear Information System (INIS)

    Highlights: • Sensitive residual solvents detection in ADCs. • 125 ppm QL for common conjugation solvents. • Generic and validatable method. - Abstract: The detection and quantification of residual solvents present in clinical and commercial pharmaceutical products is necessary from both patient safety and regulatory perspectives. Head-space gas chromatography is routinely used for quantitation of residual solvents for small molecule APIs produced through synthetic processes; however residual solvent analysis is generally not needed for protein based pharmaceuticals produced through cultured cell lines where solvents are not introduced. In contrast, antibody drug conjugates and other protein conjugates where a drug or other molecule is covalently bound to a protein typically use solvents such as N,N-dimethylacetamide (DMA), N,N‑dimethylformamide (DMF), dimethyl sulfoxide (DMSO), or propylene glycol (PG) to dissolve the hydrophobic small molecule drug for conjugation to the protein. The levels of the solvent remaining following the conjugation step are therefore important to patient safety as these parental drug products are introduced directly into the patients bloodstream. We have developed a rapid sample preparation followed by a gas chromatography separation for the detection and quantification of several solvents typically used in these conjugation reactions. This generic method has been validated and can be easily implemented for use in quality control testing for clinical or commercial bioconjugated products

  3. Synthesis and Characterization of Hapten-Protein Conjugates for Antibody Production against Cyanogenic Glycosides.

    Science.gov (United States)

    Bolarinwa, Islamiyat Folashade

    2015-07-01

    Consumption of cyanogenic plants can cause serious health problems for humans. The ability to detect and quantify cyanogenic glycosides, capable of generating cyanide, could contribute to prevention of cyanide poisoning from the consumption of improperly processed cyanogenic plants. Hapten-protein conjugates were synthesized with amygdalin and linamarin by using a novel approach. Polyclonal antibodies were generated by immunizing four New Zealand White rabbits with synthesized amygdalin-bovine serum albumin and linamarin-bovine serum albumin immunogen. This is the first time an antibody was produced against linamarin. Antibody titer curves were obtained from all the four rabbits by using a noncompetitive enzyme-linked immunosorbent assay. High antibody titer was obtained at dilutions greater than 1:50,000 from both immunogens. This new method is an important step forward in preventing ingestion of toxic cyanogenic glycosides. PMID:26197297

  4. Novel Phospholipid-Protein Conjugates Allow Improved Detection of Antibodies in Patients with Autoimmune Diseases

    DEFF Research Database (Denmark)

    Samuelsen, Simone V; Maity, Arindam; Nybo, Mads;

    2016-01-01

    structural features of biologically active antigens into account and leads to low reliability and poor scientific test value. Here we describe novel phospholipid-protein conjugates for specific detection of human autoimmune antibodies. Our synthetic approach includes mild oxidation of synthetic phospholipid......Reliable measurement of clinically relevant autoimmune antibodies toward phospholipid-protein conjugates is highly desirable in research and clinical assays. To date, the development in this field has been limited to the use of natural heterogeneous antigens. However, this approach does not take...... in Denmark (n = 34) and the USA (n = 27 and n = 14). Afterwards we analyzed correlation of the obtained autoantibody titers with clinical parameters for each patient. Our results prove that using novel antigens clinically relevant autoantibodies can be detected with high repeatability, sensitivity...

  5. Comparison of the Photobleaching and Photostability Traits of Alexa Fluor 568- and Fluorescein Isothiocyanate- conjugated Antibody

    OpenAIRE

    Ahmad Reza Mahmoudi; Mohtaram Vafakhah; Elham Shaban; Reza Hadavi; Mahmood Jeddi-Tehrani; Ali Ahmad Bayat; Maryam Darzi; Majid Tarahomi; Roya Ghods; Jafar Mahmoudian

    2011-01-01

    Objective: Synthetic fluorescent dyes that are conjugated to antibodies are useful tools to probe molecules. Based on dye chemical structures, their photobleaching and photostability indices are quite diverse. It is generally believed that among different fluorescent dyes, Alexa Fluor family has greater photostability than traditional dyes like fluorescein isothiocyanate (FITC) and Cy5. Alexa Fluor 568 is a member of Alexa Fluor family presumed to have superior photostability and photobleahin...

  6. World Antibody-Drug Conjugate Summit, October 15–16, 2013, San Francisco, CA

    OpenAIRE

    Klinguer-Hamour, Christine; Strop, Pavel; Shah, Dhaval K; Ducry, Laurent; Xu, April; Beck, Alain

    2013-01-01

    The World Antibody-Drug Conjugate (WADC) Summits organized by Hanson Wade are currently the largest meetings fully dedicated to ADCs. The first global ADC Summit was organized in Boston in October 2010. Since 2011, two WADC are held every year in Frankfurt and San Francisco, respectively. The 2013 WADC San Francisco event was structured around plenary sessions with keynote speakers from AbbVie, Agensys, ImmunoGen, Immunomedics, Genentech, Pfizer and Seattle Genetics. Parallel tracks were also...

  7. Photochemical inactivation of viruses by antibody conjugates of compounds generating singlet oxygen

    Science.gov (United States)

    Savitsky, Alexander P.; Tourkin, Andrey I.; Tourkina, Elena V.; Cherednikova, Tatyana V.; Ponomarev, Gely V.; Poglazov, Boris F.

    1994-03-01

    For the first time a method is proposed for targeted destruction of viral particles with a photoimmuno-toxin. The photoinununotoxin is a conjugate of antibodies and dimethoxy haematoporphyrin (a potent singlet oxygen generator), binding with the viral particle and upon illumination with visible light inactivating the virus by singlet oxygen. The method can be used to combat viral infection in humans and to prevent lysis of industrial micro-organisms by lysogenic bacteriophage strains.

  8. Comparative assay of fluorescent antibody test results among twelve European National Reference Laboratories using various anti-rabies conjugates.

    Science.gov (United States)

    Robardet, E; Andrieu, S; Rasmussen, T Bruun; Dobrostana, M; Horton, D L; Hostnik, P; Jaceviciene, I; Juhasz, T; Müller, T; Mutinelli, F; Servat, A; Smreczak, M; Vanek, E; Vázquez-Morón, S; Cliquet, F

    2013-07-01

    Twelve National Reference Laboratories (NRLs) for rabies have undertaken a comparative assay to assess the comparison of fluorescent antibody test (FAT) results using five coded commercial anti-rabies conjugates (Biorad, Bioveta, Fujirebio, Millipore, and SIFIN conjugates). Homogenized positive brain tissues infected with various lyssavirus species as well as negative samples were analyzed blindly using a standardized FAT procedure. Conjugates B, C, D, and E were found to be significantly more effective than conjugate A for GS7 (French RABV) diluted samples (1/8 and 1/100) while the frequency of concordant results of conjugates C and D differ significantly from conjugates A, B and E for CVS 27. For detection of EBLV-1 strains, conjugates C and D also presented a significantly lower frequency of discordant results compared to conjugates A, B and E. Conjugates B, C and D were found to be significantly more effective than conjugates E and A for EBLV-2 and ABLV samples. In view of these results, conjugates C and D set themselves apart from the others and appeared as the most effective of this 5-panel conjugates. This study clearly demonstrates that the variability of conjugates used by National Reference Laboratories can potentially lead to discordant results and influence assay sensitivity. In case of false negative results this could have a dramatic impact if the animal under investigation is responsible for human exposure. To avoid such situations, confirmatory tests should be implemented.

  9. Deferoxamine as a chelator for {sup 67}Ga in the preparation of antibody conjugates

    Energy Technology Data Exchange (ETDEWEB)

    Govindan, Serengulam V. [Immunomedics, Inc., Morris Plains, NJ 07950 (United States); Michel, Rosana B. [Center for Molecular Medicine and Immunology, Belleville, NJ 07109 (United States); Griffiths, Gary L. [Immunomedics, Inc., Morris Plains, NJ 07950 (United States); Goldenberg, David M. [Center for Molecular Medicine and Immunology, Belleville, NJ 07109 (United States); Mattes, M. Jules [Center for Molecular Medicine and Immunology, Belleville, NJ 07109 (United States)]. E-mail: mjmattes@gscancer.org

    2005-07-01

    {sup 67}Ga antibodies (Abs) have been shown to be effective agents for single-cell killing due to the Auger electrons emitted, but their specific activities have not been as high as desired. We therefore evaluated deferoxamine (DFO) as a chelator, as opposed to the cyclic chelator NOTA, which was used previously. Use of DFO for Ab conjugation to {sup 67}Ga was reported previously by several laboratories. DFO was conjugated to Abs by two methods, one using Ablysine conjugation and another using mild reduction of Abs to generate thiols in the hinge region. Labeling with {sup 67}Ga was efficient, and the specific activities obtained under nonoptimized conditions were twice as high as those achieved previously. However, analysis of these conjugates revealed two problems that appear to prevent their further development. First, the stability was inadequate for the 3-day half-life of the nuclide. Second, the labels were poorly retained within cells after Ab internalization and catabolism. Also, it was found that stability was significantly affected by the incubation buffer used: buffers lacking physiological concentrations of divalent cations Ca and Mg resulted in much lower stability than buffers including them. In conclusion, DFO does not seem to be a suitable chelator for {sup 67}Ga conjugation for our purposes.

  10. Development of antibody-siRNA conjugate targeted to cardiac and skeletal muscles.

    Science.gov (United States)

    Sugo, Tsukasa; Terada, Michiko; Oikawa, Tatsuo; Miyata, Kenichi; Nishimura, Satoshi; Kenjo, Eriya; Ogasawara-Shimizu, Mari; Makita, Yukimasa; Imaichi, Sachiko; Murata, Shumpei; Otake, Kentaro; Kikuchi, Kuniko; Teratani, Mika; Masuda, Yasushi; Kamei, Takayuki; Takagahara, Shuichi; Ikeda, Shota; Ohtaki, Tetsuya; Matsumoto, Hirokazu

    2016-09-10

    Despite considerable efforts to develop efficient carriers, the major target organ of short-interfering RNAs (siRNAs) remains limited to the liver. Expanding the application outside the liver is required to increase the value of siRNAs. Here we report on a novel platform targeted to muscular organs by conjugation of siRNAs with anti-CD71 Fab' fragment. This conjugate showed durable gene-silencing in the heart and skeletal muscle for one month after intravenous administration in normal mice. In particular, 1μg siRNA conjugate showed significant gene-silencing in the gastrocnemius when injected intramuscularly. In a mouse model of peripheral artery disease, the treatment with myostatin-targeting siRNA conjugate by intramuscular injection resulted in significant silencing of myostatin and hypertrophy of the gastrocnemius, which was translated into the recovery of running performance. These data demonstrate the utility of antibody conjugation for siRNA delivery and the therapeutic potential for muscular diseases. PMID:27369865

  11. A covalent and cleavable antibody-DNA conjugation strategy for sensitive protein detection via immuno-PCR

    Science.gov (United States)

    van Buggenum, Jessie A. G. L.; Gerlach, Jan P.; Eising, Selma; Schoonen, Lise; van Eijl, Roderick A. P. M.; Tanis, Sabine E. J.; Hogeweg, Mark; Hubner, Nina C.; van Hest, Jan C.; Bonger, Kimberly M.; Mulder, Klaas W.

    2016-01-01

    Immuno-PCR combines specific antibody-based protein detection with the sensitivity of PCR-based quantification through the use of antibody-DNA conjugates. The production of such conjugates depends on the availability of quick and efficient conjugation strategies for the two biomolecules. Here, we present an approach to produce cleavable antibody-DNA conjugates, employing the fast kinetics of the inverse electron-demand Diels-Alder reaction between tetrazine and trans-cyclooctene (TCO). Our strategy consists of three steps. First, antibodies are functionalized with chemically cleavable NHS-s-s-tetrazine. Subsequently, double-stranded DNA is functionalized with TCO by enzymatic addition of N3-dATP and coupling to trans-Cyclooctene-PEG12-Dibenzocyclooctyne (TCO-PEG12-DBCO). Finally, conjugates are quickly and efficiently obtained by mixing the functionalized antibodies and dsDNA at low molar ratios of 1:2. In addition, introduction of a chemically cleavable disulphide linker facilitates release and sensitive detection of the dsDNA after immuno-staining. We show specific and sensitive protein detection in immuno-PCR for human epidermal stem cell markers, ITGA6 and ITGB1, and the differentiation marker Transglutaminase 1 (TGM1). We anticipate that the production of chemically cleavable antibody-DNA conjugates will provide a solid basis for the development of multiplexed immuno-PCR experiments and immuno-sequencing methodologies. PMID:26947912

  12. Simple Method To Prepare Oligonucleotide-Conjugated Antibodies and Its Application in Multiplex Protein Detection in Single Cells.

    Science.gov (United States)

    Gong, Haibiao; Holcomb, Ilona; Ooi, Aik; Wang, Xiaohui; Majonis, Daniel; Unger, Marc A; Ramakrishnan, Ramesh

    2016-01-20

    The diversity of nucleic acid sequences enables genomics studies in a highly multiplexed format. Since multiplex protein detection is still a challenge, it would be useful to use genomics tools for this purpose. This can be accomplished by conjugating specific oligonucleotides to antibodies. Upon binding of the oligonucleotide-conjugated antibodies to their targets, the protein levels can be converted to oligonucleotide levels. In this report we describe a simple method for preparing oligonucleotide-conjugated antibodies and discuss this method's application in oligonucleotide extension reaction (OER) for multiplex protein detection. Conjugation is based on strain-promoted alkyne-azide cycloaddition (the Cu-free click reaction), in which the antibody is activated with a dibenzocyclooctyne (DBCO) moiety and subsequently linked covalently with an azide-modified oligonucleotide. In the functional test, the reaction conditions and purification processes were optimized to achieve maximum yield and best performance. The OER assay employs a pair of antibody binders (two antibodies, each conjugated with its own oligonucleotide) developed for each protein target. The two oligonucleotides contain unique six-base complementary regions at their 3' prime ends to allow annealing and extension by DNA synthesis enzymes to form a DNA template. Following preamplification, the DNA template is detected by qPCR. Distinct oligonucleotide sequences are assigned to different antibody binders to enable multiplex protein detection. When tested using recombinant proteins, some antibody binders, such as those specific to CSTB, MET, EpCAM, and CASP3, had dynamic ranges of 5-6 logs. The antibody binders were also used in a multiplexed format in OER assays, and the binders successfully detected their protein targets in cell lysates, and in single cells in combination with the C1 system. This click reaction-based antibody conjugation procedure is cost-effective, needs minimal hands-on time, and

  13. Properties of surface functionalized iron oxide nanoparticles (ferrofluid) conjugated antibody for lateral flow immunoassay application

    Energy Technology Data Exchange (ETDEWEB)

    Nor, Noorhashimah Mohamad [School of Materials and Mineral Resources Engineering, Engineering Campus, Universiti Sains Malaysia, 14300 Nibong Tebal, Penang (Malaysia); Abdul Razak, Khairunisak, E-mail: khairunisak@eng.usm.my [School of Materials and Mineral Resources Engineering, Engineering Campus, Universiti Sains Malaysia, 14300 Nibong Tebal, Penang (Malaysia); NanoBiotechnology Research and Innovation (NanoBRI), Institute for Research in Molecular Medicine (INFORMM), 11800 USM, Penang (Malaysia); Tan, Soo Choon; Noordin, Rahmah [NanoBiotechnology Research and Innovation (NanoBRI), Institute for Research in Molecular Medicine (INFORMM), 11800 USM, Penang (Malaysia)

    2012-10-15

    Highlights: Black-Right-Pointing-Pointer The effects of acid functionalization and biocompatible polymer on iron oxide nanoparticles (IONPs) ferrofluid were studied. Black-Right-Pointing-Pointer The IONPs functionalized using citric acid (IONPs-CA) is the most stable ferrofluid with zeta potential value of -49 mV. Black-Right-Pointing-Pointer IONPs-CA can be directly conjugated with antibody without biocompatible polymer coating. Black-Right-Pointing-Pointer IONPs-CA had optimum detection efficiency of 15 min assay time. Black-Right-Pointing-Pointer IONPs-CA showed the highest colour intensity in labelling lateral flow immunoassay. - Abstract: In this study, colloidal stability of iron oxide nanoparticles (IONPs) with several acid functionalizations and biocompatible polymer coating were compared for use as labelling agent in lateral flow immunoassay (LFIA). IONPs were synthesized using the precipitation method and peptized using perchloric acid (PA), nitric acid (NA) and citric acid (CA) to form a stable IONPs ferrofluid. Steric stabilization of IONPs using silane polyethelene glycol (SiPEG) was developed to improve biocompatibility and provide spaces for subsequent conjugation process. From the transmission electron microscopy (TEM) images, the sizes of IONPs obtained with different acids peptization were in range of 11-17 nm. The IONPs peptized using citric acid showed the most stable ferrofluid condition at physiological condition with zeta potential value of -49 mV. The LFIA was also developed to examine the conjugation properties of IONPs to mouse anti-human IgG{sub 4} antibody (M{alpha}HIgG{sub 4}). IONPs functionalized with citric acid can be directly conjugated with the M{alpha}HIgG{sub 4} without the need of SiPEG addition. This is due to the presence of the carboxylic group that acted as a ligand to the extended bond formation with the antibody. Moreover, the conjugation of IONPs with M{alpha}HIgG{sub 4} was also tested in a LFIA to detect brugian

  14. Compact quantum dot-antibody conjugates for FRET immunoassays with subnanomolar detection limits

    Science.gov (United States)

    Mattera, Lucia; Bhuckory, Shashi; Wegner, K. David; Qiu, Xue; Agnese, Fabio; Lincheneau, Christophe; Senden, Tim; Djurado, David; Charbonnière, Loïc J.; Hildebrandt, Niko; Reiss, Peter

    2016-05-01

    A novel two-step approach for quantum dot (QD) functionalization and bioconjugation is presented, which yields ultra-compact, stable, and highly luminescent antibody-QD conjugates suitable for use in FRET immunoassays. Hydrophobic InPZnS/ZnSe/ZnS (emission wavelength: 530 nm), CdSe/ZnS (605 nm), and CdSeTe/ZnS (705 nm) QDs were surface functionalized with zwitterionic penicillamine, enabling aqueous phase transfer under conservation of the photoluminescence properties. Post-functionalization with a heterobifunctional crosslinker, containing a lipoic acid group and a maleimide function, enabled the subsequent coupling to sulfhydryl groups of proteins. This was demonstrated by QD conjugation with fragmented antibodies (F(ab)). The obtained F(ab)-QD conjugates range among the smallest antibody-functionalized nanoprobes ever reported, with a hydrodynamic diameter detection (LOD) for PSA compared to commercially available hydrophilic QDs emitting at 605 and 705 nm, respectively. While the commercial QDs contain identical inorganic cores responsible for their fluorescence, they are coated with a comparably thick amphiphilic polymer layer leading to much larger hydrodynamic diameters (>26 nm without biomolecules). The LODs of 0.8 and 3.7 ng mL-1 obtained in 50 μL serum samples are below the clinical cut-off level of PSA (4 ng mL-1) and demonstrate their direct applicability in clinical diagnostics.A novel two-step approach for quantum dot (QD) functionalization and bioconjugation is presented, which yields ultra-compact, stable, and highly luminescent antibody-QD conjugates suitable for use in FRET immunoassays. Hydrophobic InPZnS/ZnSe/ZnS (emission wavelength: 530 nm), CdSe/ZnS (605 nm), and CdSeTe/ZnS (705 nm) QDs were surface functionalized with zwitterionic penicillamine, enabling aqueous phase transfer under conservation of the photoluminescence properties. Post-functionalization with a heterobifunctional crosslinker, containing a lipoic acid group and a maleimide

  15. Anti-Endosialin Antibody-Drug Conjugate: Potential in Sarcoma and Other Malignancies.

    Science.gov (United States)

    Rouleau, Cecile; Gianolio, Diego A; Smale, Robert; Roth, Stephanie D; Krumbholz, Roy; Harper, Jay; Munroe, Kenneth J; Green, Tessa L; Horten, Bruce C; Schmid, Steven M; Teicher, Beverly A

    2015-09-01

    Endosialin/TEM1/CD248 is a cell surface protein expressed at high levels by the malignant cells of about 50% of sarcomas and neuroblastomas. The antibody-drug conjugate (ADC) anti-endosialin-MC-VC-PABC-MMAE was selectively cytotoxic to endosialin-positive cells in vitro and achieved profound and durable antitumor efficacy in preclinical human tumor xenograft models of endosialin-positive disease. MC-VC-PABC-MMAE was conjugated with anti-endosialin with 3-4 MMAE molecules per ADC. The anti-endosialin-MC-VC-PABC-MMAE conjugate was tested for activity in four human cell lines with varied endosialin levels. The HT-1080 fibrosarcoma cells do not express endosialin, A-673 Ewing sarcoma cells and SK-N-AS neuroblastoma cells are moderate expressers of endosialin, and SJSA-1 osteosarcoma cells express very high levels of endosialin. To determine whether endosialin expression was maintained in vivo, A-673 Ewing sarcoma, SK-N-AS neuroblastoma, and SJSA-1 osteosarcoma cells were grown as xenograft tumors in nude mice. The SK-N-AS neuroblastoma and the A-673 Ewing sarcoma lines were selected for in vivo efficacy testing of the anti-endosialin-MC-VC-PABC-MMAE conjugate. The treatment groups included a vehicle control, unconjugated anti-endosialin, an admix control consisting of anti-endosialin and a dose of free MMAE equivalent to the dose administered as the ADC, and the anti-endosialin-MC-VC-PABC-MMAE conjugate. The unconjugated anti-endosialin had no antitumor activity and resulted in similar tumor growth as the vehicle control. The admix control produced a modest tumor growth delay. Administration of the anti-endosialin-MC-VC-PABC-MMAE conjugate resulted in a marked prolonged tumor response of both xenograts. These proof-of-concept results break new ground and open a promising drug discovery approach to these rare and neglected tumors. PMID:26184481

  16. Immunophotosensitizer: the preparation and antitumor properties of monoclonal antibody-hematoporphyrin conjugate

    Science.gov (United States)

    Bai, Shan; Liu, Cheng-gui; Guo, Zhong-He

    1993-03-01

    The immunophotosensitizer was prepared by conjugating hematoporphyrin (HP) with anti-CEA and anti-colonic cancer monoclonal antibody (McAb), respectively. In vitro, the anti-CEA McAb-HP conjugate showed cytotoxicity for human colonic cancer cell line SW1116 which was CEA positive, but no cytotoxicity for the CEA-negative Hep-2 cell line. The cytotoxicity of immunophotosensitizer was much higher than the McAb alone, the HP alone, or the mixture of McAb and HP. In vivo experiments, the nude mice bearing the xenografted human colonic cancer were used to test the activity of the anti-colonic cancer McAb-HP conjugate. The results demonstrated that the tumor necrotic areas of the conjugate-treated animals were notably larger than those of the free HP-treated animals. The specificity offered by the McAb permits increase of the aggregation of the drug at the tumor site. This property makes it possible to use a lower effective dosage of the drug, which minimizes undesired side effects. Further experiments are now in progress.

  17. Rational design, biophysical and biological characterization of site-specific antibody-tubulysin conjugates with improved stability, efficacy and pharmacokinetics.

    Science.gov (United States)

    Thompson, Pamela; Fleming, Ryan; Bezabeh, Binyam; Huang, Fengying; Mao, Shenlan; Chen, Cui; Harper, Jay; Zhong, Haihong; Gao, Xizhe; Yu, Xiang-Qing; Hinrichs, Mary Jane; Reed, Molly; Kamal, Adeela; Strout, Patrick; Cho, Song; Woods, Rob; Hollingsworth, Robert E; Dixit, Rakesh; Wu, Herren; Gao, Changshou; Dimasi, Nazzareno

    2016-08-28

    Antibody-drug conjugates (ADCs) are among the most promising empowered biologics for cancer treatment. ADCs are commonly prepared by chemical conjugation of small molecule cytotoxic anti-cancer drugs to antibodies through either lysine side chains or cysteine thiols generated by the reduction of interchain disulfide bonds. Both methods yield heterogeneous conjugates with complex biophysical properties and suboptimal serum stability, efficacy, and pharmacokinetics. To limit the complexity of cysteine-based ADCs, we have engineered and characterized in vitro and in vivo antibody cysteine variants that allow precise control of both site of conjugation and drug load per antibody molecule. We demonstrate that the chemically-defined cysteine-engineered antibody-tubulysin conjugates have improved ex vivo and in vivo stability, efficacy, and pharmacokinetics when compared to conventional cysteine-based ADCs with similar drug-to-antibody ratios. In addition, to limit the non-target FcγRs mediated uptake of the ADCs by cells of the innate immune system, which may result in off-target toxicities, the ADCs have been engineered to lack Fc-receptor binding. The strategies described herein are broadly applicable to any full-length IgG or Fc-based ADC and have been incorporated into an ADC that is in phase I clinical development.

  18. Rational design, biophysical and biological characterization of site-specific antibody-tubulysin conjugates with improved stability, efficacy and pharmacokinetics.

    Science.gov (United States)

    Thompson, Pamela; Fleming, Ryan; Bezabeh, Binyam; Huang, Fengying; Mao, Shenlan; Chen, Cui; Harper, Jay; Zhong, Haihong; Gao, Xizhe; Yu, Xiang-Qing; Hinrichs, Mary Jane; Reed, Molly; Kamal, Adeela; Strout, Patrick; Cho, Song; Woods, Rob; Hollingsworth, Robert E; Dixit, Rakesh; Wu, Herren; Gao, Changshou; Dimasi, Nazzareno

    2016-08-28

    Antibody-drug conjugates (ADCs) are among the most promising empowered biologics for cancer treatment. ADCs are commonly prepared by chemical conjugation of small molecule cytotoxic anti-cancer drugs to antibodies through either lysine side chains or cysteine thiols generated by the reduction of interchain disulfide bonds. Both methods yield heterogeneous conjugates with complex biophysical properties and suboptimal serum stability, efficacy, and pharmacokinetics. To limit the complexity of cysteine-based ADCs, we have engineered and characterized in vitro and in vivo antibody cysteine variants that allow precise control of both site of conjugation and drug load per antibody molecule. We demonstrate that the chemically-defined cysteine-engineered antibody-tubulysin conjugates have improved ex vivo and in vivo stability, efficacy, and pharmacokinetics when compared to conventional cysteine-based ADCs with similar drug-to-antibody ratios. In addition, to limit the non-target FcγRs mediated uptake of the ADCs by cells of the innate immune system, which may result in off-target toxicities, the ADCs have been engineered to lack Fc-receptor binding. The strategies described herein are broadly applicable to any full-length IgG or Fc-based ADC and have been incorporated into an ADC that is in phase I clinical development. PMID:27327768

  19. Specific Conjugation of the Hinge Region for Homogeneous Preparation of Antibody Fragment-Drug Conjugate: A Case Study for Doxorubicin-PEG-anti-CD20 Fab' Synthesis.

    Science.gov (United States)

    Zhou, Zhan; Zhang, Jing; Zhang, Yan; Ma, Guanghui; Su, Zhiguo

    2016-01-20

    Conventional preparation strategies for antibody-drug conjugates (ADCs) result in heterogeneous products with various molecular sizes and species. In this study, we developed a homogeneous preparation strategy by site-specific conjugation of the anticancer drug with an antibody fragment. The model drug doxorubicin (DOX) was coupled to the Fab' fragment of anti-CD20 IgG at its permissive sites through a heterotelechelic PEG linker, generating an antibody fragment-drug conjugate (AFDC). Anti-CD20 IgG was digested and reduced specifically with β-mercaptoethylamine to generate the Fab' fragment with two free mercapto groups in its hinge region. Meanwhile, DOX was conjugated with α-succinimidylsuccinate ω-maleimide polyethylene glycol (NHS-PEG-MAL) to form MAL-PEG-DOX, which was subsequently linked to the free mercapto containing Fab' fragment to form a Fab'-PEG-DOX conjugate. The dual site-specific bioconjugation was achieved through the combination of highly selective reduction of IgG and introduction of heterotelechelic PEG linker. The resulting AFDC provides an utterly homogeneous product, with a definite ratio of one fragment to two drugs. Laser confocal microscopy and cell ELISA revealed that the AFDC could accumulate in the antigen-positive Daudi tumor cell. In addition, the Fab'-PEG-DOX retained appreciable targeting ability and improved antitumor activity, demonstrating an excellent therapeutic effect on the lymphoma mice model for better cure rate and significantly reduced side effects.

  20. Photocleavable DNA barcode-antibody conjugates allow sensitive and multiplexed protein analysis in single cell

    Science.gov (United States)

    Agasti, Sarit S.; Liong, Monty; Peterson, Vanessa M.; Lee, Hakho; Weissleder, Ralph

    2012-01-01

    DNA barcoding is an attractive technology as it allows sensitive and multiplexed target analysis. However, DNA barcoding of cellular proteins remains challenging, primarily because barcode amplification and readout techniques are often incompatible with the cellular microenvironment. Here, we describe the development and validation of a photocleavable DNA barcode-antibody conjugate method for rapid, quantitative and multiplexed detection of proteins in single live cells. Following target binding, this method allows DNA barcodes to be photoreleased in solution, enabling easy isolation, amplification and readout. As a proof of principle, we demonstrate sensitive and multiplexed detection of protein biomarkers in a variety of cancer cells. PMID:23092113

  1. Radioimmunotherapy: A Specific Treatment Protocol for Cancer by Cytotoxic Radioisotopes Conjugated to Antibodies

    Directory of Open Access Journals (Sweden)

    Hidekazu Kawashima

    2014-01-01

    Full Text Available Radioimmunotherapy (RIT represents a selective internal radiation therapy, that is, the use of radionuclides conjugated to tumor-directed monoclonal antibodies (including those fragments or peptides. In a clinical field, two successful examples of this treatment protocol are currently extended by 90Y-ibritumomab tiuxetan (Zevalin and 131I-tositumomab (Bexxar, both of which are anti-CD20 monoclonal antibodies coupled to cytotoxic radioisotopes and are approved for the treatment of non-Hodgkin lymphoma patients. In addition, some beneficial observations are obtained in preclinical studies targeting solid tumors. To date, in order to reduce the unnecessary exposure and to enhance the therapeutic efficacy, various biological, chemical, and treatment procedural improvements have been investigated in RIT. This review outlines the fundamentals of RIT and current knowledge of the preclinical/clinical trials for cancer treatment.

  2. Effect of previous vaccination with pneumococcal conjugate vaccine on pneumococcal polysaccharide vaccine antibody responses.

    Science.gov (United States)

    Schaballie, H; Wuyts, G; Dillaerts, D; Frans, G; Moens, L; Proesmans, M; Vermeulen, F; De Boeck, K; Meyts, I; Bossuyt, X

    2016-08-01

    During the past 10 years, pneumococcal conjugate vaccine (PCV) has become part of the standard childhood vaccination programme. This may impact upon the diagnosis of polysaccharide antibody deficiency by measurement of anti-polysaccharide immunoglobulin (Ig)G after immunization with unconjugated pneumococcal polysaccharide vaccine (PPV). Indeed, contrary to PPV, PCV induces a T-dependent, more pronounced memory response. The antibody response to PPV was studied retrospectively in patients referred for suspected humoral immunodeficiency. The study population was divided into four subgroups based on age (2-5 years versus ≥ 10 years) and time tested (1998-2005 versus 2010-12). Only 2-5-year-old children tested in 2010-12 had been vaccinated with PCV prior to PPV. The PCV primed group showed higher antibody responses for PCV-PPV shared serotypes 4 and 18C than the unprimed groups. To a lesser extent, this was also found for non-PCV serotype 9N, but not for non-PCV serotypes 19A and 8. Furthermore, PCV-priming elicited a higher IgG2 response. In conclusion, previous PCV vaccination affects antibody response to PPV for shared serotypes, but can also influence antibody response to some non-PCV serotypes (9N). With increasing number of serotypes included in PCV, the diagnostic assessment for polysaccharide antibody deficiency requires careful selection of serotypes that are not influenced by prior PCV (e.g. serotype 8). Further research is needed to identify more serotypes that are not influenced. PMID:26939935

  3. Indication of viruses and virus-specific antibodies by ELISA using conjugates based on. beta. -lactamase obtained by genetic engineering

    Energy Technology Data Exchange (ETDEWEB)

    Kharitonenkov, I.G.; Kordym, V.A.; Khristova, M.L.; Leonov, S.V.; Kirillova, V.S.; Chernykh, S.I.

    1987-10-01

    The method of enzyme-linked immunosorbent assay (ELISA), by means of which antigens and antibodies of different origin can be detected with high sensitivity and specificity, is an immunoenzymatic technique based on the use of conjugates, or macromolecular complexes formed by covalent attachment of enzyme molecules to antigen or antibody molecules. Conjugates based on peroxidase, alkaline phosphatase, and beta-galactosidase are most frequently used to construct immunoenzymatic test systems. The use of these enzymes in ELISA, however, is complicated by the fact that they are often present in free or bound form in the biological material under study, and that their substrates either possess low stability, are difficult to synthesize, or are toxic. In this paper, in order to avoid these shortcomings, the authors develop a method for the biosynthesis of lactamase conjugates which is based on genetic engineering, and demonstrate the viability and stability of these conjugates in radioimmunoenzymatic assay of viruses.

  4. Exploring the effects of linker composition on site-specifically modified antibody-drug conjugates.

    Science.gov (United States)

    Albers, Aaron E; Garofalo, Albert W; Drake, Penelope M; Kudirka, Romas; de Hart, Gregory W; Barfield, Robyn M; Baker, Jeanne; Banas, Stefanie; Rabuka, David

    2014-12-17

    In the context of antibody-drug conjugates (ADCs), noncleavable linkers provide a means to deliver cytotoxic small molecules to cell targets while reducing systemic toxicity caused by nontargeted release of the free drug. Additionally, noncleavable linkers afford an opportunity to change the chemical properties of the small molecule to improve potency or diminish affinity for multidrug transporters, thereby improving efficacy. We employed the aldehyde tag coupled with the hydrazino-iso-Pictet-Spengler (HIPS) ligation to generate a panel of site-specifically conjugated ADCs that varied only in the noncleavable linker portion. The ADC panel comprised antibodies carrying a maytansine payload ligated through one of five different linkers. Both the linker-maytansine constructs alone and the resulting ADC panel were characterized in a variety of in vitro and in vivo assays measuring biophysical and functional properties. We observed that slight differences in linker design affected these parameters in disparate ways, and noted that efficacy could be improved by selecting for particular attributes. These studies serve as a starting point for the exploration of more potent noncleavable linker systems. PMID:25176286

  5. The study of conjugation of anti-CD20 monoclonal antibody for labeling with metallic or lanthanides radionuclides

    International Nuclear Information System (INIS)

    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®). 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®) 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 the influence of different

  6. Effect of different hapten-carrier conjugation ratios and molecular orientations on antibody affinity against a peptide antigen

    DEFF Research Database (Denmark)

    Pedersen, M. K.; Sørensen, Nanna Skall; Heegaard, Peter M. H.;

    2006-01-01

    -based assay systems and in deciding whether a vaccine-induced antibody response will be protective. With ovalbumin as a carrier protein and a peptide (7.2NY) representing a 19 ammo acid sequence from the E. coli-derived Verotoxin 2e as a model hapten we investigated whether it was possible to influence...... the affinity and titre of antibodies raised against the hapten using different conjugation ratios and orientations. The peptide was coupled to ovalbumin in four Conjugation ratios and two molecular orientations - terminal and central - and the Conjugates were verified by mass spectrometry. Mice were immunised......, the molecular orientation of the Coupled peptide has a major effect on the anti-peptide antibody titres induced....

  7. Enzymatic synthesis of antibody-human serum albumin conjugate for targeted drug delivery using tyrosinase from Agaricus bisporus

    OpenAIRE

    Rollett, Alexandra; Thallinger, Barbara; Ohradanova-Repic, Anna; Machacek, Christian; Walenta, Evelyn; Paulo, Artur Cavaco; Birner-Gruenberger, Ruth; Bogner-Strauss, Juliane G.; Stockinger, Hannes; Guebitz, G.M.

    2013-01-01

    Highly specific targeted drug delivery devices can be obtained with antibody-human serum albumin (mAb-HSA) conjugates. However, their conventional production involves several reaction steps including chemical modification and activation of both proteins followed by cross-linking often involving toxic chemicals. Here, we describe the enzymatic synthesis of mAb-HSA conjugates for targeted drug delivery devices using tyrosinase from Agaricus bisporus under mild reaction conditions (pH 6.8, 25 [d...

  8. Synthesis and Preliminary Biological Evaluation of High-drug Load Paclitaxel-Antibody Conjugates for Tumor-targeted Chemotherapy1

    OpenAIRE

    Quiles, Sherly; Raisch, Kevin P.; Sanford, Leisa L.; Bonner, James A.; Safavy, Ahmad

    2010-01-01

    The goal of this study was to design paclitaxel (PTX)-monoclonal antibody (MAb) prodrug conjugates (PTXMAbs) with the ability to deliver therapeutically significant doses of the drug to the tumor while avoiding the previously observed solubility limitations of conjugates with PTX : MAb molar ratios of >3. New PTX conjugates were synthesized using the discrete poly(ethylene glycol) (dPEG) as linkers. These compounds, PTX-L-Lys[(dPEG12)3-dPEG4]-dPEG6-NHS (9a and 9b, for L=GL or SX, respectively...

  9. Nano-Magnetic Immunosensor Based on Staphylococcus Protein A and the Amplification Effect of HRP-Conjugated Phage Antibody

    Directory of Open Access Journals (Sweden)

    Xihui Mu

    2015-02-01

    Full Text Available In this research, super-paramagnetic Fe3O4 nanoparticles (magnetic particles were coated with Staphylococcus protein A (SPA and coupled with polyclonal antibody (pcAb to construct magnetic capturing probes, and HRP-conjugated phage antibody was then used as specific detecting probe to design a labeled immunosensor for trace detection of Staphylococcus aureus enterotoxin B (SEB. The linear detection range of the sensor was 0.008~125 µg/L, the regression equation was Y = 0.487X + 1.2 (R = 0.996, N = 15, p < 0.0001, the limit of detection (LOD was 0.008 µg/L, and the limit of quantification (LOQ was 0.008 µg/L. HRP-conjugated phage antibody, SPA and magnetic particles can enhance the sensitivity 4-fold, 3-fold and 2.6-fold higher, respectively. Compared with conventional double-antibody sandwich ELISA, the detection sensitivity of the sensor was 31-fold higher resulting from the integrated amplifying effect. The immunosensor integrates the unique advantages of SPA-oriented antibody as magnetic capturing probe, HRP-conjugated phage antibody as detecting probe, magnetic separation immunoassay technique, and several other advanced techniques, so it achieves high sensitivity, specificity and interference-resistance. It is proven to be well suited for analysis of trace SEB in various environmental samples with high recovery rate and reproducibility.

  10. Antibody-Conjugated Paramagnetic Nanobeads: Kinetics of Bead-Cell Binding

    Directory of Open Access Journals (Sweden)

    Shahid Waseem

    2014-05-01

    Full Text Available Specific labelling of target cell surfaces using antibody-conjugated paramagnetic nanobeads is essential for efficient magnetic cell separation. However, studies examining parameters determining the kinetics of bead-cell binding are scarce. The present study determines the binding rates for specific and unspecific binding of 150 nm paramagnetic nanobeads to highly purified target and non-target cells. Beads bound to cells were enumerated spectrophotometrically. Results show that the initial bead-cell binding rate and saturation levels depend on initial bead concentration and fit curves of the form A(1 − exp(−kt. Unspecific binding within conventional experimental time-spans (up to 60 min was not detectable photometrically. For CD3-positive cells, the probability of specific binding was found to be around 80 times larger than that of unspecific binding.

  11. Aggregation of Antibody Drug Conjugates at Room Temperature: SAXS and Light Scattering Evidence for Colloidal Instability of a Specific Subpopulation.

    Science.gov (United States)

    Frka-Petesic, B; Zanchi, D; Martin, N; Carayon, S; Huille, S; Tribet, C

    2016-05-17

    Coupling a hydrophobic drug onto monoclonal antibodies via lysine residues is a common route to prepare antibody-drug conjugates (ADC), a promising class of biotherapeutics. But a few chemical modifications on protein surface often increase aggregation propensity, without a clear understanding of the aggregation mechanisms at stake (loss of colloidal stability, self-assemblies, denaturation, etc.), and the statistical nature of conjugation introduces polydispersity in the ADC population, which raises questions on whether the whole ADC population becomes unstable. To characterize the average interactions between ADC, we monitored small-angle X-ray scattering in solutions of monoclonal IgG1 human antibody drug conjugate, with average degree of conjugation of 0, 2, or 3 drug molecules per protein. To characterize stability, we studied the kinetics of aggregation at room temperature. The intrinsic Fuchs stability ratio of the ADC was estimated from the variation over time of scattered light intensity and hydrodynamic radius, in buffers of varying pH, and at diverse sucrose (0% or 10%) and NaCl (0 or 100 mM) concentrations. We show that stable ADC stock solutions became unstable upon pH shift, well below the pH of maximum average attraction between IgGs. Data indicate that aggregation can be ascribed to a fraction of ADC population usually representing less than 30 mol % of the sample. In contrast to the case of (monodisperse) monoclonal antibodies, our results suggest that a poor correlation between stability and average interaction parameters should be expected as a corollary of dispersity of ADC conjugation. In practice, the most unstable fraction of the ADC population can be removed by filtration, which affects remarkably the apparent stability of the samples. Finally, the lack of correlation between the kinetic stability and variations of the average inter-ADC interactions is tentatively attributed to the uneven nature of charge distributions and the presence of

  12. Emission variation in infrared (CdSeTe)/ZnS quantum dots conjugated to antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Jaramillo Gómez, J.A. [UPIITA – Instituto Politécnico Nacional, México D. F. 07320, México (Mexico); Casas Espinola, J.L., E-mail: jlcasas@esfm.ipn.mx [ESFM – Instituto Politécnico Nacional, México D. F. 07738, México (Mexico); Douda, J. [UPIITA – Instituto Politécnico Nacional, México D. F. 07320, México (Mexico)

    2014-11-15

    The paper presents the photoluminescence (PL) and Raman scattering investigations of infrared CdSeTe/ZnS quantum dots (QDs) with emission at 800 nm (1.60 eV) in nonconjugated states and after the conjugation to the anti-papilloma virus antibodies (Ab). The Raman scattering study has shown that the CdSeTe core includes two layers with different material compositions such as: CdSe{sub 0.5}Te{sub 0.5} and CdSe{sub 0.7}Te{sub 0.3}. PL spectra of nonconjugated CdSeTe/ZnS QDs are characterized by two Gaussian shape PL bands related to exciton emission in the CdSeTe core and in intermediate layer at the core/shell interface. PL spectra of bioconjugated QDs have changed essentially: the main PL band related to the core emission shifts into high energy and become asymmetric. The energy diagram of double core/shell CdSeTe/ZnS QDs has been analyzed to explain the PL spectrum of nonconjugated QDs and its transformation at the bioconjugation to the papiloma virus antibodies. It is shown that the PL spectrum transformation in bioconjugated QDs can be a powerful technique for biology and medicine.

  13. Antibody-antigen-adjuvant conjugates enable co-delivery of antigen and adjuvant to dendritic cells in cis but only have partial targeting specificity.

    Directory of Open Access Journals (Sweden)

    Martin Kreutz

    Full Text Available Antibody-antigen conjugates, which promote antigen-presentation by dendritic cells (DC by means of targeted delivery of antigen to particular DC subsets, represent a powerful vaccination approach. To ensure immunity rather than tolerance induction the co-administration of a suitable adjuvant is paramount. However, co-administration of unlinked adjuvant cannot ensure that all cells targeted by the antibody conjugates are appropriately activated. Furthermore, antigen-presenting cells (APC that do not present the desired antigen are equally strongly activated and could prime undesired responses against self-antigens. We, therefore, were interested in exploring targeted co-delivery of antigen and adjuvant in cis in form of antibody-antigen-adjuvant conjugates for the induction of anti-tumour immunity. In this study, we report on the assembly and characterization of conjugates consisting of DEC205-specific antibody, the model antigen ovalbumin (OVA and CpG oligodeoxynucleotides (ODN. We show that such conjugates are more potent at inducing cytotoxic T lymphocyte (CTL responses than control conjugates mixed with soluble CpG. However, our study also reveals that the nucleic acid moiety of such antibody-antigen-adjuvant conjugates alters their binding and uptake and allows delivery of the antigen and the adjuvant to cells partially independently of DEC205. Nevertheless, antibody-antigen-adjuvant conjugates are superior to antibody-free antigen-adjuvant conjugates in priming CTL responses and efficiently induce anti-tumour immunity in the murine B16 pseudo-metastasis model. A better understanding of the role of the antibody moiety is required to inform future conjugate vaccination strategies for efficient induction of anti-tumour responses.

  14. In vitro photothermal destruction of neuroblastoma cells using carbon nanotubes conjugated with GD2 monoclonal antibody

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Chung-Hao; Huang, Yao-Jhang; Chang, Chia-Wei; Peng, Ching-An [Department of Chemical Engineering, National Taiwan University, Taipei, Taiwan (China); Hsu, Wen-Ming, E-mail: cpeng@mtu.ed [Department of Surgery, National Taiwan University, Taipei, Taiwan (China)

    2009-08-05

    Despite aggressive multimodality therapy, most neuroblastoma-bearing patients relapse and survival rate remains poor. Exploration of alternative therapeutic modalities is needed. Carbon nanotubes (CNTs), revealing optical absorbance in the near-infrared region, warrant their merits in photothermal therapy. In order to specifically target disialoganglioside (GD2) overexpressed on the surface of neuroblastoma stNB-V1 cells, GD2 monoclonal antibody (anti-GD2) was conjugated to acidified CNTs. To examine the fate of anti-GD2 bound CNTs after incubation with stNB-V1 cells, rhodamine B was labeled on carboxylated CNTs functionalized with and without anti-GD2. Our results illustrated that anti-GD2-linked CNTs were extensively internalized by neuroblastoma cells via GD2-mediated endocytosis. In addition, we showed that anti-GD2 bound CNTs were not ingested by PC12 cells without GD2 expression. After anti-GD2 conjugated CNTs were incubated with neuroblastoma cells for 6 h and endocytosed by the cells, CNT-laden neuroblastoma cells were further irradiated with an 808 nm near-infrared (NIR) laser with intensity ramping from 0.6 to 6 W cm{sup -2} for 10 min which was then maintained at 6 W cm{sup -2} for an additional 5 min. Post-NIR laser exposure, and after being examined by calcein-AM dye, stNB-V1 cells were all found to undergo necrosis, while non-GD2 expressing PC12 cells all remained viable. Based on the in vitro study, CNTs bound with anti-GD2 have the potential to be utilized as a therapeutic thermal coupling agent that generates heat sufficient to selectively kill neuroblastoma cells under NIR laser light exposure.

  15. Non-invasive tumor detection in small animals using novel functional Pluronic nanomicelles conjugated with anti-mesothelin antibody

    Science.gov (United States)

    Ding, Hong; Yong, Ken-Tye; Law, Wing-Chueng; Roy, Indrajit; Hu, Rui; Wu, Fang; Zhao, Weiwei; Huang, Kun; Erogbogbo, Folarin; Bergey, Earl J.; Prasad, Paras N.

    2011-04-01

    In this study QDs were encapsulated in carboxylated PluronicF127 (F127COOH) triblock polymeric micelles and conjugated with anti-mesothelin antibody for the purpose of alleviating potential toxicity, enhancing the stability and improving targeting efficiency of CdTe/ZnS quantum dots (QDs) in tumors. The amphiphilic triblock polymer of F127COOH contains hydrophilic carboxylated poly(ethylene oxide) (PEO) and hydrophobic poly(propylene oxide) (PPO) units. After encapsulating QDs into carboxylated F127 (F127COOH-QD) micelles, the particles were conjugated with anti-mesothelin antibodies to allow targeting of cancerous areas. The size of the monodispersed spherical QD-containing micelles was determined to be ~120 nm by dynamic light scattering (DLS). The critical micelle concentration (CMC) was estimated to be 4.7 × 10-7 M. In an in vitro study, the anti-methoselin antibody conjugated F127COOH (Me-F127COOH-QD) nanomicelles showed negligible cytotoxicity to pancreatic cancer cells (Panc-1). Confocal microscopy demonstrated that the Me-F127COOH-QD nanomicelles were taken up more efficiently by Panc-1cells, due to antibody mediated targeting. An in vivo imaging study showed that Me-F127COOH-QD nanomicelles accumulated at the pancreatic tumor site 15 min after intravenous injection. In addition, the low in vivo toxicity of the nanomicellar formulation was evaluated by pathological assays. These results suggest that anti-mesothein antibody conjugated carboxylated F127 nanomicelles may serve as a promising nanoscale platform for early human pancreatic cancer detection and targeted drug delivery.

  16. Altering Antibody-Drug Conjugate Binding to the Neonatal Fc Receptor Impacts Efficacy and Tolerability.

    Science.gov (United States)

    Hamblett, Kevin J; Le, Tiep; Rock, Brooke M; Rock, Dan A; Siu, Sophia; Huard, Justin N; Conner, Kip P; Milburn, Robert R; O'Neill, Jason W; Tometsko, Mark E; Fanslow, William C

    2016-07-01

    Antibody-drug conjugates (ADC) rely on the target-binding specificity of an antibody to selectively deliver potent drugs to cancer cells. IgG antibody half-life is regulated by neonatal Fc receptor (FcRn) binding. Histidine 435 of human IgG was mutated to alanine (H435A) to explore the effect of FcRn binding on the pharmacokinetics, efficacy, and tolerability of two separate maytansine-based ADC pairs with noncleavable linkers, (c-DM1 and c-H435A-DM1) and (7v-Cys-may and 7v-H435A-Cys-may). The in vitro cell-killing potency of each pair of ADCs was similar, demonstrating that H435A showed no measurable impact on ADC bioactivity. The H435A mutant antibodies showed no detectable binding to human or mouse FcRn in vitro, whereas their counterpart wild-type IgG ADCs were found to bind to FcRn at pH = 6.0. In xenograft bearing SCID mice expressing mouse FcRn, the AUC of 7v-Cys-may was 1.6-fold higher than that of 7v-H435A-may, yet the observed efficacy was similar. More severe thrombocytopenia was observed with 7v-H435A-Cys-may as compared to 7v-Cys-may at multiple dose levels. The AUC of c-DM1 was approximately 3-fold higher than that of c-H435A-DM1 in 786-0 xenograft bearing SCID mice, which led to a 3-fold difference in efficacy by dose. Murine FcRn knockout, human FcRn transgenic line 32 SCID animals bearing 786-0 xenografts showed an amplified exposure difference between c-DM1 and c-H435A-DM1 as compared to murine FcRn expressing SCID mice, leading to a 10-fold higher dose required for efficacy despite a 6-fold higher AUC of the c-H435A-DM1. The accelerated clearance observed for the noncleavable maytansine ADCs with the H435A FcRn mutation led to reduced efficacy at equivalent doses and exacerbation of clinical pathology parameters (decreased tolerability) at equivalent doses. The results show that reduced ADC clearance mediated by FcRn modulation can improve therapeutic index. PMID:27248573

  17. Association of Serotype-Specific Antibody Concentrations and Functional Antibody Titers with Subsequent Pneumococcal Carriage in Toddlers Immunized with a 9-Valent Pneumococcal Conjugate Vaccine

    OpenAIRE

    Simell, Birgit; Nurkka, Anu; Lahdenkari, Mika; Givon-Lavi, Noga; Käyhty, Helena; Dagan, Ron; Jokinen, Jukka

    2012-01-01

    Association of pneumococcal nasopharyngeal carriage with the concentration and opsonophagocytic activity (OPA) of serum serotype-specific antibodies was determined for toddlers 1 month after immunization with a 9-valent pneumococcal conjugate vaccine. Higher anti-serotype 14 and anti-serotype 19F IgG and anti-serotype 14 IgM correlated with a lowered probability of pneumococcal acquisition. Postvaccination OPA did not correlate with pneumococcal carriage.

  18. Blood stage merozoite surface protein conjugated to nanoparticles induce potent parasite inhibitory antibodies.

    Science.gov (United States)

    Pusic, Kae; Xu, Hengyi; Stridiron, Andrew; Aguilar, Zoraida; Wang, Andrew; Hui, George

    2011-11-01

    In this proof-of-concept study we report the use of nanoparticles as a vaccine delivery system for a blood stage malaria vaccine. The recombinant malarial antigen, Merozoite Surface Protein 1 (rMSP1) of Plasmodium falciparum served as the model vaccine. The rMSP1 was covalently conjugated to polymer-coated quantum dot CdSe/ZnS nanoparticles (QDs) via surface carboxyl groups, forming rMSP1-QDs. Anti-MSP1 antibody responses induced by rMSP1-QDs were found to have 2-3 log higher titers than those obtained with rMSP1 administered with the conventional adjuvants, Montanide ISA51 and CFA. Moreover, the immune responsiveness and the induction of parasite inhibitory antibodies were significantly superior in mice injected with rMSP1-QDs. The rMSP1-QDs delivered via intra-peritoneal (i.p.), intra-muscular (i.m.), and subcutaneous (s.c.) routes were equally efficacious. The high level of immunogenicity exhibited by the rMSP1-QDs was achieved without further addition of other adjuvant components. Bone marrow derived dendritic cells were shown to efficiently take up the nanoparticles leading to their activation and the expression/secretion of key cytokines, suggesting that this may be a mode of action for the enhanced immunogenicity. This study provides promising results for the use of water soluble, inorganic nanoparticles (<15 nm) as potent vehicles/platforms to enhance the immunogenicity of polypeptide antigens in adjuvant-free immunizations.

  19. Imaging of Hsp70-positive tumors with cmHsp70.1 antibody-conjugated gold nanoparticles

    Directory of Open Access Journals (Sweden)

    Gehrmann MK

    2015-09-01

    Full Text Available Mathias K Gehrmann,1 Melanie A Kimm,2 Stefan Stangl,1 Thomas E Schmid,1 Peter B Noël,2 Ernst J Rummeny,2 Gabriele Multhoff11Department of Radiation Oncology, 2Department of Diagnostic and Interventional Radiology, Klinikum rechts der Isar, Technische Universität München, Munich, GermanyAbstract: Real-time imaging of small tumors is still one of the challenges in cancer diagnosis, prognosis, and monitoring of clinical outcome. Targeting novel biomarkers that are selectively expressed on a large variety of different tumors but not normal cells has the potential to improve the imaging capacity of existing methods such as computed tomography. Herein, we present a novel technique using cmHsp70.1 monoclonal antibody-conjugated spherical gold nanoparticles for quantification of the targeted uptake of gold nanoparticles into membrane Hsp70-positive tumor cells. Upon binding, cmHsp70.1-conjugated gold nanoparticles but not nanoparticles coupled to an isotype-matched IgG1 antibody or empty nanoparticles are rapidly taken up by highly malignant Hsp70 membrane-positive mouse tumor cells. After 24 hours, the cmHsp70.1-conjugated gold nanoparticles are found to be enriched in the perinuclear region. Specificity for membrane Hsp70 was shown by using an Hsp70 knockout tumor cell system. Toxic side effects of the cmHsp70.1-conjugated nanoparticles are not observed at a concentration of 1–10 µg/mL. Experiments are ongoing to evaluate whether cmHsp70.1 antibody-conjugated gold nanoparticles are suitable for the detection of membrane-Hsp70-positive tumors in vivo.Keywords: heat shock protein 70, tumor biomarker, theranostics, multimodal CT, multispectral CT, k-edge

  20. World Antibody-Drug Conjugate Summit, October 15–16, 2013, San Francisco, CA

    Science.gov (United States)

    Klinguer-Hamour, Christine; Strop, Pavel; Shah, Dhaval K; Ducry, Laurent; Xu, April; Beck, Alain

    2014-01-01

    The World Antibody-Drug Conjugate (WADC) Summits organized by Hanson Wade are currently the largest meetings fully dedicated to ADCs. The first global ADC Summit was organized in Boston in October 2010. Since 2011, two WADC are held every year in Frankfurt and San Francisco, respectively. The 2013 WADC San Francisco event was structured around plenary sessions with keynote speakers from AbbVie, Agensys, ImmunoGen, Immunomedics, Genentech, Pfizer and Seattle Genetics. Parallel tracks were also organized addressing ADC discovery, development and optimization of chemistry, manufacturing and control (CMC) issues. Discovery and process scientists, regulatory experts (US Food and Drug Administration), academics and clinicians were present, including representatives from biotechnology firms (Concortis, CytomX Therapeutics, Glykos, Evonik, Igenica, Innate Pharma, Mersana Therapeutics, Polytherics, Quanta Biodesign, Redwood Bioscience, Sutro Biopharma, SynAffix), pharmaceutical companies (Amgen, Genmab, Johnson and Johnson, MedImmune, Novartis, Progenics, Takeda) and contract research or manufacturing organizations (Baxter, Bayer, BSP Pharmaceuticals, Fujifilm/Diosynth, Lonza, Pierre Fabre Contract Manufacturing, Piramal, SAFC, SafeBridge). PMID:24423618

  1. World Antibody-Drug Conjugate Summit, October 15-16, 2013, San Francisco, CA.

    Science.gov (United States)

    Klinguer-Hamour, Christine; Strop, Pavel; Shah, Dhaval K; Ducry, Laurent; Xu, April; Beck, Alain

    2014-01-01

    The World Antibody-Drug Conjugate (WADC) Summits organized by Hanson Wade are currently the largest meetings fully dedicated to ADCs. The first global ADC Summit was organized in Boston in October 2010. Since 2011, two WADC are held every year in Frankfurt and San Francisco, respectively. The 2013 WADC San Francisco event was structured around plenary sessions with keynote speakers from AbbVie, Agensys, ImmunoGen, Immunomedics, Genentech, Pfizer and Seattle Genetics. Parallel tracks were also organized addressing ADC discovery, development and optimization of chemistry, manufacturing and control (CMC) issues. Discovery and process scientists, regulatory experts (US Food and Drug Administration), academics and clinicians were present, including representatives from biotechnology firms (Concortis, CytomX Therapeutics, Glykos, Evonik, Igenica, Innate Pharma, Mersana Therapeutics, Polytherics, Quanta Biodesign, Redwood Bioscience, Sutro Biopharma, SynAffix), pharmaceutical companies (Amgen, Genmab, Johnson and Johnson, MedImmune, Novartis, Progenics, Takeda) and contract research or manufacturing organizations (Baxter, Bayer, BSP Pharmaceuticals, Fujifilm/Diosynth, Lonza, Pierre Fabre Contract Manufacturing, Piramal, SAFC, SafeBridge). PMID:24423618

  2. Anti-neuropilin 1 antibody Fab' fragment conjugated liposomal docetaxel for active targeting of tumours.

    Science.gov (United States)

    Manjappa, Arehalli S; Goel, Peeyush N; Gude, Rajiv P; Ramachandra Murthy, Rayasa S

    2014-09-01

    Neuropilin-1, a transmembrane receptor entailed in wide range of human tumour cell lines and diverse neoplasms, mediates the effects of VEGF and Semaphorins during the processes of cellular proliferation, survival and migration. In view of this, we had developed and evaluated in vitro and in vivo efficacy of anti-neuropilin-1 immunoliposomes against neuropilin-1 receptor expressing tumours. The PEGylated liposomes loaded with docetaxel were prepared using thin film hydration method. Functionalised PEGylated liposomes were prepared using post-insertion technique. Anti-neuropilin-1 immunoliposomes were prepared by covalently conjugating Fab' fragments of neuropilin-1 antibody to functionalised PEGylated liposomes via thioether linkage. In vivo evaluation of Taxotere and liposomal formulations was performed using intradermal tumour model to demonstrate anti-angiogenic and tumour regression ability. The modified Fab' fragments and immunoliposomes were found to be immunoreactive against A549 cells. Further, docetaxel loaded PEGylated liposomes and PEGylated immunoliposomes demonstrated higher in vitro cytotoxicity than Taxotere formulation at the same drug concentration and exposure time. The live imaging showed distinctive cellular uptake of functional immunoliposomes. Further, significant decrease in micro-blood vessel density and tumour volumes was observed using bio-engineered liposomes. The results clearly highlight the need to seek neuropilin-1 as one of the prime targets in developing an anti-angiogenic therapy.

  3. Two-color SERS microscopy for protein co-localization in prostate tissue with primary antibody-protein A/G-gold nanocluster conjugates

    Science.gov (United States)

    Salehi, Mohammad; Schneider, Lilli; Ströbel, Philipp; Marx, Alexander; Packeisen, Jens; Schlücker, Sebastian

    2014-01-01

    SERS microscopy is a novel staining technique in immunohistochemistry, which is based on antibodies labeled with functionalized noble metal colloids called SERS labels or nanotags for optical detection. Conventional covalent bioconjugation of these SERS labels cannot prevent blocking of the antigen recognition sites of the antibody. We present a rational chemical design for SERS label-antibody conjugates which addresses this issue. Highly sensitive, silica-coated gold nanoparticle clusters as SERS labels are non-covalently conjugated to primary antibodies by using the chimeric protein A/G, which selectively recognizes the Fc part of antibodies and therefore prevents blocking of the antigen recognition sites. In proof-of-concept two-color imaging experiments for the co-localization of p63 and PSA on non-neoplastic prostate tissue FFPE specimens, we demonstrate the specificity and signal brightness of these rationally designed primary antibody-protein A/G-gold nanocluster conjugates.SERS microscopy is a novel staining technique in immunohistochemistry, which is based on antibodies labeled with functionalized noble metal colloids called SERS labels or nanotags for optical detection. Conventional covalent bioconjugation of these SERS labels cannot prevent blocking of the antigen recognition sites of the antibody. We present a rational chemical design for SERS label-antibody conjugates which addresses this issue. Highly sensitive, silica-coated gold nanoparticle clusters as SERS labels are non-covalently conjugated to primary antibodies by using the chimeric protein A/G, which selectively recognizes the Fc part of antibodies and therefore prevents blocking of the antigen recognition sites. In proof-of-concept two-color imaging experiments for the co-localization of p63 and PSA on non-neoplastic prostate tissue FFPE specimens, we demonstrate the specificity and signal brightness of these rationally designed primary antibody-protein A/G-gold nanocluster conjugates

  4. TLR9-adjuvanted pneumococcal conjugate vaccine induces antibody-independent memory responses in HIV-infected adults

    OpenAIRE

    Offersen, Rasmus; Melchjorsen, Jesper; Paludan, Søren R; Østergaard, Lars; Tolstrup, Martin; Søgaard, Ole S.

    2012-01-01

    HIV-patients have excess of pneumococcal infection. We immunized 40 HIV-patients twice with pneumococcal conjugate vaccine (Prevnar, Pfizer) +/− a TLR9 agonist (CPG 7909). Peripheral blood mononuclear cells were stimulated with pneumococcal polysaccharides and cytokine concentrations measured. The CPG 7909 adjuvant group had significantly higher relative cytokine responses than the placebo group for IL-1β, IL-2R, IL-6, IFN-γ and MIP-β, which, did not correlate with IgG antibody responses. The...

  5. Demonstration of Actinomyces and Arachnia species in cervicovaginal smears by direct staining with species-specific fluorescent-antibody conjugate.

    OpenAIRE

    Pine, L; Malcolm, G B; Curtis, E M; Brown, J. M.

    1981-01-01

    For direct observation of microaerophilic actinomycetes by fluorescent antibody, a procedure was developed in which pepsin treatment and rhodamine conjugate of normal serum were used to reduce nonspecific staining in cervicovaginal smears. Actinomyces israelii, Actinomyces naeslundii, and Arachnia propionica were observed in cervicovaginal smears from women who did use and who did not use an intrauterine contraceptive device. A. israelii was found more commonly in women with an intrauterine c...

  6. Engineering of ultra-small diagnostic nanoprobes through oriented conjugation of single-domain antibodies and quantum dots

    OpenAIRE

    sprotocols

    2015-01-01

    Authors: Alyona Sukhanova, Klervi Even-Desrumeaux, Patrick Chames, Daniel Baty, Mikhail Artemyev, Vladimir Oleinikov & Igor Nabiev ### Abstract Nanoparticle-based biodetection commonly employs monoclonal antibodies (mAbs) for targeting. Although several types of conjugates have been used for biomarker labeling, the large size of mAbs limits the number of ligands per nanoparticle, impedes their intratumoral distribution, and limits intracellular penetration. Furthermore, the condi...

  7. PEG-coated gold nanorod monoclonal antibody conjugates in preclinical research with optoacoustic tomography, photothermal therapy, and sensing

    Science.gov (United States)

    Liopo, Anton V.; Conjusteau, André; Oraevsky, Alexander A.

    2012-02-01

    Gold nanorods (GNR) with a peak absorption wavelength of 760 nm were prepared using a seed-mediated method. A novel protocol has been developed to replace hexadecyltrimethylammonium bromide (CTAB) on the surface of GNR with 16-mercaptohexadecanoic acid (MHDA) and metoxy-poly(ethylene glycol)-thiol (PEG), and the monoclonal antibodies: HER2 or CD33. The physical chemistry property of the conjugates was monitored through optical and zetapotential measurements to confirm surface chemistry. The plasmon resonance is kept in the near infrared area, and changes from strong positive charge for GNR-CTAB to slightly negative for GNR-PEG-mAb conjugates are observed. The conjugates were investigated for different cells lines: breast cancer cells and human leukemia lines in vivo applications. These results demonstrate successful tumor accumulation of our modified PEG-MHDA conjugates of GNR for HER2/neu in both overexpressed breast tumors in nude mice, and for thermolysis of human leukemia cells in vitro. The conjugates are non-toxic and can be used in pre-clinical applications, as well as molecular and optoacoustic imaging, and quantitative sensing of biological substrates.

  8. Multifunctional HER2-antibody conjugated polymeric nanocarrier-based drug delivery system for multi-drug-resistant breast cancer therapy.

    Science.gov (United States)

    Vivek, Raju; Thangam, Ramar; NipunBabu, Varukattu; Rejeeth, Chandrababu; Sivasubramanian, Srinivasan; Gunasekaran, Palani; Muthuchelian, Krishnasamy; Kannan, Soundarapandian

    2014-05-14

    Nanotechnology-based medical approaches have made tremendous potential for enhancing the treatment efficacy with minimal doses of chemotherapeutic drugs against cancer. In this study, using tamoxifen (Tam), biodegradable antibody conjugated polymeric nanoparticles (NPs) was developed to achieve targeted delivery as well as sustained release of the drug against breast cancer cells. Poly(D,L-lactic-co-glycolic acid) (PLGA) NPs were stabilized by coating with poly(vinyl alcohol) (PVA), and copolymer polyvinyl-pyrrolidone (PVP) was used to conjugate herceptin (antibody) with PLGA NPs for promoting the site-specific intracellular delivery of Tam against HER2 receptor overexpressed breast cancer (MCF-7) cells. The Tam-loaded PVP-PLGA NPs and herceptin-conjugated Tam-loaded PVP-PLGA NPs were characterized in terms of morphology, size, surface charge, and structural chemistry by dynamic light scattering (DLS), Transmission electron microscopy (TEM), ζ potential analysis, 1H nuclear magnetic resonance (NMR), and Fourier transform infrared (FT-IR) spectroscopy. pH-based drug release property and the anticancer activity (in vitro and in vivo models) of the herceptin conjugated polymeric NPs were evaluated by flow cytometry and confocal image analysis. Besides, the extent of cellular uptake of drug via HER2 receptor-mediated endocytosis by herceptin-conjugated Tam-loaded PVP-PLGA NPs was examined. Furthermore, the possible signaling pathway of apoptotic induction in MCF-7 cells was explored by Western blotting, and it was demonstrated that drug-loaded PLGA NPs were capable of inducing apoptosis in a caspase-dependent manner. Hence, this nanocarrier drug delivery system (DDS) not only actively targets a multidrug-resistance (MDR) associated phenotype (HER2 receptor overexpression) but also improves therapeutic efficiency by enhancing the cancer cell targeted delivery and sustained release of therapeutic agents. PMID:24780315

  9. Aldehyde tag coupled with HIPS chemistry enables the production of ADCs conjugated site-specifically to different antibody regions with distinct in vivo efficacy and PK outcomes.

    Science.gov (United States)

    Drake, Penelope M; Albers, Aaron E; Baker, Jeanne; Banas, Stefanie; Barfield, Robyn M; Bhat, Abhijit S; de Hart, Gregory W; Garofalo, Albert W; Holder, Patrick; Jones, Lesley C; Kudirka, Romas; McFarland, Jesse; Zmolek, Wes; Rabuka, David

    2014-07-16

    It is becoming increasingly clear that site-specific conjugation offers significant advantages over conventional conjugation chemistries used to make antibody-drug conjugates (ADCs). Site-specific payload placement allows for control over both the drug-to-antibody ratio (DAR) and the conjugation site, both of which play an important role in governing the pharmacokinetics (PK), disposition, and efficacy of the ADC. In addition to the DAR and site of conjugation, linker composition also plays an important role in the properties of an ADC. We have previously reported a novel site-specific conjugation platform comprising linker payloads designed to selectively react with site-specifically engineered aldehyde tags on an antibody backbone. This chemistry results in a stable C-C bond between the antibody and the cytotoxin payload, providing a uniquely stable connection with respect to the other linker chemistries used to generate ADCs. The flexibility and versatility of the aldehyde tag conjugation platform has enabled us to undertake a systematic evaluation of the impact of conjugation site and linker composition on ADC properties. Here, we describe the production and characterization of a panel of ADCs bearing the aldehyde tag at different locations on an IgG1 backbone conjugated using Hydrazino-iso-Pictet-Spengler (HIPS) chemistry. We demonstrate that in a panel of ADCs with aldehyde tags at different locations, the site of conjugation has a dramatic impact on in vivo efficacy and pharmacokinetic behavior in rodents; this advantage translates to an improved safety profile in rats as compared to a conventional lysine conjugate. PMID:24924618

  10. Development and validation of the 57Co assay for determining the ligand to antibody ratio in bifunctional chelate/antibody conjugates for use in radioimmunotherapy

    International Nuclear Information System (INIS)

    Introduction: The ligand to antibody ratio is an important characteristic of a chelate/antibody conjugate. It has been widely reported that if the ratio is too high, there will be detrimental effects on immunoreactivity and biodistribution; conversely, if the ratio is too low, the radionuclide may not bind efficiently, and the stability and the specific activity will be reduced. There are little published data on the accuracy or precision of the 57Co assay. The UK Clinical Trials Regulations state that “systems with procedures that assure the quality of every aspect of the trial should be implemented”. The aims of this study were to assess the reliability and accuracy of the 57Co binding assay and validate it against defined criteria. Method: Thirty-two serial assays were assessed for reliability. Two batches of conjugated antibody were also analysed by matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF) mass spectrometry (MS) to allow the comparison of the functional test with a physical method. Results: Reliability: The coefficient of variation was 0.13. Accuracy: There was 9% variation between the 57Co binding assay and MALDI-TOF MS results. Conclusion: A detailed method for the 57Co ligand to antibody test is described that allows a discrete value to be obtained. The assay was validated as fit for purpose against target values of coefficient of variation <0.20, accuracy±10%, over a permissive range of 0.5–3.0 ligand to antibody ratio.

  11. Characterization of the antibody response to a Haemophilus influenzae type b conjugate vaccine in children with recurrent lower respiratory tract infection

    DEFF Research Database (Denmark)

    Kristensen, K; Barington, T; Pressler, T;

    1995-01-01

    once with a Haemophilus influenzae type b (Hib) conjugate vaccine. Total IgG subclasses, total antipolysaccharide Hib antibodies, and antipolysaccharide Hib antibodies of IgM, IgG, IgA, and IgG1-4 specificity were determined by ELISA. There were no significant differences between the two groups in any...

  12. Genetically Encoded Azide Containing Amino Acid in Mammalian Cells Enables Site-Specific Antibody-Drug Conjugates Using Click Cycloaddition Chemistry.

    Science.gov (United States)

    VanBrunt, Michael P; Shanebeck, Kurt; Caldwell, Zachary; Johnson, Jeffrey; Thompson, Pamela; Martin, Thomas; Dong, Huifang; Li, Gary; Xu, Hengyu; D'Hooge, Francois; Masterson, Luke; Bariola, Pauline; Tiberghien, Arnaud; Ezeadi, Ebele; Williams, David G; Hartley, John A; Howard, Philip W; Grabstein, Kenneth H; Bowen, Michael A; Marelli, Marcello

    2015-11-18

    Antibody-drug conjugates (ADC) have emerged as potent antitumor drugs that provide increased efficacy, specificity, and tolerability over chemotherapy for the treatment of cancer. ADCs generated by targeting cysteines and lysines on the antibody have shown efficacy, but these products are heterogeneous, and instability may limit their dosing. Here, a novel technology is described that enables site-specific conjugation of toxins to antibodies using chemistry to produce homogeneous, potent, and highly stable conjugates. We have developed a cell-based mammalian expression system capable of site-specific integration of a non-natural amino acid containing an azide moiety. The azide group enables click cycloaddition chemistry that generates a stable heterocyclic triazole linkage. Antibodies to Her2/neu were expressed to contain N6-((2-azidoethoxy)carbonyl)-l-lysine at four different positions. Each site allowed over 95% conjugation efficacy with the toxins auristatin F or a pyrrolobenzodiazepine (PBD) dimer to generate ADCs with a drug to antibody ratio of >1.9. The ADCs were potent and specific in in vitro cytotoxicity assays. An anti Her2/neu conjugate demonstrated stability in vivo and a PBD containing ADC showed potent efficacy in a mouse tumor xenograph model. This technology was extended to generate fully functional ADCs with four toxins per antibody. The high stability of the azide-alkyne linkage, combined with the site-specific nature of the expression system, provides a means for the generation of ADCs with optimized pharmacokinetic, biological, and biophysical properties. PMID:26332743

  13. Cationic liposomes enhance targeted delivery and expression of exogenous DNA mediated by N-terminal modified poly(L-lysine)-antibody conjugate in mouse lung endothelial cells.

    Science.gov (United States)

    Trubetskoy, V S; Torchilin, V P; Kennel, S; Huang, L

    1992-07-15

    A new and improved system for targeted gene delivery and expression is described. Transfection efficiency of N-terminal modified poly(L-lysine) (NPLL) conjugated with anti-thrombomodulin antibody 34A can be improved by adding to the system a lipophilic component, cationic liposomes. DNA, antibody conjugate and cationic liposomes form a ternary electrostatic complex which preserves the ability to bind specifically to the target cells. At the same time the addition of liposomes enhance the specific transfection efficiency of antibody-polylysine/DNA binary complex by 10 to 20-fold in mouse lung endothelial cells in culture.

  14. An aptamer-based colorimetric assay for chloramphenicol using a polymeric HRP-antibody conjugate for signal amplification

    International Nuclear Information System (INIS)

    We describe an aptamer-based colorimetric assay for chloramphenicol (CAP) based on the ability of anti-single-stranded DNA antibody (anti-ssDNA Ab) to recognize ssDNA, and the catalytic ability of PowerVision (PV), which is a polymeric conjugate of horseradish peroxidase and antibody with a high enzyme-to-antibody ratio. The complementary DNA of the aptamer (cDNA) was immobilized on magnetic gold nanoparticles (Fe3O4-Au) and used as a capture probe (AuMNPs-cDNA). The ssDNA Ab and PV were conjugated to AuNPs to form signal tags that recognize ssDNA with anti-ssDNA Ab to form beads containing the amplified probe (AuMNPs-cDNA-anti-ssDNA Ab/PV-AuNPs). The PV on their surface catalyzes the oxidation of the substrate 3,3’,5,5’-tetramethylbenzidine to produce a color change which is quantified by absorptiometry at 652 nm. The assay has a linear calibration plot for CAP in the 0.01 to 100 ng mL−1 range, with a detection limit as low as 3 pg mL−1. The method was successfully employed to detect CAP in real samples. Results were consistent with data obtained using a conventional enzyme-linked immunosorbent assay. (author)

  15. A SPR biosensor based on signal amplification using antibody-QD conjugates for quantitative determination of multiple tumor markers.

    Science.gov (United States)

    Wang, Huan; Wang, Xiaomei; Wang, Jue; Fu, Weiling; Yao, Chunyan

    2016-01-01

    The detection of tumor markers is very important in early cancer diagnosis; however, tumor markers are usually present at very low concentrations, especially in the early stages of tumor development. Surface plasmon resonance (SPR) is widely used to detect biomolecular interactions; it has inherent advantages of being high-throughput, real-time, and label-free technique. However, its sensitivity needs essential improvement for practical applications. In this study, we developed a signal amplification strategy using antibody-quantum dot (QD) conjugates for the sensitive and quantitative detection of α-fetoprotein (AFP), carcinoembryonic antigen (CEA) and cytokeratin fragment 21-1 (CYFRA 21-1) in clinical samples. The use of a dual signal amplification strategy using AuNP-antibody and antibody-QD conjugates increased the signal amplification by 50-folds. The constructed SPR biosensor showed a detection limit as low as 0.1 ng/mL for AFP, CEA, and CYFRA 21-1. Moreover, the results obtained using this SPR biosensor were consistent with those obtained using the electrochemiluminescence method. Thus, the constructed SPR biosensor provides a highly sensitive and specific approach for the detection of tumor markers. This SPR biosensor can be expected to be readily applied for the detection of other tumor markers and can offer a potentially powerful solution for tumor screening. PMID:27615417

  16. Stimulation of protective antibodies against type Ia and Ib group B streptococci by a type Ia polysaccharide-tetanus toxoid conjugate vaccine.

    OpenAIRE

    Wessels, M R; Paoletti, L C; Rodewald, A K; Michon, F; DiFabio, J; Jennings, H J; Kasper, D L

    1993-01-01

    Antisera elicited by type Ia group B streptococci (GBS) contain antibodies that react with both type Ia and type Ib strains. Previous studies suggested that antibodies elicited by type Ia organisms recognized a carbohydrate antigen or epitope common to Ia and Ib strains. We now report the synthesis and immunogenicity testing of a type Ia polysaccharide-tetanus toxoid (Ia-TT) conjugate vaccine. Ia-TT elicited type Ia polysaccharide-specific immunoglobulin G antibodies in all three of the rabbi...

  17. Comparison of Gold Nanoparticle Conjugated Secondary Antibody with Non-Gold Secondary Antibody in an ELISA Kit Model.

    Science.gov (United States)

    Emami, Tara; Madani, Rasool; Golchinfar, Fariba; Shoushtary, Abdolhamid; Amini, Seyed Mohammad

    2015-10-01

    In this study, gold nanoparticles (AuNPs) were used as carriers of the signaling anti-chicken antibody peroxidase in comparison with anti-chicken antibody peroxidase without gold nanoparticle in a commercial avian influenza kit. AuNPs enhanced the absorbance and shortened the assay time. AuNPs act as a carrier of many enzymes and multiply the effect of enzyme when reacting with substrate. They amplify optical signal, while keeping low background signals.

  18. An enzymatic deconjugation method for the analysis of small molecule active drugs on antibody-drug conjugates.

    Science.gov (United States)

    Li, Yi; Gu, Christine; Gruenhagen, Jason; Yehl, Peter; Chetwyn, Nik P; Medley, Colin D

    2016-01-01

    Antibody-drug conjugates (ADCs) are complex therapeutic agents that use the specific targeting properties of antibodies and the highly potent cytotoxicity of small molecule drugs to selectively eliminate tumor cells while limiting the toxicity to normal healthy tissues. Two critical quality attributes of ADCs are the purity and stability of the active small molecule drug linked to the ADC, but these are difficult to assess once the drug is conjugated to the antibody. In this study, we report a enzyme deconjugation approach to cleave small molecule drugs from ADCs, which allows the drugs to be subsequently characterized by reversed-phase high performance liquid chromatography. The model ADC we used in this study utilizes a valine-citrulline linker that is designed to be sensitive to endoproteases after internalization by tumor cells. We screened several proteases to determine the most effective enzyme. Among the 3 cysteine proteases evaluated, papain had the best efficiency in cleaving the small molecule drug from the model ADC. The deconjugation conditions were further optimized to achieve complete cleavage of the small molecule drug. This papain deconjugation approach demonstrated excellent specificity and precision. The purity and stability of the active drug on an ADC drug product was evaluated and the major degradation products of the active drug were identified. The papain deconjugation method was also applied to several other ADCs, with the results suggesting it could be applied generally to ADCs containing a valine-citrulline linker. Our results indicate that the papain deconjugation method is a powerful tool for characterizing the active small molecule drug conjugated to an ADC, and may be useful in ensuring the product quality, efficacy and the safety of ADCs. PMID:26891281

  19. Triggered Drug Release from an Antibody-Drug Conjugate Using Fast "Click-to-Release" Chemistry in Mice.

    Science.gov (United States)

    Rossin, Raffaella; van Duijnhoven, Sander M J; Ten Hoeve, Wolter; Janssen, Henk M; Kleijn, Laurens H J; Hoeben, Freek J M; Versteegen, Ron M; Robillard, Marc S

    2016-07-20

    The use of a bioorthogonal reaction for the selective cleavage of tumor-bound antibody-drug conjugates (ADCs) would represent a powerful new tool for ADC therapy, as it would not rely on the currently used intracellular biological activation mechanisms, thereby expanding the scope to noninternalizing cancer targets. Here we report that the recently developed inverse-electron-demand Diels-Alder pyridazine elimination reaction can provoke rapid and self-immolative release of doxorubicin from an ADC in vitro and in tumor-bearing mice. PMID:27306828

  20. CD33 monoclonal antibody conjugated Au cluster nano-bioprobe for targeted flow-cytometric detection of acute myeloid leukaemia

    Science.gov (United States)

    Retnakumari, Archana; Jayasimhan, Jasusri; Chandran, Parwathy; Menon, Deepthy; Nair, Shantikumar; Mony, Ullas; Koyakutty, Manzoor

    2011-07-01

    Protein stabilized gold nanoclusters (Au-NCs) are biocompatible, near-infrared (NIR) emitting nanosystems having a wide range of biomedical applications. Here, we report the development of a Au-NC based targeted fluorescent nano-bioprobe for the flow-cytometric detection of acute myeloid leukaemia (AML) cells. Au-NCs with ~ 25-28 atoms showing bright red-NIR fluorescence (600-750 nm) and average size of ~ 0.8 nm were prepared by bovine serum albumin assisted reduction-cum-stabilization in aqueous phase. The protein protected clusters were conjugated with monoclonal antibody against CD33 myeloid antigen, which is overexpressed in ~ 99.2% of the primitive population of AML cells, as confirmed by immunophenotyping using flow cytometry. Au-NC-CD33 conjugates having average size of ~ 12 nm retained bright fluorescence over an extended duration of ~ a year, as the albumin protein protects Au-NCs against degradation. Nanotoxicity studies revealed excellent biocompatibility of Au-NC conjugates, as they showed no adverse effect on the cell viability and inflammatory response. Target specificity of the conjugates for detecting CD33 expressing AML cells (KG1a) in flow cytometry showed specific staining of ~ 95.4% of leukaemia cells within 1-2 h compared to a non-specific uptake of ~ 8.2% in human peripheral blood cells (PBMCs) which are CD33low. The confocal imaging also demonstrated the targeted uptake of CD33 conjugated Au-NCs by leukaemia cells, thus confirming the flow cytometry results. This study demonstrates that novel nano-bioprobes can be developed using protein protected fluorescent nanoclusters of Au for the molecular receptor targeted flow cytometry based detection and imaging of cancer cells.

  1. CD33 monoclonal antibody conjugated Au cluster nano-bioprobe for targeted flow-cytometric detection of acute myeloid leukaemia

    Energy Technology Data Exchange (ETDEWEB)

    Retnakumari, Archana; Jayasimhan, Jasusri; Chandran, Parwathy; Menon, Deepthy; Nair, Shantikumar; Mony, Ullas; Koyakutty, Manzoor, E-mail: manzoork@aims.amrita.edu, E-mail: ullasmony@aims.amrita.edu [Amrita Centre for Nanoscience and Molecular Medicine, Amrita Institute of Medical Science, Cochin 682 041 (India)

    2011-07-15

    Protein stabilized gold nanoclusters (Au-NCs) are biocompatible, near-infrared (NIR) emitting nanosystems having a wide range of biomedical applications. Here, we report the development of a Au-NC based targeted fluorescent nano-bioprobe for the flow-cytometric detection of acute myeloid leukaemia (AML) cells. Au-NCs with {approx} 25-28 atoms showing bright red-NIR fluorescence (600-750 nm) and average size of {approx} 0.8 nm were prepared by bovine serum albumin assisted reduction-cum-stabilization in aqueous phase. The protein protected clusters were conjugated with monoclonal antibody against CD33 myeloid antigen, which is overexpressed in {approx} 99.2% of the primitive population of AML cells, as confirmed by immunophenotyping using flow cytometry. Au-NC-CD33 conjugates having average size of {approx} 12 nm retained bright fluorescence over an extended duration of {approx} a year, as the albumin protein protects Au-NCs against degradation. Nanotoxicity studies revealed excellent biocompatibility of Au-NC conjugates, as they showed no adverse effect on the cell viability and inflammatory response. Target specificity of the conjugates for detecting CD33 expressing AML cells (KG1a) in flow cytometry showed specific staining of {approx} 95.4% of leukaemia cells within 1-2 h compared to a non-specific uptake of {approx} 8.2% in human peripheral blood cells (PBMCs) which are CD33{sup low}. The confocal imaging also demonstrated the targeted uptake of CD33 conjugated Au-NCs by leukaemia cells, thus confirming the flow cytometry results. This study demonstrates that novel nano-bioprobes can be developed using protein protected fluorescent nanoclusters of Au for the molecular receptor targeted flow cytometry based detection and imaging of cancer cells.

  2. Immunosuppressive drugs impairs antibody response of the polysaccharide and conjugated pneumococcal vaccines in patients with Crohn's disease

    DEFF Research Database (Denmark)

    Kantsø, Bjørn; Halkjær, Sofie Ingdam; Thomsen, Ole Østergaard;

    2015-01-01

    BACKGROUND: Patients with Crohn's disease (CD) have a higher risk of infectious diseases including pneumococcal infections, and the risk increases with immunotherapy. The primary endpoint of this study was to investigate the specific antibody response to two pneumococcal vaccines in CD patients...... with and without immunosuppressive treatment four weeks post vaccination. METHODS: In a randomized trial of the 23-valent pneumococcal polysaccharide vaccine (PPV23) and the 13-valent pneumococcal conjugated vaccine (PCV13), a group of CD patients treated with immunosuppressive drugs (IS) alone or in combination...... with TNF-α antagonists were compared to a group of CD patients not treated with any of these drugs (untreated). Specific pneumococcal antibody concentrations were measured against 12 serotypes common to the two vaccines before and 4 week after vaccination. RESULTS: PCV13 induced a significantly higher...

  3. A new bifunctional chelate, BrMe sub 2 HBED: An effective conjugate for radiometals and antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Mathias, C.J.; Connett, J.M.; Philpott, G.W.; Welch, M.J. (Washington Univ. School of Medicine, St. Louis, MO (USA)); Sun, Yizhen; Martell, A.E. (Texas A and M Univ., College Station (USA))

    1990-04-18

    A new bifunctional chelate, N-(2-hydroxy-3,5-dimethylbenzyl)-N{prime}-(2-hydroxy-5-(bromoacetamido)benzyl)ethylenediamine-N,-N{prime}-diacetic acid (BrMe{sub 2}HBED), was designed and synthesized to bind trivalent cationic metals with monoclonal antibodies. The stability constants (log values) for indium complexed with a similar ligand, HBED, were increased over those of more commonly used ligands DTPA and EDTA. Predictably, the increased metal-ligand complex stability would expedite the in vivo clearance from nontarget regions and perhaps enhance the localization of the radiolabeled antibody (Ab). BrMe{sub 2}HBED was conjugated with the Ab (24 h) and then radiolabeled with indium-111 citrate (24 h). Additionally, the Ab was radiolabeled by using conventional methods ({sup 111}In-DTPA and {sup 125}I-lactoperoxidase) and then compared by measuring the in vitro stability, in vitro immunoreactivity(IR), and in vivo distribution and clearance. A 10:1 BrMe{sub 2} HBED:Ab mole ratio resulted in good labeling efficiency with {sup 111}In and more importantly a very high IR. In a hamster tumor model, {sup 111}In-BrMe{sub 2} HBED-labeled monoclonal antibody (1A3) had high uptake in the tumor tissue and preferable blood clearance compared to either of the more conventional radiolabeled 1A3 monoclonal antibodies ({sup 111}In-DTPA or {sup 125}I-lactoperoxidase). 49 refs., 4 figs., 8 tabs.

  4. Indication of viruses and virus-specific antibodies by ELISA using conjugates based on β-lactamase obtained by genetic engineering

    International Nuclear Information System (INIS)

    The method of enzyme-linked immunosorbent assay (ELISA), by means of which antigens and antibodies of different origin can be detected with high sensitivity and specificity, is an immunoenzymatic technique based on the use of conjugates, or macromolecular complexes formed by covalent attachment of enzyme molecules to antigen or antibody molecules. Conjugates based on peroxidase, alkaline phosphatase, and beta-galactosidase are most frequently used to construct immunoenzymatic test systems. The use of these enzymes in ELISA, however, is complicated by the fact that they are often present in free or bound form in the biological material under study, and that their substrates either possess low stability, are difficult to synthesize, or are toxic. In this paper, in order to avoid these shortcomings, the authors develop a method for the biosynthesis of lactamase conjugates which is based on genetic engineering, and demonstrate the viability and stability of these conjugates in radioimmunoenzymatic assay of viruses

  5. Oriented conjugates of monoclonal and single-domain antibodies with quantum dots for flow cytometry and immunohistochemistry diagnostic applications

    Science.gov (United States)

    Sukhanova, Alyona; Even-Desrumeaux, Klervi; Millot, Jean-Marc; Chames, Patrick; Baty, Daniel; Artemyev, Mikhail; Oleinikov, Vladimir; Cohen, Jacques H. M.; Nabiev, Igor

    2012-03-01

    Ideal diagnostic nanoprobes should not exceed 15 nm in size and should contain high-affinity homogeneously oriented capture molecules on their surface. An advanced procedure for antibody (Ab) reduction was used to cleave each Ab into two functional half-Abs, 75-kDa heavy-light chain fragments, each containing an intact antigen-binding site. Affinity purification of half-Abs followed by their linkage to quantum dots (QDs) yielded oriented QD-Ab conjugates whose functionality was considerably improved compared to those obtained using the standard protocols. Ultrasmall diagnostic nanoprobes were engineered through oriented conjugation of QDs with 13-kDa single-domain Abs (sdAbs) derived from llama IgG. sdAbs were tagged with QDs via an additional cysteine residue specifically integrated into the C-terminal region of sdAb using genetic engineering. This approach made it possible to obtain sdAb-QD nanoprobes protocols of anatomo-pathological practice. The nano-bioengineering approaches developed can be extended to oriented conjugation of Abs and sdAbs with different semiconductor, noble metal, or magnetic nanoparticles.

  6. Tetrafluorophenolate of HBED-CC: a versatile conjugation agent for 68Ga-labeled small recombinant antibodies

    International Nuclear Information System (INIS)

    The success of 68Ga-labeled peptides for positron emission tomography of neuroendocrine tumors is mainly depending on the complex chemistry of this radioisotope. 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), the chelator of choice has however limitations if its application is expanded to heat-sensitive proteins. Recombinant antibodies like single chain Fv or diabodies belong to this class of proteins. They are suited to provide imaging contrast despite the short-lived 68Ga because of their rapid blood clearances and nanomolar affinities. The heterobifunctional agent N,N'-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N'-diacetic acid (HBED-CC) was chosen as an alternative ligand because this agent is complexing [68Ga]Ga3+ much faster than DOTA at ambient temperatures. A versatile technology for HBED-CC conjugation of proteins and 68Ga-labeling has been developed. This included HBED-CC-tetrafluorophenol (TFP) ester synthesis, coupling to the antibody at various pH and complexation reactions performed in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer under different conditions. The synthesis of the monoreactive 2,3,5,6-tetrafluorophenolate of HBED-CC at a carboxyl group not participating in complex formation used [Fe(HBED-CC)]- for ester formation. The removal of Fe3+ from purified (HBED-CC)TFP ester was achieved with RP18 cartridge technology. The conjugation chemistry was performed with mAb425 which binds to the epidermal growth factor receptor (EGFR). This protein was used for optimizing purposes only. The influence of complexation parameters like temperature, pH, reaction time, and HBED-CC/antibody ratio on the biological activity of this model antibody was investigated. Furthermore, the outcome of this labeling procedure on the biological activity of a recombinant diabody (50 kDa) was studied. It is known that small HBED-CC/antibody ratios are prerequisites for minimal interference of labels with antigen

  7. Study of conjugation and radiolabeling of monoclonal antibody rituximab for use in radionuclide therapy; Estudo da conjugacao e radiomarcacao do anticorpo monoclonal rituximab para aplicacao em terapia radionuclidica

    Energy Technology Data Exchange (ETDEWEB)

    Massicano, Adriana Vidal Fernandes

    2011-07-01

    Lymphomas are tumors originated from the transformation of a lymphocyte in the lymphatic system. The most common lymphoma is the Non-Hodgkin Lymphoma (NHL). Advances in immunology and molecular biology have been improving NHL's detection and treatment strategies development, such as Radioimmunotherapy (RIT). Rituximab is an anti-CD20 monoclonal antibody used as immunotherapeutic to treat refractory or relapsed NHL. The goal of the present work was to conjugate this antibody to DOTA-NHS-ester bifunctional chelator and to radiolabel it with {sup 177}Lu radioisotope in order to develop a radio immunotherapeutic agent for NHL's treatment. Different rituximab to DOTA molar ratios (1:5, 1:10, 1:20, 1:50, 1:250, 1:500 and 1:1000) were evaluated in order to determine the best condition for obtaining the highest radiochemical purity of radio immunotherapeutic. The stability of the unlabeled immuno conjugated was evaluated by high performance liquid chromatography (HPLC) for up to 240 days in different storage conditions. The stability of the labeled preparations was evaluated either after storing at 2-8 degree C or incubation in human serum at 37 degree C. The binding to serum proteins was also determined. In vivo studies were performed in healthy Swiss mice, in order to characterize the biological properties of labeled conjugate. Finally, preliminary studies of radio immuno conjugated competitive binding to CD20 positive Raji cells were carried out in order to analyze if the process of conjugation and radiolabeling compromises the immunoreactivity of the antibody. The conjugation applying lower antibody to chelator molar ratios (1:5, 1:10 and 1:20) showed high stability when stored for up to 240 days in different conditions. The HPLC analysis showed that the monoclonal antibody conjugated in molar ratio 1:50 was labeled with higher radiochemical purity (> 95%) when purified in PD-10 column. This conjugate showed reasonable stability at 2-8 degree C. The analysis

  8. Use of N-terminal modified poly(L-lysine)-antibody conjugate as a carrier for targeted gene delivery in mouse lung endothelial cells.

    Science.gov (United States)

    Trubetskoy, V S; Torchilin, V P; Kennel, S J; Huang, L

    1992-01-01

    A DNA targeted delivery and expression system has been designed based on an N-terminal modified poly(L-lysine) (NPLL)-antibody conjugate, which readily forms a complex with plasmid DNA. Monoclonal antibodies against the cell-surface thrombomodulin conjugated with NPLL were used for targeted delivery of foreign plasmid DNA to an antigen-expressing mouse lung endothelial cell line in vitro and to mouse lungs in vivo. In both cases significant amounts of DNA can be specifically bound to the target cells or tissues. Specific gene expression was observed in the treated mouse lung endothelial cells.

  9. Amperometric Immunosensor for Carbofuran Detection Based on MWCNTs/GS-PEI-Au and AuNPs-Antibody Conjugate

    Directory of Open Access Journals (Sweden)

    Xiangyou Wang

    2013-04-01

    Full Text Available In this paper, an amperometric immunosensor for the detection of carbofuran was developed. Firstly, multiwall carbon nanotubes (MWCNTs and graphene sheets-ethyleneimine polymer-Au (GS-PEI-Au nanocomposites were modified onto the surface of a glass carbon electrode (GCE via self-assembly. The nanocomposites can increase the surface area of the GCE to capture a large amount of antibody, as well as produce a synergistic effect in the electrochemical performance. Then the modified electrode was coated with gold nanoparticles-antibody conjugate (AuNPs-Ab and blocked with BSA. The monoclonal antibody against carbofuran was covalently immobilized on the AuNPs with glutathione as a spacer arm. The morphologies of the GS-PEI-Au nanocomposites and the fabrication process of the immunosensor were characterized by X-ray diffraction (XRD, ultraviolet and visible absorption spectroscopy (UV-vis and scanning electron microscopy (SEM, respectively. Under optimal conditions, the immunosensor showed a wide linear range, from 0.5 to 500 ng/mL, with a detection limit of 0.03 ng/mL (S/N = 3. The as-constructed immunosensor exhibited notable performance features such as high specificity, good reproducibility, acceptable stability and regeneration performance. The results are mainly due to the excellent properties of MWCNTs, GS-PEI-Au nanocomposites and the covalent immobilization of Ab with free hapten binding sites for further immunoreaction. It provides a new avenue for amperometric immunosensor fabrication.

  10. Amperometric immunosensor for carbofuran detection based on MWCNTs/GS-PEI-Au and AuNPs-antibody conjugate.

    Science.gov (United States)

    Zhu, Ying; Cao, Yaoyao; Sun, Xia; Wang, Xiangyou

    2013-04-19

    In this paper, an amperometric immunosensor for the detection of carbofuran was developed. Firstly, multiwall carbon nanotubes (MWCNTs) and graphene sheets-ethyleneimine polymer-Au (GS-PEI-Au) nanocomposites were modified onto the surface of a glass carbon electrode (GCE) via self-assembly. The nanocomposites can increase the surface area of the GCE to capture a large amount of antibody, as well as produce a synergistic effect in the electrochemical performance. Then the modified electrode was coated with gold nanoparticles-antibody conjugate (AuNPs-Ab) and blocked with BSA. The monoclonal antibody against carbofuran was covalently immobilized on the AuNPs with glutathione as a spacer arm. The morphologies of the GS-PEI-Au nanocomposites and the fabrication process of the immunosensor were characterized by X-ray diffraction (XRD), ultraviolet and visible absorption spectroscopy (UV-vis) and scanning electron microscopy (SEM), respectively. Under optimal conditions, the immunosensor showed a wide linear range, from 0.5 to 500 ng/mL, with a detection limit of 0.03 ng/mL (S/N = 3). The as-constructed immunosensor exhibited notable performance features such as high specificity, good reproducibility, acceptable stability and regeneration performance. The results are mainly due to the excellent properties of MWCNTs, GS-PEI-Au nanocomposites and the covalent immobilization of Ab with free hapten binding sites for further immunoreaction. It provides a new avenue for amperometric immunosensor fabrication.

  11. Pneumococcal Conjugate Vaccines Overcome Splenic Dependency of Antibody Response to Pneumococcal Polysaccharides

    OpenAIRE

    Breukels, Mijke A.; Zandvoort, Andre; van den Dobbelsteen, Germie P. J. M.; van den Muijsenberg, Adrie; Lodewijk, Monique E.; Beurret, Michel; Pieter A Klok; Timens, Wim; Rijkers, Ger T.

    2001-01-01

    Protection against infections with Streptococcus pneumoniae depends on the presence of antibodies against capsular polysaccharides that facilitate phagocytosis. Asplenic patients are at increased risk for pneumococcal infections, since both phagocytosis and the initiation of the antibody response to polysaccharides take place in the spleen. Therefore, vaccination with pneumococcal polysaccharide vaccines is recommended prior to splenectomy, which, as in the case of trauma, is not always feasi...

  12. Synthesis and Characterization of Anti-HER2 Antibody Conjugated CdSe/CdZnS Quantum Dots for Fluorescence Imaging of Breast Cancer Cells

    OpenAIRE

    Takashi Jin; Watanabe, Tomonobu M.; Keiko Yoshizawa; Dhermendra K Tiwari; Yasushi Inouye; Shin-Ichi Tanaka

    2009-01-01

    The early detection of HER2 (human epidermal growth factor receptor 2) status in breast cancer patients is very important for the effective implementation of anti-HER2 antibody therapy. Recently, HER2 detections using antibody conjugated quantum dots (QDs) have attracted much attention. QDs are a new class of fluorescent materials that have superior properties such as high brightness, high resistance to photo-bleaching, and multi-colored emission by a single-light source excitation. In this s...

  13. Understanding How the Stability of the Thiol-Maleimide Linkage Impacts the Pharmacokinetics of Lysine-Linked Antibody-Maytansinoid Conjugates.

    Science.gov (United States)

    Ponte, Jose F; Sun, Xiuxia; Yoder, Nicholas C; Fishkin, Nathan; Laleau, Rassol; Coccia, Jennifer; Lanieri, Leanne; Bogalhas, Megan; Wang, Lintao; Wilhelm, Sharon; Widdison, Wayne; Pinkas, Jan; Keating, Thomas A; Chari, Ravi; Erickson, Hans K; Lambert, John M

    2016-07-20

    Antibody-drug conjugates (ADCs) have become a widely investigated modality for cancer therapy, in part due to the clinical findings with ado-trastuzumab emtansine (Kadcyla). Ado-trastuzumab emtansine utilizes the Ab-SMCC-DM1 format, in which the thiol-functionalized maytansinoid cytotoxic agent, DM1, is linked to the antibody (Ab) via the maleimide moiety of the heterobifunctional SMCC linker. The pharmacokinetic (PK) data for ado-trastuzumab emtansine point to a faster clearance for the ADC than for total antibody. Cytotoxic agent release in plasma has been reported with nonmaytansinoid, cysteine-linked ADCs via thiol-maleimide exchange, for example, brentuximab vedotin. For Ab-SMCC-DM1 ADCs, however, the main catabolite reported is lysine-SMCC-DM1, the expected product of intracellular antibody proteolysis. To understand these observations better, we conducted a series of studies to examine the stability of the thiol-maleimide linkage, utilizing the EGFR-targeting conjugate, J2898A-SMCC-DM1, and comparing it with a control ADC made with a noncleavable linker that lacked a thiol-maleimide adduct (J2898A-(CH2)3-DM). We employed radiolabeled ADCs to directly measure both the antibody and the ADC components in plasma. The PK properties of the conjugated antibody moiety of the two conjugates, J2898A-SMCC-DM1 and J2898A-(CH2)3-DM (each with an average of 3.0 to 3.4 maytansinoid molecules per antibody), appear to be similar to that of the unconjugated antibody. Clearance values of the intact conjugates were slightly faster than those of the Ab components. Furthermore, J2898A-SMCC-DM1 clears slightly faster than J2898A-(CH2)3-DM, suggesting that there is a fraction of maytansinoid loss from the SMCC-DM1 ADC, possibly through a thiol-maleimide dependent mechanism. Experiments on ex vivo stability confirm that some loss of maytansinoid from Ab-SMCC-DM1 conjugates can occur via thiol elimination, but at a slower rate than the corresponding rate of loss reported for thiol

  14. Quantitation of antibody-secreting cells in the blood after vaccination with Haemophilus influenzae type b conjugate vaccine

    DEFF Research Database (Denmark)

    Barington, T; Heilmann, C; Andersen, V

    1990-01-01

    -specific antibody-secreting cells (AbSC) of the isotypes IgM, IgG, and IgA. The appearance of AbSC in the blood after vaccination of adults with diphtheria toxoid-conjugated Hib polysaccharide was investigated. AbSC were detected from post-vaccination day 5 to day 14. IgA was the predominant isotype among...... these cells. IgM AbSC peaked slightly earlier (median day 7) than IgG and IgA AbSC (both day 8). On post-vaccination day 8 the numbers of AbSC were: IgA, 1217/10(6) mononuclear cells (median); IgG, 211; and IgM, 30 (n = 11). Similar isotype distribution has earlier been found after vaccination with pure...... capsular polysaccharides from Hib and pneumococci. The predominance of IgA AbSC in response to both conjugate and pure polysaccharide vaccines is probably due to reactivation of the same clones of IgA-committed memory B cells originally primed at the mucosa by natural exposure to the polysaccharide...

  15. Antibody-antigen-adjuvant conjugates enable co-delivery of antigen and adjuvant to dendritic cells in cis but only have partial targeting specificity

    NARCIS (Netherlands)

    Kreutz, M.; Giquel, B.; Hu, Q.; Abuknesha, R.; Uematsu, S.; Akira, S.; Nestle, F.O.; Diebold, S.S.

    2012-01-01

    Antibody-antigen conjugates, which promote antigen-presentation by dendritic cells (DC) by means of targeted delivery of antigen to particular DC subsets, represent a powerful vaccination approach. To ensure immunity rather than tolerance induction the co-administration of a suitable adjuvant is par

  16. Site-specific antibody-liposome conjugation through copper-free click chemistry: a molecular biology approach for targeted photodynamic therapy (Conference Presentation)

    Science.gov (United States)

    Obaid, Girgis; Wang, Yucheng; Kuriakose, Jerrin; Broekgaarden, Mans; Alkhateeb, Ahmed; Bulin, Anne-Laure; Hui, James; Tsourkas, Andrew; Hasan, Tayyaba

    2016-03-01

    Nanocarriers, such as liposomes, have the ability to potentiate photodynamic therapy (PDT) treatment regimens by the encapsulation of high payloads of photosensitizers and enhance their passive delivery to tumors through the enhanced permeability and retention effect. By conjugating targeting moieties to the surface of the liposomal nanoconstructs, cellular selectivity is imparted on them and PDT-based therapies can be performed with significantly higher dose tolerances, as off-target toxicity is simultaneously reduced.1 However, the maximal benefits of conventional targeted nanocarriers, including liposomes, are hindered by practical limitations including chemical instability, non-selective conjugation chemistry, poor control over ligand orientation, and loss of ligand functionality following conjugation, amongst others.2 We have developed a robust, physically and chemically stable liposomal nanoplatform containing benzoporphyrin derivative photosensitizer molecules within the phospholipid bilayer and an optimized surface density of strained cyclooctyne moieties for `click' conjugation to azido-functionalized antibodies.3 The clinical chimeric anti-EGFR antibody Cetuximab is site-specifically photocrosslinked to a recombinant bioengineered that recognizes the antibody's Fc region, containing a terminal azide.4 The copper-free click conjugation of the bioengineered Cetuximab derivative to the optimized photosensitizing liposome provides exceptional control over the antibody's optimal orientation for cellular antigen binding. Importantly, the reaction occurs rapidly under physiological conditions, bioorthogonally (selectively in the presence of other biomolecules) and without the need for toxic copper catalysis.3 Such state-of-the-art conjugation strategies push the boundaries of targeted photodynamic therapy beyond the limitations of traditional chemical coupling techniques to produce more robust and effective targeted therapeutics with applications beyond

  17. Visualization of Pseudomonas aeruginosa O antigens by using a protein A-dextran-colloidal gold conjugate with both immunoglobulin G and immunoglobulin M monoclonal antibodies.

    OpenAIRE

    Lam, J S; Lam, M. Y.; MacDonald, L A; Hancock, R E

    1987-01-01

    Two lipopolysaccharide O-antigen-specific monoclonal antibodies, MA1-8 (an immunoglobulin G1 [IgG1]) and MF15-4 (an IgM), were used to localize the O antigen of the lipopolysaccharide of Pseudomonas aeruginosa PAO1. A protein A-dextran-gold conjugate with an average particle diameter of 12.5 nm was used to label bacterial cells treated with MA1-8, while a second antibody (goat anti-mouse IgM) was required before the same probe could interact with cells treated with the IgM antibody MF15-4. Bo...

  18. Antibody Persistence in Young Children 5 Years after Vaccination with a Combined Haemophilus influenzae Type b-Neisseria meningitidis Serogroup C Conjugate Vaccine Coadministered with Diphtheria-Tetanus-Acellular Pertussis-Based and Pneumococcal Conjugate Vaccines

    Science.gov (United States)

    Tejedor, Juan Carlos; Brzostek, Jerzy; Konior, Ryszard; Grunert, Detlef; Kolhe, Devayani; Baine, Yaela

    2016-01-01

    We evaluated antibody persistence in children up to 5 years after administration of a combined Haemophilus influenzae type b (Hib)-Neisseria meningitidis serogroup C (MenC)-tetanus toxoid (TT) conjugate vaccine coadministered with a pneumococcal conjugate vaccine. This is the follow-up study of a randomized trial (ClinicalTrials.gov registration no. NCT00334334/00463437) in which healthy children were vaccinated (primary vaccinations at 2, 4, and 6 months of age and booster vaccination at 11 to 18 months of age) with Hib-MenC-TT or a control MenC conjugate vaccine, coadministered with diphtheria-tetanus-acellular pertussis (DTPa)-based combination vaccines (DTPa/Hib for control groups) and a pneumococcal conjugate vaccine (10-valent pneumococcal nontypeable H. influenzae protein D conjugate vaccine [PHiD-CV] or 7-valent cross-reacting material 197 [CRM197] conjugate vaccine [7vCRM]). MenC antibody titers were measured with a serum bactericidal antibody (SBA) assay using rabbit complement (i.e., rabbit SBA [rSBA]), and antibodies against Hib polyribosylribitol phosphate (PRP) were measured with an enzyme-linked immunosorbent assay. Antibody persistence up to 5 years after booster vaccination is reported for 530 children ∼6 years of age. The percentages of children with seroprotective rSBA-MenC titers were between 24.2% and 40.1% in all groups approximately 5 years after booster vaccination. More than 98.5% of children in each group retained seroprotective anti-PRP concentrations. No vaccine-related serious adverse events and no events related to a lack of vaccine efficacy were reported. Approximately 5 years after booster vaccination, the majority of children retained seroprotective anti-PRP antibody concentrations. The percentage of children retaining seroprotective rSBA-MenC titers was low (≤40%), suggesting that a significant proportion of children may be unprotected against MenC disease. (This study has been registered at ClinicalTrials.gov under

  19. Antibody Persistence in Young Children 5 Years after Vaccination with a Combined Haemophilus influenzae Type b-Neisseria meningitidis Serogroup C Conjugate Vaccine Coadministered with Diphtheria-Tetanus-Acellular Pertussis-Based and Pneumococcal Conjugate Vaccines.

    Science.gov (United States)

    Tejedor, Juan Carlos; Brzostek, Jerzy; Konior, Ryszard; Grunert, Detlef; Kolhe, Devayani; Baine, Yaela; Van Der Wielen, Marie

    2016-07-01

    We evaluated antibody persistence in children up to 5 years after administration of a combined Haemophilus influenzae type b (Hib)-Neisseria meningitidis serogroup C (MenC)-tetanus toxoid (TT) conjugate vaccine coadministered with a pneumococcal conjugate vaccine. This is the follow-up study of a randomized trial (ClinicalTrials.gov registration no. NCT00334334/00463437) in which healthy children were vaccinated (primary vaccinations at 2, 4, and 6 months of age and booster vaccination at 11 to 18 months of age) with Hib-MenC-TT or a control MenC conjugate vaccine, coadministered with diphtheria-tetanus-acellular pertussis (DTPa)-based combination vaccines (DTPa/Hib for control groups) and a pneumococcal conjugate vaccine (10-valent pneumococcal nontypeable H. influenzae protein D conjugate vaccine [PHiD-CV] or 7-valent cross-reacting material 197 [CRM197] conjugate vaccine [7vCRM]). MenC antibody titers were measured with a serum bactericidal antibody (SBA) assay using rabbit complement (i.e., rabbit SBA [rSBA]), and antibodies against Hib polyribosylribitol phosphate (PRP) were measured with an enzyme-linked immunosorbent assay. Antibody persistence up to 5 years after booster vaccination is reported for 530 children ∼6 years of age. The percentages of children with seroprotective rSBA-MenC titers were between 24.2% and 40.1% in all groups approximately 5 years after booster vaccination. More than 98.5% of children in each group retained seroprotective anti-PRP concentrations. No vaccine-related serious adverse events and no events related to a lack of vaccine efficacy were reported. Approximately 5 years after booster vaccination, the majority of children retained seroprotective anti-PRP antibody concentrations. The percentage of children retaining seroprotective rSBA-MenC titers was low (≤40%), suggesting that a significant proportion of children may be unprotected against MenC disease. (This study has been registered at ClinicalTrials.gov under

  20. Antibody Persistence in Young Children 5 Years after Vaccination with a Combined Haemophilus influenzae Type b-Neisseria meningitidis Serogroup C Conjugate Vaccine Coadministered with Diphtheria-Tetanus-Acellular Pertussis-Based and Pneumococcal Conjugate Vaccines.

    Science.gov (United States)

    Tejedor, Juan Carlos; Brzostek, Jerzy; Konior, Ryszard; Grunert, Detlef; Kolhe, Devayani; Baine, Yaela; Van Der Wielen, Marie

    2016-07-01

    We evaluated antibody persistence in children up to 5 years after administration of a combined Haemophilus influenzae type b (Hib)-Neisseria meningitidis serogroup C (MenC)-tetanus toxoid (TT) conjugate vaccine coadministered with a pneumococcal conjugate vaccine. This is the follow-up study of a randomized trial (ClinicalTrials.gov registration no. NCT00334334/00463437) in which healthy children were vaccinated (primary vaccinations at 2, 4, and 6 months of age and booster vaccination at 11 to 18 months of age) with Hib-MenC-TT or a control MenC conjugate vaccine, coadministered with diphtheria-tetanus-acellular pertussis (DTPa)-based combination vaccines (DTPa/Hib for control groups) and a pneumococcal conjugate vaccine (10-valent pneumococcal nontypeable H. influenzae protein D conjugate vaccine [PHiD-CV] or 7-valent cross-reacting material 197 [CRM197] conjugate vaccine [7vCRM]). MenC antibody titers were measured with a serum bactericidal antibody (SBA) assay using rabbit complement (i.e., rabbit SBA [rSBA]), and antibodies against Hib polyribosylribitol phosphate (PRP) were measured with an enzyme-linked immunosorbent assay. Antibody persistence up to 5 years after booster vaccination is reported for 530 children ∼6 years of age. The percentages of children with seroprotective rSBA-MenC titers were between 24.2% and 40.1% in all groups approximately 5 years after booster vaccination. More than 98.5% of children in each group retained seroprotective anti-PRP concentrations. No vaccine-related serious adverse events and no events related to a lack of vaccine efficacy were reported. Approximately 5 years after booster vaccination, the majority of children retained seroprotective anti-PRP antibody concentrations. The percentage of children retaining seroprotective rSBA-MenC titers was low (≤40%), suggesting that a significant proportion of children may be unprotected against MenC disease. (This study has been registered at ClinicalTrials.gov under

  1. A Reduction-Based Sensor for Acrolein Conjugates with the Inexpensive Nitrobenzene as an Alternative to Monoclonal Antibody

    Science.gov (United States)

    Takamatsu, Masayuki; Fukase, Koichi; Oka, Ritsuko; Kitazume, Shinobu; Taniguchi, Naoyuki; Tanaka, Katsunori

    2016-01-01

    Acrolein, a highly toxic α, β–unsaturated aldehyde, has been a longstanding key biomarker associated with a range of disorders related to oxidative stresses. One of the most promising methods for detecting acrolein involves the use of antibodies that can recognize the acrolein–lysine conjugate, 3-formyl-3, 4-dehydropiperidines (FDP), within oxidatively stressed cells and tissues from various disease states. We have uncovered here that FDP could reduce nitroarenes in high yields at 100 °C in the presence of excess CaCl2 as a Lewis acid promoter. This unique transformation allowed for the development of a de novo method for detecting levels of FDPs generated from proteins in urine or blood serum samples. Thus we successfully converted a non-fluorescent and inexpensive 4-nitrophthalonitrile probe to the corresponding fluorescent aniline, thereby constituting the concept of fluorescent switching. Its sensitivity level (0.84 nmol/mL) is more than that of ELISA assays (3.13 nmol/mL) and is already equally reliable and reproducible at this early stage of development. More importantly, this method is cost effective and simple to operate, requiring only mixing of samples with a kit solution. Our method thus possesses potential as a future alternative to the more costly and operatively encumbered conventional antibody-based methods. PMID:27782170

  2. Human alpha-fetal protein immunoassay using fluorescence suppression with fluorescent-bead/antibody conjugate and enzymatic reaction.

    Science.gov (United States)

    Ahn, Junhyoung; Shin, Yong-Beom; Lee, JaeJong; Kim, Min-Gon

    2015-09-15

    The aim of the study was to develop a simple and rapid immunoassay using fluorescent microbeads and enzyme-substrate reactions to measure alpha-fetal protein (AFP) concentrations. We demonstrated the functionality of the fluorescent immunosensor using antibody-conjugated fluorescent latex beads (AB-FLBs) and horseradish peroxidase (HRP) to catalyze a reaction, where the products would precipitate and suppress the fluorescence of AB-FLBs. First, the AB-FLBs were incubated with antigen, biotinylated antibodies (bABs), and streptavidin-HRP (SAv-HRP) to form a sandwich-type immunoreaction. The mixture was then filtered through a membrane to concentrate the beads on a small area. After washing to remove unbound bABs and SAv-HRP, a chromogenic HRP substrate and H2O2 were added to form precipitates on the FLB surface. The suppression of the fluorescence was measured with a fluorescent image analyzer system. Under optimized conditions, AFP could be measured at concentrations as low as 1 pg mL(-1) with a dynamic range up to 100 ng mL(-1).

  3. Production and Evaluation of Immunoreactivity of Poly Lysine-Tagged Single Chain Fragment Variable (ScFv) Lym-1 Antibody for Direct Conjugation to Fluorescence Dye

    Energy Technology Data Exchange (ETDEWEB)

    Jung, Jae Ho; Choi, Tae Hyun; Woo, Kwang Sun; Chung, Wee Sup; Kang, Joo Hyun; Jeong, Su Young; Choi, Chang Woon; Lim, Sang Moo; Cheon, Gi Jeong [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

    2009-10-15

    Small size of recombinant scFv antibody has many advantages such as rapid blood clearances and improved targeting antibodies to tumor region. On the other hand owing to small size, number of amino group is insufficient in conjugation with chelator and fluorescence labeling. This study is to introduce poly lysine tag to the C-terminal end of scFv lym-1 sequence for fluorescence chelator conjugation. Poly lysine scFv lym-1 gene, cloned into pET-22b (+) vector, was expressed in E. coli BL21 (DE3) strain. Antibody purification was performed with Ni-NTA column and then size exclusion column chromatography. Expression and purification levels of poly lysine tagged scFv lym-1 antibody were confirmed by western blot analysis. I-124, I-125, I-131 and Tc-99m were used for radiolabeling of purified poly lysine scFv lym-1. Flow cytometry analysis of FITC conjugated poly lysine scFv lym-1 was performed for confirmation of immunoreactivity of human Burkitt's lymphoma cells. Poly lysine scFv lym-1 antibody was purified through two steps and identified as molecular weight of 48 KDa. Radiolabeling yields of I-124, I-125, I-131 and Tc-99m into poly lysine scFv lym-1 were >99%, >99%, >95% and >99%, respectively. Flow cytometry analysis of poly lysine scFv and scFv lym-1 was showed similar immunoreactivity to human Burkitt's lymphoma cells. Poly lysine tag was useful for the sufficient number of amino groups to scFv lym-1 antibody for chelator conjugation with minimizing loss of immunoreactivity

  4. Production and Evaluation of Immunoreactivity of Poly Lysine-Tagged Single Chain Fragment Variable (ScFv) Lym-1 Antibody for Direct Conjugation to Fluorescence Dye

    International Nuclear Information System (INIS)

    Small size of recombinant scFv antibody has many advantages such as rapid blood clearances and improved targeting antibodies to tumor region. On the other hand owing to small size, number of amino group is insufficient in conjugation with chelator and fluorescence labeling. This study is to introduce poly lysine tag to the C-terminal end of scFv lym-1 sequence for fluorescence chelator conjugation. Poly lysine scFv lym-1 gene, cloned into pET-22b (+) vector, was expressed in E. coli BL21 (DE3) strain. Antibody purification was performed with Ni-NTA column and then size exclusion column chromatography. Expression and purification levels of poly lysine tagged scFv lym-1 antibody were confirmed by western blot analysis. I-124, I-125, I-131 and Tc-99m were used for radiolabeling of purified poly lysine scFv lym-1. Flow cytometry analysis of FITC conjugated poly lysine scFv lym-1 was performed for confirmation of immunoreactivity of human Burkitt's lymphoma cells. Poly lysine scFv lym-1 antibody was purified through two steps and identified as molecular weight of 48 KDa. Radiolabeling yields of I-124, I-125, I-131 and Tc-99m into poly lysine scFv lym-1 were >99%, >99%, >95% and >99%, respectively. Flow cytometry analysis of poly lysine scFv and scFv lym-1 was showed similar immunoreactivity to human Burkitt's lymphoma cells. Poly lysine tag was useful for the sufficient number of amino groups to scFv lym-1 antibody for chelator conjugation with minimizing loss of immunoreactivity

  5. Complement C3d conjugation to anthrax protective antigen promotes a rapid, sustained, and protective antibody response.

    Directory of Open Access Journals (Sweden)

    Ravi V Kolla

    Full Text Available B. anthracis is the causative agent of anthrax. Pathogenesis is primarily mediated through the exotoxins lethal factor and edema factor, which bind protective antigen (PA to gain entry into the host cell. The current anthrax vaccine (AVA, Biothrax consists of aluminum-adsorbed cell-free filtrates of unencapsulated B. anthracis, wherein PA is thought to be the principle target of neutralization. In this study, we evaluated the efficacy of the natural adjuvant, C3d, versus alum in eliciting an anti-PA humoral response and found that C3d conjugation to PA and emulsion in incomplete Freund's adjuvant (IFA imparted superior protection from anthrax challenge relative to PA in IFA or PA adsorbed to alum. Relative to alum-PA, immunization of mice with C3d-PA/IFA augmented both the onset and sustained production of PA-specific antibodies, including neutralizing antibodies to the receptor-binding portion (domain 4 of PA. C3d-PA/IFA was efficacious when administered either i.p. or s.c., and in adolescent mice lacking a fully mature B cell compartment. Induction of PA-specific antibodies by C3d-PA/IFA correlated with increased efficiency of germinal center formation and plasma cell generation. Importantly, C3d-PA immunization effectively protected mice from intranasal challenge with B. anthracis spores, and was approximately 10-fold more effective than alum-PA immunization or PA/IFA based on dose challenge. These data suggest that incorporation of C3d as an adjuvant may overcome shortcomings of the currently licensed aluminum-based vaccine, and may confer protection in the early days following acute anthrax exposure.

  6. Detection of Chlamydia trachomatis inclusions in Mccoy cell cultures with fluorescein-conjugated monoclonal antibodies.

    OpenAIRE

    Stamm, W. E.; Tam, M; Koester, M; Cles, L

    1983-01-01

    We compared two methods for identification of Chlamydia trachomatis inclusions in McCoy cell monolayers: conventional iodine staining and immunofluorescence staining with monoclonal antibodies against the species-specific major outer membrane protein antigen of C. trachomatis. Among 878 urethral and cervical specimens tested in parallel, the immunofluorescence method detected eightfold more inclusions per monolayer, identified a higher proportion of positive specimens on first passage (98 ver...

  7. Radionuclide antibody-conjugates: developments and applications to obtain a targeted cancer therapy

    OpenAIRE

    Gjorgieva Ackova, Darinka; Smilkov, Katarina; Makreski, Petre; Stafilov, Trajče; Duatti, Adriano; Janevik-Ivanovska, Emilija

    2015-01-01

    Understanding the behaviour and function of biomolecules at the molecular level is key to the discovery and development of new drugs, as well as diagnostic techniques. The characterization of therapeutic monoclonal antibodies (mAbs) poses many challenges compared to those of low-molecular mass drugs because of their inherent complexity due to their protein nature. Achievements in this field of science have changed the way that drugs are being designed and developed nowadays. Vibrational spect...

  8. Prodrugs of anthracyclines for chemotherapy via enzyme-monoclonal antibody conjugates.

    Science.gov (United States)

    Gesson, J P; Jacquesy, J C; Mondon, M; Petit, P; Renoux, B; Andrianomenjanahary, S; Dufat-Trinh Van, H; Koch, M; Michel, S; Tillequin, F

    1994-10-01

    New prodrugs of daunorubicin, 1c, 1e and 2c, including a galactopyranosyl residue linked to the N-3' of the daunosaminyl moiety through substituted o- or p-benzyloxycarbonyl groups were synthesized. Their low cytotoxicity and high stability in plasma fulfil the conditions for antibody-directed enzyme prodrug therapy (ADEPT). Enzymatic hydrolysis using alpha-D-galactosidase gives rise to daunorubicin by subsequent self-elimination of the spacers. However, elimination clearly depends on the aromatic substitution pattern, as demonstrated especially by comparison with non-substituted analogues. PMID:7945725

  9. Fluorophore-conjugated antibodies for imaging and resection of GI tumors

    Science.gov (United States)

    Bouvet, Michael; Hoffman, Robert M.

    2016-03-01

    Negative surgical margins are critical to prevent recurrence in cancer surgery. This is because with current technology in many cases negative margins are impossible due the inability of the surgeon to detect the margin. Our laboratory has developed fluorophore-labeled monoclonal antibodies to aid in cancer visualization in orthotopic nude mouse models of human gastrointestinal (GI) cancer in order to achieve negative margins in fluorescence-guided surgery (FGS). The technologies described herein have the potential to change the paradigm of surgical oncology to engender significantly improved outcomes.

  10. Elevated levels of maternal anti-tetanus toxin antibodies do not suppress the immune response to a Haemophilus influenzae type b polyribosylphosphate-tetanus toxoid conjugate vaccine.

    OpenAIRE

    Panpitpat, C.; Thisyakorn, U.; Chotpitayasunondh, T; Fürer, E; Que, J U; Hasler, T.; Cryz, S J

    2000-01-01

    Reported are the effects of elevated levels of anti-tetanus antibodies on the safety and immune response to a Haemophilus influenzae type b polyribosylphosphate (PRP)-tetanus toxoid conjugate (PRP-T) vaccine. A group of Thai infants (n = 177) born to women immunized against tetanus during pregnancy were vaccinated with either a combined diphtheria-tetanus-pertussis (DTP) PRP-T vaccine or DTP and a PRP-conjugate vaccine using Neisseria meningitidis group B outer-membrane proteins as a carrier ...

  11. Effect of charge localization on the in vivo optical imaging properties of near-infrared cyanine dye/monoclonal antibody conjugates.

    Science.gov (United States)

    Sato, Kazuhide; Gorka, Alexander P; Nagaya, Tadanobu; Michie, Megan S; Nakamura, Yuko; Nani, Roger R; Coble, Vince L; Vasalatiy, Olga V; Swenson, Rolf E; Choyke, Peter L; Schnermann, Martin J; Kobayashi, Hisataka

    2016-10-20

    Near-infrared (NIR) fluorophores show superior in vivo imaging properties than visible-light fluorophores because of the increased light penetration in tissue and lower autofluorescence of these wavelengths. We have recently reported that new NIR cyanine dyes containing a novel C4'-O-alkyl linker exhibit greater chemical stability and excellent optical properties relative to existing C4'-O-aryl variants. In this study, we synthesized two NIR cyanine dyes with the same core structure and charge but different indolenine substituents: FNIR-Z-759 bearing a combination of two sulfonates and two quaternary ammonium cations, and FNIR-G-765 bearing a combination of two sulfonates and two guanidines, resulting in zwitterionic charge with distinct cationic moieties. In this study, we compare the in vitro and in vivo optical imaging properties of monoclonal antibody (mAb) conjugates of FNIR-Z-759 and FNIR-G-765 with panitumumab (pan) at antibody-to-dye ratios of 1 : 2 or 1 : 5. One-to-five conjugation of pan-to-FNIR-G-765 was not successful due to aggregate formation during the conjugation reaction. Conjugates of both dyes to pan (2 : 1) demonstrated similar quenching capacity, stability, and brightness in target cells in vitro. However, FNIR-Z-759 conjugates showed significantly lower accumulation in the mouse liver, resulting in higher tumor-to-liver ratio. Thus, FNIR-Z-759 conjugates appear to have superior in vivo imaging characteristics compared with FNIR-G-765 conjugates, especially in the abdominal region. Moreover, from a chemistry point of view, mAb conjugation with FNIR-Z-759 has an advantage over FNIR-G-765, because it does not form aggregates at high dye-to-mAb ratio. These results suggest that zwitterionic cyanine dyes are a superior class of fluorophores for conjugating with mAbs for fluorescence imaging applications due to improving target-to-background contrast in vivo. However, zwitterionic cyanine dyes should be designed carefully, as small

  12. Use of a charge reducing agent to enable intact mass analysis of cysteine-linked antibody-drug-conjugates by native mass spectrometry

    Directory of Open Access Journals (Sweden)

    Kamila J. Pacholarz

    2016-06-01

    Full Text Available Antibody-drug-conjugates (ADC are a growing class of anticancer biopharmaceuticals. Conjugation of cysteine linked ADCs, requires initial reduction of mAb inter-chain disulfide bonds, as the drugs are attached via thiol chemistry. This results in the active mAb moiety being transformed from a covalently linked tetramer to non-covalently linked complexes, which hinders precise determination of drug load with LC–MS. Here, we show how the addition of the charge reducing agent triethylammonium acetate (TEAA preserves the intact mAb structure, is well suited to the study of cysteine linked conjugates and facilitates easy drug load determination by direct infusion native MS.

  13. Non-epitope-specific suppression of the antibody response to Haemophilus influenzae type b conjugate vaccines by preimmunization with vaccine components

    DEFF Research Database (Denmark)

    Barington, T; Skettrup, M; Juul, L;

    1993-01-01

    Recently, conjugate vaccines containing Haemophilus influenzae type b capsular polysaccharide (HibCP) coupled to protein carriers were introduced for use in infants and certain adult risk groups. Similar conjugate vaccines against other capsulated bacteria are currently under development for both...... children and adults. Despite its potential importance, the possible influence of preexisting immunity to the components of such conjugates on the vaccination response in humans has been addressed by few studies. To study this issue, we randomized 82 healthy adult volunteers into six groups and vaccinated...... them twice, with a 4-week interval between immunizations. Four groups received tetanus toxoid (TT) or diphtheria toxoid (DT) and then HibCP coupled to TT (HibCP-TT) or DT (HibCP-DT). Two groups received HibCP-TT followed by HibCP-DT or vice versa. The total antibody levels to HibCP, TT, and DT...

  14. DOTA-Functionalized Polylysine: A High Number of DOTA Chelates Positively Influences the Biodistribution of Enzymatic Conjugated Anti-Tumor Antibody chCE7agl

    OpenAIRE

    Jürgen Grünberg; Simone Jeger; Dikran Sarko; Patrick Dennler; Kurt Zimmermann; Walter Mier; Roger Schibli

    2013-01-01

    Site-specific enzymatic reactions with microbial transglutaminase (mTGase) lead to a homogenous species of immunoconjugates with a defined ligand/antibody ratio. In the present study, we have investigated the influence of different numbers of 1,4,7,10-tetraazacyclododecane-N-N'-N''-N'''-tetraacetic acid (DOTA) chelats coupled to a decalysine backbone on the in vivo behavior of the chimeric monoclonal anti-L1CAM antibody chCE7agl. The enzymatic conjugation of (DOTA)1-decalysine, (DOTA)3-decaly...

  15. Heavy-chain isotype patterns of human antibody-secreting cells induced by Haemophilus influenzae type b conjugate vaccines in relation to age and preimmunity

    DEFF Research Database (Denmark)

    Barington, T; Juul, Lars; Gyhrs, A;

    1994-01-01

    The influence of preexisting immunity on the heavy-chain isotypes of circulating antibody-secreting cells (AbSC) induced by vaccination with Haemophilus influenzae type b (Hib) capsular polysaccharide (HibCP) coupled to tetanus toxoid (TT) or diphtheria toxoid (DT) and by vaccination with TT or DT...... of natural HibCP antibodies (r = 0.59; P = 0.00002). A possible role of natural exposure for Hib or cross-reactive bacteria on the mucosal surfaces in the shaping of the isotype response to HibCP conjugate vaccines is discussed....

  16. Detection of MUC1-Expressing Ovarian Cancer by C595 Monoclonal Antibody-Conjugated SPIONs Using MR Imaging

    Directory of Open Access Journals (Sweden)

    Daryoush Shahbazi-Gahrouei

    2013-01-01

    Full Text Available The aim of this study is to find out the development and application of MUC1-expressing ovarian cancer (OVCAR3 by C595 monoclonal antibody-conjugated superparamagnetic iron oxide nanoparticles (SPIONs using MR imaging. At the end, its use as a nanosized contrast agent MR imaging probe for ovarian cancer detection was investigated. The strategy is to use SPIONs attached to C595 mAb that binds to the MUC1, to specifically detect ovarian cancer cells. Anticancer effects and MR imaging parameters of the prepared nanoconjugate was investigated both under in vitro and in vivo experiments. The characterization of nanoconjugate includes its size, cell toxicity, flow cytometry, Prussian blue staining test and its cellular uptake as well as its biodistribution, and MR imaging was also investigated. The findings of the study showed good tumor accumulation and detection, no in vivo toxicity, and potential selective antiovarian cancer activity. Overall, based on the findings SPIONs-C595 nanosized probe is a selective ovarian molecular imaging modality. Further subsequent clinical trials appear warranted.

  17. Effect of polyethylene glycol conjugation on conformational and colloidal stability of a monoclonal antibody antigen-binding fragment (Fab').

    Science.gov (United States)

    Roque, Cristopher; Sheung, Anthony; Rahman, Nausheen; Ausar, S Fernando

    2015-02-01

    We have investigated the effects of site specific "hinge" polyethylene glycol conjugation (PEGylation) on thermal, pH, and colloidal stability of a monoclonal antibody antigen-binding fragment (Fab') using a variety of biophysical techniques. The results obtained by circular dichroism (CD), ultraviolet (UV) absorbance, and fluorescence spectroscopy suggested that the physical stability of the Fab' is maximized at pH 6-7 with no apparent differences due to PEGylation. Temperature-induced aggregation experiments revealed that PEGylation was able to increase the transition temperature, as well as prevent the formation of visible and subvisible aggregates. Statistical comparison of the three-index empirical phase diagram (EPD) revealed significant differences in thermal and pH stability signatures between Fab' and PEG-Fab'. Upon mechanical stress, micro-flow imaging (MFI) and measurement of the optical density at 360 nm showed that the PEG-Fab' had significantly higher resistance to surface-induced aggregation compared to the Fab'. Analysis of the interaction parameter, kD, indicated repulsive intermolecular forces for PEG-Fab' and attractive forces for Fab'. In conclusion, PEGylation appears to protect Fab' against thermal and mechanical stress-induced aggregation, likely due to a steric hindrance mechanism.

  18. Antibody-conjugated gold-gold sulfide nanoparticles as multifunctional agents for imaging and therapy of breast cancer

    Directory of Open Access Journals (Sweden)

    Emily S Day

    2010-06-01

    Full Text Available Emily S Day, Lissett R Bickford, John H Slater, Nicholas S Riggall, Rebekah A Drezek, Jennifer L WestDepartment of Bioengineering, Rice University, Houston, TX, USAAbstract: The goal of this study was to develop near-infrared (NIR resonant gold-gold sulfide nanoparticles (GGS-NPs as dual contrast and therapeutic agents for cancer management via multiphoton microscopy followed by higher intensity photoablation. We demonstrate that GGS-NPs exposed to a pulsed, NIR laser exhibit two-photon induced photoluminescence that can be utilized to visualize cancerous cells in vitro. When conjugated with anti-HER2 antibodies, these nanoparticles specifically bind SK-BR-3 breast carcinoma cells that overexpress the HER2 receptor, enabling the cells to be imaged via multiphoton microscopy with an incident laser power of 1 mW. Higher excitation power (50 mW could be employed to induce thermal damage to the cancerous cells, producing extensive membrane blebbing within seconds leading to cell death. GGS-NPs are ideal multifunctional agents for cancer management because they offer the ability to pinpoint precise treatment sites and perform subsequent thermal ablation in a single setting.Keywords: cancer, nanomedicine, multiphoton microscopy, photoluminescence, photothermal therapy, theranostics

  19. Novel antibody conjugates for enhanced tumor uptake. Final report, September 1, 1992--August 31, 1997

    International Nuclear Information System (INIS)

    Progress in three areas of research is summarized. These are as follows: Labeling Monoclonal antibodies (MAbs) with Tc-99m and Re-186; human melanoma tumors and specific MAbs; evaluation of biological response modifiers (BRM). The techniques of labeling MAbs (IgM, IgG, F(ab')2 or F(ab')) with Tc-99m was developed in the author's laboratory in 1989 and that with Re-186 in 1992. The techniques are in daily use in the laboratory since then and are adapted to a convenient kit formulation. The metal ions are bound at MAb sulfhydryls generated by a controlled reduction of a pair of disulfide groups. At least two types of MAbs labeled with Tc-99m by this method have been administered into patients and excellent diagnostic results have been obtained. Over the past two and a half years the author has been successfully growing human melanoma tumors in athymic Balb/c nude mice. The cell LINE, WM-9, was obtained from Dr. D Herlyn's laboratory at Wistar Institute in Philadelphia. Sufficient quantities of antihuman melanoma specific antibodies ME 31.3 (Wistar, IgG-1) and MEM-136 (Hybritech, IgG-2A) and their F(ab')2 fragments are also available in the laboratory. The use of BRM is a rapidly evolving field. Over the past four years, the author has evaluated a number of BRMs in a quest for agents that may augment MAb tumor uptake. These included interferon-α; a pokeweed mitogen and Ukrain, an alkaloid separated from a plant Chelideonium Majis. In these preliminary studies, normal Balb/c mice were used and the BRMs were given i.p. one hour prior to the i.v. administration of tumor necrosis factor or an MAb (TNT-F(ab')2) labeled with Tc-99m which served as an imaging agent. Animals were sacrificed at 1.5 hr or 4 hrs post-injection. Highlights of the work are given here in a table

  20. Novel antibody conjugates for enhanced tumor uptake. Final report, September 1, 1992--August 31, 1997

    Energy Technology Data Exchange (ETDEWEB)

    Thakur, M.

    1997-12-31

    Progress in three areas of research is summarized. These are as follows: Labeling Monoclonal antibodies (MAbs) with Tc-99m and Re-186; human melanoma tumors and specific MAbs; evaluation of biological response modifiers (BRM). The techniques of labeling MAbs (IgM, IgG, F(ab{prime}){sub 2} or F(ab{prime})) with Tc-99m was developed in the author`s laboratory in 1989 and that with Re-186 in 1992. The techniques are in daily use in the laboratory since then and are adapted to a convenient kit formulation. The metal ions are bound at MAb sulfhydryls generated by a controlled reduction of a pair of disulfide groups. At least two types of MAbs labeled with Tc-99m by this method have been administered into patients and excellent diagnostic results have been obtained. Over the past two and a half years the author has been successfully growing human melanoma tumors in athymic Balb/c nude mice. The cell LINE, WM-9, was obtained from Dr. D Herlyn`s laboratory at Wistar Institute in Philadelphia. Sufficient quantities of antihuman melanoma specific antibodies ME 31.3 (Wistar, IgG-1) and MEM-136 (Hybritech, IgG-2A) and their F(ab{prime}){sub 2} fragments are also available in the laboratory. The use of BRM is a rapidly evolving field. Over the past four years, the author has evaluated a number of BRMs in a quest for agents that may augment MAb tumor uptake. These included interferon-{alpha}; a pokeweed mitogen and Ukrain, an alkaloid separated from a plant Chelideonium Majis. In these preliminary studies, normal Balb/c mice were used and the BRMs were given i.p. one hour prior to the i.v. administration of tumor necrosis factor or an MAb (TNT-F(ab{prime}){sub 2}) labeled with Tc-99m which served as an imaging agent. Animals were sacrificed at 1.5 hr or 4 hrs post-injection. Highlights of the work are given here in a table.

  1. Preparation of HIV monoclonal antibody-conjugated pulchellin in order to study its intracellular trafficking pathway in HIV-infected cells by confocal microscopy

    Science.gov (United States)

    Sadraeian, M.; Tsutae, F. M.; Moreira, H. H. T.; Araujo, A. P. U.; Guimarães, F. E. G.; Pincus, S. H.

    2015-06-01

    Pulchellin is a type 2 of ribosome-inactivating proteins isolated from some seeds significantly growing in Brazil. It is a potent agent to inhibit the protein synthesis in cancer cells and also HIV-infected cells. Pulchellin can be conjugated to HIV monoclonal antibodies to specifically target the HIV-infected cells. To analyze the protein synthesis inhibition by Pulchellin, the intracellular localization of the immunoconjugate should be compared to Pulchellin. In this case, the intracellular trafficking of this protein in cells can be determined by confocal microscopy. In our study, we utilized Pulchellin to construct HIV monoclonal antibody-conjugated Pulchellin A chain in order to target HIV-infected lymphocyte cells. Afterward the conjugation was labeled with the superior Alexa Fluor 488 dye. As a subsequent step, we are interested in studying the intracellular trafficking pathway of this novel conjugation in HIV-infected cells by confocal microscopy. Moreover, possible quantitative methods for fluorescent labeling of the immunoconjugate during confocal microscopy will be investigated.

  2. Co-association of methotrexate and SPIONs into anti-CD64 antibody-conjugated PLGA nanoparticles for theranostic application

    Directory of Open Access Journals (Sweden)

    Moura CC

    2014-10-01

    significantly affect the properties of the nanoparticles. Conjugation with the anti-CD64 antibody, in turn, caused a slight increase in size and surface charge. Transmission electron microscopy confirmed the association of SPIONs within the poly(lactic-co-glycolic acid matrix. Both anti-CD64 and methotrexate association were confirmed by Fourier transform infrared spectroscopy, and quantified yielding values as high as 36% and 79%, respectively. In vitro toxicity studies confirmed the methotrexate-loaded nanosystem to be more effective than the free drug.Conclusion: Multifunctional anti-CD64-conjugated poly(lactic-co-glycolic acid nanoparticles for the combined delivery of methotrexate and SPIONs were successfully prepared and characterized. This nanosystem has the potential to provide a new theranostic approach for the management of RA. Keywords: FcγRI, methotrexate, poly(lactic-co-glycolic acid, superparamagnetic iron oxide nanoparticles, targeted drug delivery

  3. SGN-CD33A: a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML.

    Science.gov (United States)

    Kung Sutherland, May S; Walter, Roland B; Jeffrey, Scott C; Burke, Patrick J; Yu, Changpu; Kostner, Heather; Stone, Ivan; Ryan, Maureen C; Sussman, Django; Lyon, Robert P; Zeng, Weiping; Harrington, Kimberly H; Klussman, Kerry; Westendorf, Lori; Meyer, David; Bernstein, Irwin D; Senter, Peter D; Benjamin, Dennis R; Drachman, Jonathan G; McEarchern, Julie A

    2013-08-22

    Outcomes in acute myeloid leukemia (AML) remain unsatisfactory, and novel treatments are urgently needed. One strategy explores antibodies and their drug conjugates, particularly those targeting CD33. Emerging data with gemtuzumab ozogamicin (GO) demonstrate target validity and activity in some patients with AML, but efficacy is limited by heterogeneous drug conjugation, linker instability, and a high incidence of multidrug resistance. We describe here the development of SGN-CD33A, a humanized anti-CD33 antibody with engineered cysteines conjugated to a highly potent, synthetic DNA cross-linking pyrrolobenzodiazepine dimer via a protease-cleavable linker. The use of engineered cysteine residues at the sites of drug linker attachment results in a drug loading of approximately 2 pyrrolobenzodiazepine dimers per antibody. In preclinical testing, SGN-CD33A is more potent than GO against a panel of AML cell lines and primary AML cells in vitro and in xenotransplantation studies in mice. Unlike GO, antileukemic activity is observed with SGN-CD33A in AML models with the multidrug-resistant phenotype. Mechanistic studies indicate that the cytotoxic effects of SGN-CD33A involve DNA damage with ensuing cell cycle arrest and apoptotic cell death. Together, these data suggest that SGN-CD33A has CD33-directed antitumor activity and support clinical testing of this novel therapeutic in patients with AML.

  4. Synthesis and Characterization of Anti-HER2 Antibody Conjugated CdSe/CdZnS Quantum Dots for Fluorescence Imaging of Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Takashi Jin

    2009-11-01

    Full Text Available The early detection of HER2 (human epidermal growth factor receptor 2 status in breast cancer patients is very important for the effective implementation of anti-HER2 antibody therapy. Recently, HER2 detections using antibody conjugated quantum dots (QDs have attracted much attention. QDs are a new class of fluorescent materials that have superior properties such as high brightness, high resistance to photo-bleaching, and multi-colored emission by a single-light source excitation. In this study, we synthesized three types of anti-HER2 antibody conjugated QDs (HER2Ab-QDs using different coupling agents (EDC/sulfo-NHS, iminothiolane/sulfo-SMCC, and sulfo-SMCC. As water-soluble QDs for the conjugation of antibody, we used glutathione coated CdSe/CdZnS QDs (GSH-QDs with fluorescence quantum yields of 0.23~0.39 in aqueous solution. Dispersibility, hydrodynamic size, and apparent molecular weights of the GSH-QDs and HER2Ab-QDs were characterized by using dynamic light scattering, fluorescence correlation spectroscopy, atomic force microscope, and size-exclusion HPLC. Fluorescence imaging of HER2 overexpressing cells (KPL-4 human breast cancer cell line was performed by using HER2Ab-QDs as fluorescent probes. We found that the HER2Ab-QD prepared by using SMCC coupling with partially reduced antibody is a most effective probe for the detection of HER2 expression in KPL-4 cells. We have also studied the size dependency of HER2Ab-QDs (with green, orange, and red emission on the fluorescence image of KPL-4 cells.

  5. Synthesis and Characterization of Anti-HER2 Antibody Conjugated CdSe/CdZnS Quantum Dots for Fluorescence Imaging of Breast Cancer Cells.

    Science.gov (United States)

    Tiwari, Dhermendra K; Tanaka, Shin-Ichi; Inouye, Yasushi; Yoshizawa, Keiko; Watanabe, Tomonobu M; Jin, Takashi

    2009-01-01

    The early detection of HER2 (human epidermal growth factor receptor 2) status in breast cancer patients is very important for the effective implementation of anti-HER2 antibody therapy. Recently, HER2 detections using antibody conjugated quantum dots (QDs) have attracted much attention. QDs are a new class of fluorescent materials that have superior properties such as high brightness, high resistance to photo-bleaching, and multi-colored emission by a single-light source excitation. In this study, we synthesized three types of anti-HER2 antibody conjugated QDs (HER2Ab-QDs) using different coupling agents (EDC/sulfo-NHS, iminothiolane/sulfo-SMCC, and sulfo-SMCC). As water-soluble QDs for the conjugation of antibody, we used glutathione coated CdSe/CdZnS QDs (GSH-QDs) with fluorescence quantum yields of 0.23∼0.39 in aqueous solution. Dispersibility, hydrodynamic size, and apparent molecular weights of the GSH-QDs and HER2Ab-QDs were characterized by using dynamic light scattering, fluorescence correlation spectroscopy, atomic force microscope, and size-exclusion HPLC. Fluorescence imaging of HER2 overexpressing cells (KPL-4 human breast cancer cell line) was performed by using HER2Ab-QDs as fluorescent probes. We found that the HER2Ab-QD prepared by using SMCC coupling with partially reduced antibody is a most effective probe for the detection of HER2 expression in KPL-4 cells. We have also studied the size dependency of HER2Ab-QDs (with green, orange, and red emission) on the fluorescence image of KPL-4 cells. PMID:22291567

  6. A sensitive multidimensional method for the detection, characterization, and quantification of trace free drug species in antibody-drug conjugate samples using mass spectral detection.

    Science.gov (United States)

    Birdsall, Robert E; McCarthy, Sean M; Janin-Bussat, Marie Claire; Perez, Michel; Haeuw, Jean-François; Chen, Weibin; Beck, Alain

    2016-01-01

    Conjugation processes and stability studies associated with the production and shelf life of antibody-drug conjugates (ADCs) can result in free (non-conjugated) drug species. These free drug species can increase the risk to patients and reduce the efficacy of the ADC. Despite stringent purification steps, trace levels of free drug species may be present in formulated ADCs, reducing the therapeutic window. The reduction of sample preparation steps through the incorporation of multidimensional techniques has afforded analysts more efficient methods to assess trace drug species. Multidimensional methods coupling size-exclusion and reversed phase liquid chromatography with ultra-violet detection (SEC-RPLC/UV) have been reported, but offer limited sensitivity and can limit method optimization. The current study addresses these challenges with a multidimensional method that is specific, sensitive, and enables method control in both dimensions via coupling of an on-line solid phase extraction column to RPLC with mass spectral detection (SPE-RPLC/MS). The proposed method was evaluated using an antibody-fluorophore conjugate (AFC) as an ADC surrogate to brentuximab vedotin and its associated parent maleimide-val-cit-DSEA payload and the derived N-acetylcysteine adduct formed during the conjugation process. Assay sensitivity was found to be 2 orders more sensitive using MS detection in comparison to UV-based detection with a nominal limit of quantitation of 0.30 ng/mL (1.5 pg on-column). Free-drug species were present in an unadulterated ADC surrogate sample at concentrations below 7 ng/mL, levels not detectable by UV alone. The proposed SPE-RPLC/MS method provides a high degree of specificity and sensitivity in the assessment of trace free drug species and offers improved control over each dimension, enabling straightforward integration into existing or novel workflows. PMID:26651262

  7. Novel Hybrid Compound of a Plinabulin Prodrug with an IgG Binding Peptide for Generating a Tumor Selective Noncovalent-Type Antibody-Drug Conjugate.

    Science.gov (United States)

    Muguruma, Kyohei; Yakushiji, Fumika; Kawamata, Ryosuke; Akiyama, Daichi; Arima, Risako; Shirasaka, Takuya; Kikkawa, Yamato; Taguchi, Akihiro; Takayama, Kentaro; Fukuhara, Takeshi; Watabe, Tetsuro; Ito, Yuji; Hayashi, Yoshio

    2016-07-20

    Although several approaches for making antibody-drug conjugates (ADC) have been developed, it has yet to be reported that an antibody binding peptide such as Z33 from protein A is utilized as the pivotal unit to generate the noncovalent-type ADC (NC-ADC). Herein we aim to establish a novel probe for NC-ADC by synthesizing the Z33-conjugated antitumor agent, plinabulin. Due to the different solubility of two components, including hydrophobic plinabulin and hydrophilic Z33, an innovative method with a solid-supported disulfide coupling reagent is required for the synthesis of the target compounds with prominent efficiency (29% isolated yield). We demonstrate that the synthesized hybrid exhibits a binding affinity against the anti-HER2 antibody (Herceptin) and the anti-CD71 antibody (6E1) (Kd = 46.6 ± 0.5 nM and 4.5 ± 0.56 μM, respectively) in the surface plasmon resonance (SPR) assay. In the cell-based assays, the hybrid provides a significant cytotoxicity in the presence of Herceptin against HER2 overexpressing SKBR-3 cells, but not against HER2 low-expressing MCF-7 cells. Further, it is noteworthy that the hybrid in combination with Herceptin induces cytotoxicity against Herceptin-resistant SKBR-3 (SKBR-3HR) cells. Similar results are obtained with the 6E1 antibody, suggesting that the synthesized hybrid can be widely applicable for NC-ADC using the antibody of interest. In summary, a series of evidence presented here strongly indicate that NC-ADCs have high potential for the next generation of antitumor agents. PMID:27304609

  8. Cancer Cell Targeting Using Folic Acid/Anti-HER2 Antibody Conjugated Fluorescent CdSe/CdS/ZnS-MPA and CdTe-MSA Quantum Dots.

    Science.gov (United States)

    Singh, Gurpal; Kumar, Manoj; Soni, Udit; Arora, Vikas; Bansal, Vivek; Gupta, Dikshi; Bhat, Madhusudan; Dinda, Amit K; Sapra, Sameer; Singh, Harpal

    2015-12-01

    CdSe/CdS/ZnS and CdTe quantum dots (QDs) were synthesized by successive ion layer adsorption and reaction (SILAR) technique and direct aqueous synthesis respectively using thiol stabilizers. Synthesized CdSe/CdS/ZnS and CdTe QDs stabilized with 3-mercaptopropionic acid (MPA) and mercaptosuccinic acid (MSA) were used as fluorescent labels after conjugation with folic acid (FA) and anti-HER2 antibodies. Photoluminescence quantum yield of folated CdSe/CdS/ZnS-MPA and CdTe-MSA QDs was 59% and 77% than that of non-folated hydrophilic QDs. The folate receptor-mediated delivery of folic acid-conjugated CdTe-MSA and CdSe/CdS/ZnS-MPA QDs showed higher cellular internalization as observed by confocal laser scanning microscopic studies. Folated and non-folated CdTe-MSA QDs were highly toxic and exhibited only 10% cell viability as compared to > 80% cell viability with CdSe/CdS/ZnS-MPA QDs over the concentration ranging from 3.38 to 50 pmoles. Immunohistochemistry (IHC) results of human breast cancer tissue samples showed positive results with anti-HER2 antibody conjugated CdSe/CdS/ZnS-MPA QDs with better sensitivity and specificity as compared to conventional IHC analysis using diaminobenzedene staining. PMID:26682358

  9. The Methylene Alkoxy Carbamate Self-Immolative Unit: Utilization for the Targeted Delivery of Alcohol-Containing Payloads with Antibody-Drug Conjugates.

    Science.gov (United States)

    Kolakowski, Robert V; Haelsig, Karl T; Emmerton, Kim K; Leiske, Chris I; Miyamoto, Jamie B; Cochran, Julia H; Lyon, Robert P; Senter, Peter D; Jeffrey, Scott C

    2016-07-01

    A strategy for the conjugation of alcohol-containing payloads to antibodies has been developed and involves the methylene alkoxy carbamate (MAC) self-immolative unit. A series of MAC β-glucuronide model constructs were prepared to evaluate stability and enzymatic release, and the results demonstrated high stability at physiological pH in a substitution-dependent manner. All the MAC model compounds efficiently released alcohol drug surrogates under the action of β-glucuronidase. To assess the MAC technology for ADCs, the potent microtubule-disrupting agent auristatin E (AE) was incorporated through the norephedrine alcohol. Conjugation of the MAC β-glucuronide AE drug linker to the anti-CD30 antibody cAC10, and an IgG control antibody, gave potent and immunologically specific activities in vitro and in vivo. These studies validate the MAC self-immolative unit for alcohol-containing payloads within ADCs, a class that has not been widely exploited. PMID:27198854

  10. Elimination of Tumor Cells Using Folate Receptor Targeting by Antibody-Conjugated, Gold-Coated Magnetite Nanoparticles in a Murine Breast Cancer Model

    Directory of Open Access Journals (Sweden)

    Evan S. Krystofiak

    2012-01-01

    Full Text Available Background. The chemotherapeutic treatment of cancer suffers from poor specificity for targeting the tumor cells and often results in adverse effects such as systemic toxicity, damage to nontarget tissues, and development of drug-resistant tumors in patients. Increasingly, drug nanocarriers have been explored as a way of lessening or overcoming these problems. In this study, antibody-conjugated Au-coated magnetite nanoparticles, in conjunction with inductive heating produced by exposure to an oscillating magnetic field (OMF, were evaluated for their effects on the viability of tumor cells in a murine model of breast cancer. Treatment effects were evaluated by light microscopy and SEM. Results. 4T1 mammary epithelial carcinoma cells overexpressing the folate receptor were targeted with an anti-folate receptor primary antibody, followed by labeling with secondary antibody-conjugated Au-coated magnetite nanoparticles. In the absence of OMF exposure, nanoparticle labeling had no effect on 4T1 cell viability. However, following OMF treatment, many of the labeled 4T1 cells showed extensive membrane damage by SEM analysis, and dramatically reduced viability as assessed using a live/dead staining assay. Conclusions. These results demonstrate that Au-coated magnetite targeted to tumor cells through binding to an overexpressed surface receptor, in the presence of an OMF, can lead to tumor cell death.

  11. DOTA-functionalized polylysine: a high number of DOTA chelates positively influences the biodistribution of enzymatic conjugated anti-tumor antibody chCE7agl.

    Directory of Open Access Journals (Sweden)

    Jürgen Grünberg

    Full Text Available Site-specific enzymatic reactions with microbial transglutaminase (mTGase lead to a homogenous species of immunoconjugates with a defined ligand/antibody ratio. In the present study, we have investigated the influence of different numbers of 1,4,7,10-tetraazacyclododecane-N-N'-N''-N'''-tetraacetic acid (DOTA chelats coupled to a decalysine backbone on the in vivo behavior of the chimeric monoclonal anti-L1CAM antibody chCE7agl. The enzymatic conjugation of (DOTA1-decalysine, (DOTA3-decalysine or (DOTA5-decalysine to the antibody heavy chain (via Gln295/297 gave rise to immunoconjugates containing two, six or ten DOTA moieties respectively. Radiolabeling of the immunoconjugates with (177Lu yielded specific activities of approximately 70 MBq/mg, 400 MBq/mg and 700 MBq/mg with increasing numbers of DOTA chelates. Biodistribution experiments in SKOV3ip human ovarian cancer cell xenografts demonstrated a high and specific accumulation of radioactivity at the tumor site for all antibody derivatives with a maximal tumor accumulation of 43.6±4.3% ID/g at 24 h for chCE7agl-[(DOTA-decalysine]2, 30.6±12.0% ID/g at 24 h for chCE7agl-[(DOTA3-decalysine]2 and 49.9±3.1% ID/g at 48 h for chCE7agl-[(DOTA5-decalysine]2. The rapid elimination from the blood of chCE7agl-[(DOTA-decalysine]2 (1.0±0.1% ID/g at 24 h is associated with a high liver accumulation (23.2±4.6% ID/g at 24 h. This behavior changed depending on the numbers of DOTA moieties coupled to the decalysine peptide with a slower blood clearance (5.1±1.0 (DOTA3 versus 11.7±1.4% ID/g (DOTA5, p<0.005 at 24 h and lower radioactivity levels in the liver (21.4±3.4 (DOTA3 versus 5.8±0.7 (DOTA5, p<0.005 at 24 h. We conclude that the site-specific and stoichiometric uniform conjugation of the highly DOTA-substituted decalysine ((DOTA5-decalysine to an anti-tumor antibody leads to the formation of immunoconjugates with high specific activity and excellent in vivo behavior and is a valuable option for

  12. Heptavalent pneumococcal conjugate vaccine elicits similar antibody response as standard 23-valent polysaccharide vaccine in adult patients with RA treated with immunomodulating drugs.

    Science.gov (United States)

    Kapetanovic, Meliha Crnkic; Roseman, Carmen; Jönsson, Göran; Truedsson, Lennart

    2011-12-01

    The objectives of the study were to compare antibody response in immunosuppressed patients with rheumatoid arthritis (RA) after vaccination with heptavalent pneumococcal conjugate vaccine (PCV7) to that of RA patients and healthy controls vaccinated with 23-valent polysaccharide vaccine (PPV23) and to study the impact of disease and/or treatment characteristics and type of vaccine on antibody response following pneumococcal vaccination in patients with RA. In total, 253 RA patients treated with methotrexate (MTX), anti-TNF blockers as monotherapy or anti-TNF + MTX were vaccinated with a single dose (0.5 ml) of PCV7. In addition, 149 RA patients receiving corresponding treatments and 47 healthy controls were vaccinated with a single dose (0.5 ml) of PPV23. Serotype-specific IgG to 23F and 6B were measured at vaccination and 4-6 weeks after vaccination using ELISA. Antibody response ratio (ARR), i.e. ratio between post-/prevaccination antibody levels, was compared between corresponding treatment groups. Differences in ARR were analysed using analysis of variance. Positive antibody response (posAR) was defined as equal to or greater than twofold increase in prevaccination antibody levels. Possible predictors of posAR were analysed using logistic regression model. Corresponding RA treatment groups showed similar ARR and posAR for both serotypes regardless of vaccine type. Higher age at vaccination and concomitant MTX were identified as predictors of impaired posAR for both serotypes tested, whereas type of vaccine did not influence posAR significantly. PCV7 elicits similar antibody response as PPV23 in patients with RA receiving immunosuppressive treatment. In RA patients, higher age and MTX treatment but not type of vaccine predicted impaired posAR.

  13. Outer Membrane Protein Complex of Meningococcus Enhances the Antipolysaccharide Antibody Response to Pneumococcal Polysaccharide–CRM197 Conjugate Vaccine ▿

    OpenAIRE

    Lai, Zengzu; Schreiber, John R.

    2011-01-01

    Bacterial polysaccharides (PS) are T cell-independent antigens that do not induce immunologic memory and are poor immunogens in infants. Conjugate vaccines in which the PS is covalently linked to a carrier protein have enhanced immunogenicity that resembles that of T cell-dependent antigens. The Haemophilus influenzae type b (Hib) conjugate vaccine, which uses the outer membrane protein complex (OMPC) from meningococcus as a carrier protein, elicits protective levels of anti-capsular PS antib...

  14. Impact of IgM Antibodies on Cross-Protection against Pneumococcal Serogroups 6 and 19 after Immunization with 7-Valent Pneumococcal Conjugate Vaccine in Children.

    Science.gov (United States)

    Cho, Hye-Kyung; Park, In Ho; Burton, Robert L; Kim, Kyung-Hyo

    2016-06-01

    Although it is well known that pneumococcal conjugate vaccines provide cross-protection against some vaccine-related serotypes, these mechanisms are still unclear. This study was performed to investigate the role of cross-protective IgM antibodies against vaccine-related serotypes 6A, 6C, and 19A induced in children aged 12-23 months after immunization with 7-valent pneumococcal conjugate vaccine (PCV7). We obtained serum samples from 18 Korean children aged 12-23 months after a PCV7 booster immunization. The serum IgG and IgM concentrations of serotypes 6B and 19F were measured by enzyme-linked immunosorbent assay (ELISA) in serum. The opsonic indices (OIs) against vaccine serotypes 6B and 19F and vaccine-related serotypes 6A, 6C, and 19A were determined by an opsonophagocytic killing assay (OPA) in IgM-depleted and control serum. Both IgG and IgM antibodies in ELISA and opsonic indices in OPA against serotypes 6B and 19F were demonstrated in the immune serum. IgM depletion decreased the OIs against vaccine serotypes 6B (geometric means of OIs (GMIs) of 3,009 vs. 1,396, 38% reduction) and 19F (1,117 vs. 750, 36% reduction). In addition, IgM depletion markedly decreased the OIs against vaccine-related serotypes 6A (GMIs of 961 vs. 329, 70% reduction), 6C (432 vs. 185, 72% reduction), and 19A (301 vs. 166, 58% reduction). The booster immunization PCV7 induced protective antibodies in the form of both IgG and IgM isotypes. IgM antibodies contributed to eliciting cross-protection against vaccine-related serotypes as well as against vaccine serotypes. PMID:27247505

  15. Development of indirect competitive fluorescence immunoassay for 2,2′,4,4′-tetrabromodiphenyl ether using DNA/dye conjugate as antibody multiple labels

    Institute of Scientific and Technical Information of China (English)

    Zi-Yan Fan; Young Soo Keum; Qing-Xiao Li; Weilin L. Shelver; Liang-Hong Guo

    2012-01-01

    An indirect competitive fluorescence immunoassay using a DNA/dye conjugate as antibody multiple labels was developed on 96-well plates for the identification and quantification of 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) in aqueous samples.A hapten,2,4,2′-tribromodiphenyl ether-4′-aldehyde,was synthesized,and was conjugated to bovine serum albumin to form a coating antigen,Specific recognition of the antigen by anti-PBDE antiserum was confirmed by a surface plasmon resonance measurement.In the immunoassay,the coating antigen was adsorbed on a 96-well plate first,and a sample,antiserum and biotinylated goat anti-rabbit secondary antibody were then added and reacted sequentially.A biotinylated,double-stranded DNA with 219 base pairs was attached to the secondary antibody by using streptavidin as a molecular bridge.In situ multiple labeling of the antibody was accomplished after addition of a DNA-binding fluorescent dye,SYBR Green I.The working range of the immunoassay for the BDE-47 standard was 3.1-390 μg/L,with an IC50 value of 15.6 μg/L.The calculated LOD of the immunoassay is 0.73 μg/L.The immunoassay demonstrated relatively high selectivity for BDE-47,showing very low cross-reactivity (< 3%) with BDE-15,BDE-153 and BDE-209.With a spiked river water sample containing 50 μg/L BDE-47,quantification by the immunoassay was 41.9 μg/L,which compared well with the standard GC-ECD method (45.7 μg/L).The developed immunoassay provides a rapid screening tool for polybrominated diphenyl ethers in environmental samples.

  16. Comparison of a chimeric anti-carcinoembryonic antigen antibody conjugated with visible or near-infrared fluorescent dyes for imaging pancreatic cancer in orthotopic nude mouse models

    Science.gov (United States)

    Maawy, Ali A.; Hiroshima, Yukihiko; Kaushal, Sharmeela; Luiken, George A.; Hoffman, Robert M.; Bouvet, Michael

    2013-12-01

    The aim of this study was to evaluate a set of visible and near-infrared dyes conjugated to a tumor-specific chimeric antibody for high-resolution tumor imaging in orthotopic models of pancreatic cancer. BxPC-3 human pancreatic cancer was orthotopically implanted into pancreata of nude mice. Mice received a single intravenous injection of a chimeric anti-carcinoembryonic antigen antibody conjugated to one of the following fluorophores: 488-nm group (Alexa Fluor 488 or DyLight 488); 550-nm group (Alexa Fluor 555 or DyLight 550); 650-nm group (Alexa Fluor 660 or DyLight 650), or the 750-nm group (Alexa Fluor 750 or DyLight 755). After 24 h, the Olympus OV100 small-animal imaging system was used for noninvasive and intravital fluorescence imaging of mice. Dyes were compared with respect to depth of imaging, resolution, tumor-to-background ratio (TBR), photobleaching, and hemoglobin quenching. The longer wavelength dyes had increased depth of penetration and ability to detect the smallest tumor deposits and provided the highest TBRs, resistance to hemoglobin quenching, and specificity. The shorter wavelength dyes were more photostable. This study showed unique advantages of each dye for specific cancer imaging in a clinically relevant orthotopic model.

  17. The HB22.7-vcMMAE antibody-drug conjugate has efficacy against non-Hodgkin lymphoma mouse xenografts with minimal systemic toxicity.

    Science.gov (United States)

    Abuhay, Mastewal; Kato, Jason; Tuscano, Emily; Barisone, Gustavo A; Sidhu, Ranjit S; O'Donnell, Robert T; Tuscano, Joseph M

    2016-10-01

    In this study, HB22.7, an anti-CD22 monoclonal antibody, was used for specific, targeted delivery of monomethyl auristatin E (MMAE) to non-Hodgkin lymphoma (NHL). MMAE was covalently coupled to HB22.7 through a valine-citrulline peptide linker (vc). Maleimide-functionalized vcMMAE (mal-vcMMAE) was reacted with thiols of the partially reduced mAb. Approximately 4 molecules of MMAE were conjugated to HB22.7 as determined by residual thiol measurement and hydrophobic interaction chromatography-HPLC (HIC-HPLC). HB22.7-vcMMAE antibody-drug conjugate (ADC) retained its binding to Ramos NHL cells and also exhibited potent and specific in vitro cytotoxicity on a panel of B cell NHL cell lines with IC50s of 20-284 ng/ml. HB22.7-vcMMAE also showed potent efficacy in vivo against established NHL xenografts using the DoHH2 and Granta 519 cell lines. One dose of the ADC induced complete and persistent response in all DoHH2 xenografts and 90 % of Granta xenografts. Minimal toxicity was observed. In summary, HB22.7-vcMMAE is an effective ADC that should be evaluated for clinical translation. PMID:27506529

  18. Localization of radioiodine conjugated to the monoclonal antibody HMFG2 in human ovarian carcinoma: assessment of intravenous and intraperitoneal routes of administration

    Energy Technology Data Exchange (ETDEWEB)

    Ward, B.G.; Mather, S.J.; Hawkins, L.R.; Crowther, M.E.; Shepherd, J.H.; Granowska, M.; Britton, K.E.; Slevin, M.L.

    1987-09-01

    The localization of i.p. injected, radioiodine conjugated monoclonal antibody HMFG2 was studied in 18 patients with ovarian carcinoma. Patients were injected i.p. at time points up to 168 h before laparotomy, at which time tumor, ascites, normal tissue, and blood samples were removed and the contained radioactivity measured. In the first 10 patients, localization was compared with that of a simultaneously injected irrelevant (nonspecific) antibody (UJ13A) of the same immunoglobulin class and, in the subsequent 8 patients, with HMFG2 administered i.v. After i.p. injection, HMFG2-radioiodine was found in concentrations of 0.0001-0.0030% of the injected amount per gram in solid tumor, 0.0363-0.02560%/g in ascites, 0.0003-0.0017%/g in blood, and 0.001-0.0012%/g in normal tissue. Tumor:normal tissue ratios of 0.9-10.0 and tumor:blood ratios of 0.3-4.0 were seen up to 168 h after injection. Localization of the HMFG2 conjugate was consistently greater than that of the irrelevant antibody. For solid tumor, the i.v. route of administration resulted in consistently higher absolute levels of HMFG2 conjugate uptake but tumor:blood and tumor:normal tissue ratios were similar. On the other hand the i.p. route of administration offered consistent advantages of 4- to 71-fold over the i.v. route when HMFG2 conjugate localization on ascites cells was examined. Ascites:normal tissue and ascites:blood ratios of up to 512 and 448, respectively, were achieved. After i.p. injection, radioiodine was cleared from the body exponentially with a half-life of 50 h. Maximum circulating blood levels of 8.6 +/- 2.0% injected activity were seen at 48 h and these then decreased with a t 1/2 value of 38 h. Over 80% of injected activity was cleared in the urine as nonprotein bound iodine by 168 h.

  19. Localization of radioiodine conjugated to the monoclonal antibody HMFG2 in human ovarian carcinoma: assessment of intravenous and intraperitoneal routes of administration

    International Nuclear Information System (INIS)

    The localization of i.p. injected, radioiodine conjugated monoclonal antibody HMFG2 was studied in 18 patients with ovarian carcinoma. Patients were injected i.p. at time points up to 168 h before laparotomy, at which time tumor, ascites, normal tissue, and blood samples were removed and the contained radioactivity measured. In the first 10 patients, localization was compared with that of a simultaneously injected irrelevant (nonspecific) antibody (UJ13A) of the same immunoglobulin class and, in the subsequent 8 patients, with HMFG2 administered i.v. After i.p. injection, HMFG2-radioiodine was found in concentrations of 0.0001-0.0030% of the injected amount per gram in solid tumor, 0.0363-0.02560%/g in ascites, 0.0003-0.0017%/g in blood, and 0.001-0.0012%/g in normal tissue. Tumor:normal tissue ratios of 0.9-10.0 and tumor:blood ratios of 0.3-4.0 were seen up to 168 h after injection. Localization of the HMFG2 conjugate was consistently greater than that of the irrelevant antibody. For solid tumor, the i.v. route of administration resulted in consistently higher absolute levels of HMFG2 conjugate uptake but tumor:blood and tumor:normal tissue ratios were similar. On the other hand the i.p. route of administration offered consistent advantages of 4- to 71-fold over the i.v. route when HMFG2 conjugate localization on ascites cells was examined. Ascites:normal tissue and ascites:blood ratios of up to 512 and 448, respectively, were achieved. After i.p. injection, radioiodine was cleared from the body exponentially with a half-life of 50 h. Maximum circulating blood levels of 8.6 +/- 2.0% injected activity were seen at 48 h and these then decreased with a t 1/2 value of 38 h. Over 80% of injected activity was cleared in the urine as nonprotein bound iodine by 168 h

  20. Antibody-conjugated, dual-modal, near-infrared fluorescent iron oxide nanoparticles for antiamyloidgenic activity and specific detection of amyloid-β fibrils

    Directory of Open Access Journals (Sweden)

    Skaat H

    2013-10-01

    Full Text Available Hadas Skaat,1 Enav Corem-Slakmon,1 Igor Grinberg,1 David Last,2 David Goez,2 Yael Mardor,2,3 Shlomo Margel1 1Department of Chemistry, Bar-Ilan Institute of Nanotechnology and Advanced Materials, Ramat-Gan, Israel; 2Advanced Technology Center, Sheba Medical Center, Tel-Hashomer, Ramat-Gan, Israel; 3Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel Abstract: Amyloid-β (Aβ peptide is the main fibrillar component of plaque deposits found in brains affected by Alzheimer's disease (AD and is related to the pathogenesis of AD. Passive anti-Aβ immunotherapy has emerged as a promising approach for the therapy of AD, based on the administration of specific anti-Aβ monoclonal antibodies (aAβmAbs to delay Aβ aggregation in the brain. However, the main disadvantage of this approach is the required readministration of the aAβmAbs at frequent intervals. There are only a few reports describing in vitro study for the immobilization of aAβmAbs to nanoparticles as potential targeting agents of Aβ aggregates. In this article, we report the immobilization of the aAβmAb clone BAM10 to near-infrared fluorescent maghemite nanoparticles for the inhibition of Aβ40 fibrillation kinetics and the specific detection of Aβ40 fibrils. The BAM10-conjugated iron oxide nanoparticles were well-characterized, including their immunogold labeling and cytotoxic effect on PC-12 (pheochromocytoma cell line. Indeed, these antibody-conjugated nanoparticles significantly inhibit the Aβ40 fibrillation kinetics compared with the same concentration, or even five times higher, of the free BAM10. This inhibitory effect was confirmed by different assays such as the photo-induced crosslinking of unmodified proteins combined with sodium dodecyl sulfate–polyacrylamide gel electrophoresis. A cell viability assay also confirmed that these antibody-conjugated nanoparticles significantly reduced the Aβ40-induced cytotoxicity to PC-12 cells. Furthermore, the selective

  1. 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

    2012-07-01

    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

  2. Anti-HER2 antibody and ScFvEGFR-conjugated antifouling magnetic iron oxide nanoparticles for targeting and magnetic resonance imaging of breast cancer

    Directory of Open Access Journals (Sweden)

    Chen H

    2013-10-01

    Full Text Available Hongwei Chen,1,* Liya Wang,1,2,* Qiqi Yu,1,2 Weiping Qian,3 Diana Tiwari,1 Hong Yi,4 Andrew Y Wang,5 Jing Huang,1,2 Lily Yang,3 Hui Mao1,2 1Department of Radiology and Imaging Sciences, 2Center for Systems Imaging, 3Department of Surgery, Emory University School of Medicine, 4Robert Apkarian Electron Microscopy Core, Emory University, Atlanta, GA, 5Ocean NanoTech LLC, Springdale, AK, USA *These authors contributed equally to this work Abstract: Antifouling magnetic iron oxide nanoparticles (IONPs coated with block copolymer poly(ethylene oxide-block-poly(γ-methacryloxypropyltrimethoxysilane (PEO-b-PγMPS were investigated for improving cell targeting by reducing nonspecific uptake. Conjugation of a HER2 antibody, Herceptin®, or a single chain fragment (ScFv of antibody against epidermal growth factor receptor (ScFvEGFR to PEO-b-PγMPS-coated IONPs resulted in HER2-targeted or EGFR-targeted IONPs (anti-HER2-IONPs or ScFvEGFR-IONPs. The anti-HER2-IONPs bound specifically to SK-BR-3, a HER2-overexpressing breast cancer cell line, but not to MDA-MB-231, a HER2-underexpressing cell line. On the other hand, the ScFvEGFR-IONPs showed strong reactivity with MDA-MB-231, an EGFR-positive human breast cancer cell line, but not with MDA-MB-453, an EGFR-negative human breast cancer cell line. Transmission electron microscopy revealed internalization of the receptor-targeted nanoparticles by the targeted cancer cells. In addition, both antibody-conjugated and non-antibody-conjugated IONPs showed reduced nonspecific uptake by RAW264.7 mouse macrophages in vitro. The developed IONPs showed a long blood circulation time (serum half-life 11.6 hours in mice and low accumulation in both the liver and spleen. At 24 hours after systemic administration of ScFvEGFR-IONPs into mice bearing EGFR-positive breast cancer 4T1 mouse mammary tumors, magnetic resonance imaging revealed signal reduction in the tumor as a result of the accumulation of the targeted IONPs

  3. 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

    Science.gov (United States)

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

    2014-10-01

    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 pdyes). Human pancreatic tumors subcutaneously implanted into nude mice were labeled with antibody-dye conjugates and serially imaged. Labeling with conjugated PEGylated dyes resulted in significantly brighter tumors compared to the non-PEGylated dyes (pdyes; p=0.01 for 750 dyes). PEGylation of the NIR dyes also decreased 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.

  4. Antitumor activity of anti-type Ⅳcollagenase monoclonal antibody and its lidamycin conjugate against colon carcinoma

    Institute of Scientific and Technical Information of China (English)

    Liang Li; Yun-Hong Huang; Yi Li; Feng-Qiang Wang; Bo-Yang Shang; Yong-Su Zhen

    2005-01-01

    AIM: Type Ⅳ collagenase including MMP-2 and -9 plays an important role in cancer cell invasion and metastasis and is an attractive target for mAb-directed therapy. The immunoreactivity of mAb 3G11, a mAb directed against type Ⅳ collagenase in human colorectal carcinomas, was studied by immuno-histochemical (IHC) staining. mAb 3G11 was conjugated to an antitumor antibiotic lidamycin (LDM). The antitumor activity of 3G11-LDM conjugate against colon carcinoma was investigated in mice.METHODS: ELISA, gelatin zymography, and Western blot assay were used for the biological characterization of mAb 3G11. The immunoreactivity of mAb 3G11 with human colorectal carcinomas was detected by IHC staining. The cytotoxicity of LDM and 3G11-LDM conjugate to human colon carcinoma HT-29 cells was examined by clonogenic assay and MTT assay. The therapeutic effect of conjugate 3G11-LDM was evaluated with colon carcinoma 26 in mice.RESULTS: As shown in ELISA, mAb 3G11 reacted specifically with type Ⅳ collagenase, while 3G11-LDM conjugate also recognized specifically its respective antigen. In IHC assay,mAb 3G11 showed positive immunoreactivity in most cases of colorectal carcinoma, and negative immunoreactivity in the adjacent non-malignant tissues. By gelatin zymography,the inhibition effect of mAb 3G11 on the secretion activity of type Ⅳ collagenase was proved. In terms of IC50 values in MTT assay, the cytotoxicity of LDM to human colon carcinoma HT-29 cells was 10 000-fold more potent than that of mitomycin C (MMC) and adriamycin (ADM). 3G11-LDM conjugate also displayed extremely potent cytotoxicity to human colon carcinoma HT-29 cells with an ICs0 value of 5.6×10-19 mol/L. 3G11-LDM conjugate at the doses of 0.05 and 0.1 mg/kg inhibited the growth of colon carcinoma 26 in mice by 70.3 and 81.2%, respectively.CONCLUSION: mAb 3G11 is immunoreactive with human colorectal carcinoma and its conjugate with LDM is highly effective against colon carcinoma in mice.

  5. Bio-distribution and toxicity assessment of intravenously injected anti-HER2 antibody conjugated CdSe/ZnS quantum dots in Wistar rats

    Directory of Open Access Journals (Sweden)

    Dhermendra K Tiwari

    2011-02-01

    Full Text Available Dhermendra K Tiwari1, Takashi Jin2, Jitendra Behari11School of Environmental Sciences, Jawaharlal Nehru University, New Delhi, India; 2WPI-Immunology Frontier Research Center, Osaka University, Osaka, JapanAbstract: Anti-HER2 antibody conjugated with quantum dots (anti-HER2ab-QDs is a very recent fluorescent nanoprobe for HER2+ve breast cancer imaging. In this study we investigated in-vivo toxicity of anti-HER2ab conjugated CdSe/ZnS QDs in Wistar rats. For toxicity evaluation of injected QDs sample, body weight, organ coefficient, complete blood count (CBC, biochemistry panel assay (AST, ALT, ALP, and GGTP, comet assay, reactive oxygen species, histology, and apoptosis were determined. Wistar rat (8–10 weeks old were randomly divided into 4 treatment groups (n = 6. CBC and biochemistry panel assay showed nonsignificant changes in the anti-HER2ab-QDs treated group but these changes were significant (P < 0.05 in QDs treated group. No tissue damage, inflammation, lesions, and QDs deposition were found in histology and TEM images of the anti-HER2ab-QDs treated group. Apoptosis in liver and kidney was not found in the anti-HER2ab-QDs treated group. Animals treated with nonconjugated QDs showed comet formation and apoptosis. Cadmium deposition was confirmed in the QDs treated group compared with the anti-HER2ab-QDs treated group. The QDs concentration (500 nM used for this study is suitable for in-vivo imaging. The combine data of this study support the biocompatibility of anti-HER2ab-QDs for breast cancer imaging, suggesting that the antibody coating assists in controlling any possible adverse effect of quantum dots.Keywords: cancer bioimaging, HER2, anti-HER2 antibody, quantum dots, comet assay

  6. Preparation of gold nanoparticles by microwave heating and application of spectroscopy to study conjugate of gold nanoparticles with antibody E. coli O157:H7

    Science.gov (United States)

    Thanh Ngo, Vo Ke; Phuong Uyen Nguyen, Hoang; Phat Huynh, Trong; Nguyen Pham Tran, Nguyen; Lam, Quang Vinh; Dat Huynh, Thanh

    2015-09-01

    Gold nanoparticles (AuNPs) of 15-20 nm size range have attracted attention for producing smart sensing devices as diagnostic tools in biomedical sciences. Citrate capped AuNPs are negatively charged, which can be exploited for electrostatic interactions with some positively charged biomolecules like antibodies. In this paper we describe a method for the low cost synthesis of gold nanoparticles using sodium citrate (Na3Ct) reduction in chloroauric acid (HAuCl4.3H2O) by microwave heating (diameter about 13-15 nm). Gold nanoparticles were functionalized with surface activation by 3-mercaptopropionic acid for attaching antibody. These nanoparticles were then reacted with anti-E. coli O157:H7, using N-hydroxy succinimide (NHS) and carbondimide hydrochloride (EDC) coupling chemistry. The product was characterized with UV-visible spectroscopy, Fourier transform infrared (FTIR) spectroscopy and zeta potential. In addition, the binding of antibody-gold nanoparticles conjugates to E. coli O157:H7 was demonstrated using transmission electron microscopy (TEM).

  7. Social Mixing with Other Children during Infancy Enhances Antibody Response to a Pneumococcal Conjugate Vaccine in Early Childhood▿

    OpenAIRE

    Salt, Penny; Banner, Carly; Oh, Sarah; Yu, Ly-Mee; Lewis, Susan; Pan, DingXin; Griffiths, David; Ferry, Berne; Pollard, Andrew

    2007-01-01

    Children who have siblings and/or who attend day care have higher rates of nasopharyngeal colonization with pneumococci than lone children do. Pneumococcal colonization is usually asymptomatic but is a prerequisite for invasive disease. We studied the effect of social mixing with other children on immunity to a pneumococcal vaccine. One hundred sixty children aged 1 year were immunized with a 7-valent conjugate pneumococcal vaccine. A blood sample was obtained before and 9 to 11 days after th...

  8. Enhanced delivery of etoposide across the blood-brain barrier to restrain brain tumor growth using melanotransferrin antibody- and tamoxifen-conjugated solid lipid nanoparticles.

    Science.gov (United States)

    Kuo, Yung-Chih; Wang, I-Hsin

    2016-08-01

    Melanotransferrin antibody (MA) and tamoxifen (TX) were conjugated on etoposide (ETP)-entrapped solid lipid nanoparticles (ETP-SLNs) to target the blood-brain barrier (BBB) and glioblastom multiforme (GBM). MA- and TX-conjugated ETP-SLNs (MA-TX-ETP-SLNs) were used to infiltrate the BBB comprising a monolayer of human astrocyte-regulated human brain-microvascular endothelial cells (HBMECs) and to restrain the proliferation of malignant U87MG cells. TX-grafted ETP-SLNs (TX-ETP-SLNs) significantly enhanced the BBB permeability coefficient for ETP and raised the fluorescent intensity of calcein-AM when compared with ETP-SLNs. In addition, surface MA could increase the BBB permeability coefficient for ETP about twofold. The viability of HBMECs was higher than 86%, suggesting a high biocompatibility of MA-TX-ETP-SLNs. Moreover, the efficiency in antiproliferation against U87MG cells was in the order of MA-TX-ETP-SLNs  >  TX-ETP-SLNs  >  ETP-SLNs  >  SLNs. The capability of MA-TX-ETP-SLNs to target HBMECs and U87MG cells during internalization was verified by immunochemical staining of expressed melanotransferrin. MA-TX-ETP-SLNs can be a potent pharmacotherapy to deliver ETP across the BBB to GBM. PMID:26768307

  9. Salivary antibody levels in adolescents in response to a meningococcal serogroup C conjugate booster vaccination nine years after priming : systemically induced local immunity and saliva as potential surveillance tool

    NARCIS (Netherlands)

    Stoof, Susanne P; van der Klis, Fiona R M; van Rooijen, Debbie M; Bogaert, Debby; Trzcinski, Krzysztof; Sanders, Elisabeth A M; Berbers, Guy A M

    2015-01-01

    BACKGROUND: In several countries large-scale immunization of children and young adults with Meningococcal serogroup C (MenC) conjugate vaccines has induced long-standing herd protection. Salivary antibodies may play an important role in mucosal protection against meningococcal acquisition and carria

  10. Bispecific antibody complex pre-targeting and targeted delivery of polymer drug conjugates for imaging and therapy in dual human mammary cancer xenografts. Targeted polymer drug conjugates for cancer diagnosis and therapy

    Energy Technology Data Exchange (ETDEWEB)

    Khaw, Ban-An; Gada, Keyur S.; Patil, Vishwesh; Panwar, Rajiv; Mandapati, Savitri [Northeastern University, Department of Pharmaceutical Sciences, Bouve College of Health Sciences, School of Pharmacy, Boston, MA (United States); Hatefi, Arash [Rutgers University, Department of Pharmaceutics, New Brunswick, NJ (United States); Majewski, Stan [West Virginia University, Department of Radiology, Morgantown, WV (United States); Weisenberger, Andrew [Thomas Jefferson National Accelerator Facility, Jefferson Lab, Newport News, VA (United States)

    2014-08-15

    Doxorubicin, a frontline chemotherapeutic agent, limited by its cardiotoxicity and other tissue toxicities, was conjugated to N-terminal DTPA-modified polyglutamic acid (D-Dox-PGA) to produce polymer pro-drug conjugates. D-Dox-PGA or Tc-99 m labeled DTPA-succinyl-polylysine polymers (DSPL) were targeted to HER2-positive human mammary carcinoma (BT-474) in a double xenografted SCID mouse model also hosting HER2-negative human mammary carcinoma (BT-20). After pretargeting with bispecific anti-HER2-affibody-anti-DTPA-Fab complexes (BAAC), anti-DTPA-Fab or only phosphate buffered saline, D-Dox-PGA or Tc-99 m DSPL were administered. Positive therapeutic control mice were injected with Dox alone at maximum tolerated dose (MTD). Only BT-474 lesions were visualized by gamma imaging with Tc-99 m-DSPL; BT-20 lesions were not. Therapeutic efficacy was equivalent in mice pretargeted with BAAC/targeted with D-Dox-PGA to mice treated only with doxorubicin. There was no total body weight (TBW) loss at three times the doxorubicin equivalent MTD with D-Dox-PGA, whereas mice treated with doxorubicin lost 10 % of TBW at 2 weeks and 16 % after the second MTD injection leading to death of all mice. Our cancer imaging and pretargeted therapeutic approaches are highly target specific, delivering very high specific activity reagents that may result in the development of a novel theranostic application. HER/2 neu specific affibody-anti-DTPA-Fab bispecific antibody pretargeting of HER2 positive human mammary xenografts enabled exquisite targeting of polymers loaded with radioisotopes for molecular imaging and doxorubicin for effective therapy without the associating non-tumor normal tissue toxicities. (orig.)

  11. Poly(o-phenylenediamine) nanosphere-conjugated capture antibody immobilized on a glassy carbon electrode for electrochemical immunoassay of carcinoembryonic antigen

    International Nuclear Information System (INIS)

    We report on a new electrochemical immunosensor for the carcinoembryonic antigen (CEA; a model analyte). First, poly(o-phenylenediamine) nanospheres (PPDNSs) were synthesized by using a wet-chemistry method. The nanospheres were utilized as the support for immobilizing horseradish peroxidase-labeled polyclonal rabbit anti-human CEA antibody (HRP-anti-CEA) on a pretreated glassy carbon electrode (GCE) using glutaraldehyde as a crosslinker. In the presence of target CEA, an antigen-antibody immunocomplex formed on the electrode. This results in a partial inhibition of the active center of HRP and decreases the activity of HRP in terms of H2O2 reduction. The performance and factors influencing the performance of the immunoelectrode were studied. Under optimal conditions, the reduction current obtained from the anti-CEA-conjugated HRP (best at a working voltage of −265 mV vs. Ag/AgCl) is proportional to the CEA concentration in the 0.01 to 60 ng mL−1 range, with a detection limit of 3.2 pg mL−1. Non-specific adsorption was not observed. Relative standard deviations for intra-assay and inter-assay are <8.3 % and <9.7 %, respectively. The method was applied to the analysis of nine human serum samples, and a good relationship was found between the electrochemical immunoassay and the commercialized ELISA kit for human CEA. (author)

  12. Pneumococcal antibody concentrations of subjects in communities fully or partially vaccinated with a seven-valent pneumococcal conjugate vaccine.

    Directory of Open Access Journals (Sweden)

    Martin O C Ota

    Full Text Available BACKGROUND: A recent trial with PCV-7 in a rural Gambian community showed reduced vaccine-type pneumococcal carriage in fully vaccinated compared with control communities. We measured pneumococcal polysaccharide antibody concentrations in this trial to understand further the mechanisms underlying the observed changes. METHODS: A single-blind, cluster-randomized (by village trial was conducted in 21 Gambian villages. In 11 villages, all residents received PCV-7 (Vaccine group; in 10 control villages only children 5.0 µg/mL for all but serotype 9V of the PCV-7 serotypes in the older group, but not in the younger age group. CONCLUSION: Higher antibodies in vaccinated communities provide an explanation for the lower pneumococcal carriage rates in fully vaccinated compared to control communities. TRIAL REGISTRATION: Controlled-Trials.com ISRCTN51695599 51695599.

  13. Pneumococcal antibody concentrations of subjects in communities fully or partially vaccinated with a seven-valent pneumococcal conjugate vaccine.

    OpenAIRE

    Ota, Martin O. C.; Anna Roca; Christian Bottomley; Philip C. Hill; Uzochukwu Egere; Brian Greenwood; Adegbola, Richard A.

    2012-01-01

    BACKGROUND A recent trial with PCV-7 in a rural Gambian community showed reduced vaccine-type pneumococcal carriage in fully vaccinated compared with control communities. We measured pneumococcal polysaccharide antibody concentrations in this trial to understand further the mechanisms underlying the observed changes. METHODS A single-blind, cluster-randomized (by village) trial was conducted in 21 Gambian villages. In 11 villages, all residents received PCV-7 (Vaccine group); in 10 contr...

  14. Optical Imaging of Disseminated Leukemia Models in Mice with Near-Infrared Probe Conjugated to a Monoclonal Antibody

    OpenAIRE

    Sabrina Pesnel; Arnaud Pillon; Laurent Créancier; Stéphanie Lerondel; Alain Le Pape; Christian Recher; Cécile Demur; Nicolas Guilbaud; Anna Kruczynski

    2012-01-01

    BACKGROUND: The assessment of anticancer agents to treat leukemia needs to have animal models closer to the human pathology such as implantation in immunodeficient mice of leukemic cells from patient samples. A sensitive and early detection of tumor cells in these orthotopic models is a prerequisite for monitoring engraftment of leukemic cells and their dissemination in mice. Therefore, we developed a fluorescent antibody based strategy to detect leukemic foci in mice bearing patient-derived ...

  15. Platinum(II) as bifunctional linker in antibody-drug conjugate formation: coupling of a 4-nitrobenzo-2-oxa-1,3-diazole fluorophore to trastuzumab as a model.

    Science.gov (United States)

    Waalboer, Dennis C J; Muns, Joey A; Sijbrandi, Niels J; Schasfoort, Richard B M; Haselberg, Rob; Somsen, Govert W; Houthoff, Hendrik-Jan; van Dongen, Guus A M S

    2015-05-01

    The potential of platinum(II) as a bifunctional linker in the coordination of small molecules, such as imaging agents or (cytotoxic) drugs, to monoclonal antibodies (mAbs) was investigated with a 4-nitrobenzo-2-oxa-1,3-diazole (NBD) fluorophore and trastuzumab (Herceptin™) as a model antibody. The effect of ligand and reaction conditions on conjugation efficiency was explored for [Pt(en)(L-NBD)Cl](NO3 ) (en=ethylenediamine), with L=N-heteroaromatic, N-alkyl amine, or thioether. Conjugation proceeded most efficiently at pH 8.0 in the presence of NaClO4 or Na2 SO4 in tricine or HEPES buffer. Reaction of N-coordinated complexes (20 equiv) with trastuzumab at 37 °C for 2 h, followed by removal of weakly bound complexes with excess thiourea, afforded conjugates with an NBD/mAb ratio of 1.5-2.9 that were stable in phosphate-buffered saline at room temperature for at least 48 h. In contrast, thioether-coordinated complexes afforded unstable conjugates. Finally, surface plasmon resonance analysis showed no loss in binding affinity of trastuzumab after conjugation. PMID:25809281

  16. New Insights in Tissue Distribution, Metabolism, and Excretion of [3H]-Labeled Antibody Maytansinoid Conjugates in Female Tumor-Bearing Nude Rats.

    Science.gov (United States)

    Walles, Markus; Rudolph, Bettina; Wolf, Thierry; Bourgailh, Julien; Suetterlin, Martina; Moenius, Thomas; Peraus, Gisela; Heudi, Olivier; Elbast, Walid; Lanshoeft, Christian; Bilic, Sanela

    2016-07-01

    For antibody drug conjugates (ADCs), the fate of the cytotoxic payload in vivo needs to be well understood to mitigate toxicity risks and properly design the first in-patient studies. Therefore, a distribution, metabolism, and excretion (DME) study with a radiolabeled rat cross-reactive ADC ([(3)H]DM1-LNL897) targeting the P-cadherin receptor was conducted in female tumor-bearing nude rats. Although multiple components [total radioactivity, conjugated ADC, total ADC, emtansine (DM1) payload, and catabolites] needed to be monitored with different technologies (liquid scintillation counting, liquid chromatography/mass spectrometry, enzyme-linked immunosorbent assay, and size exclusion chromatography), the pharmacokinetic data were nearly superimposable with the various techniques. [(3)H]DM1-LNL897 was cleared with half-lives of 51-62 hours and LNL897-related radioactivity showed a minor extent of tissue distribution. The highest tissue concentrations of [(3)H]DM1-LNL897-related radioactivity were measured in tumor. Complimentary liquid extraction surface analysis coupled to micro-liquid chromatography-tandem mass spectrometry data proved that the lysine (LYS)-4(maleimidylmethyl) cyclohexane-1-carboxylate-DM1 (LYS-MCC-DM1) catabolite was the only detectable component distributed evenly in the tumor and liver tissue. The mass balance was complete with up to 13.8% ± 0.482% of the administered radioactivity remaining in carcass 168 hours postdose. LNL897-derived radioactivity was mainly excreted via feces (84.5% ± 3.12%) and through urine only to a minor extent (4.15% ± 0.462%). In serum, the major part of radioactivity could be attributed to ADC, while small molecule disposition products were the predominant species in excreta. We show that there is a difference in metabolite profiles depending on which derivatization methods for DM1 were applied. Besides previously published results on LYS-MCC-DM1 and MCC-DM1, maysine and a cysteine conjugate of DM1 could be

  17. ChAcNLS, a Novel Modification to Antibody-Conjugates Permitting Target Cell-Specific Endosomal Escape, Localization to the Nucleus, and Enhanced Total Intracellular Accumulation.

    Science.gov (United States)

    Beaudoin, Simon; Rondeau, Andreanne; Martel, Olivier; Bonin, Marc-Andre; van Lier, Johan E; Leyton, Jeffrey V

    2016-06-01

    The design of antibody-conjugates (ACs) for delivering molecules for targeted applications in humans has sufficiently progressed to demonstrate clinical efficacy in certain malignancies and reduced systemic toxicity that occurs with standard nontargeted therapies. One area that can advance clinical success for ACs will be to increase their intracellular accumulation. However, entrapment and degradation in the endosomal-lysosomal pathway, on which ACs are reliant for the depositing of their molecular payload inside target cells, leads to reduced intracellular accumulation. Innovative approaches that can manipulate this pathway may provide a strategy for increasing accumulation. We hypothesized that escape from entrapment inside the endosomal-lysosomal pathway and redirected trafficking to the nucleus could be an effective approach to increase intracellular AC accumulation in target cells. Cholic acid (ChAc) was coupled to the peptide CGYGPKKKRKVGG containing the nuclear localization sequence (NLS) from SV-40 large T-antigen, which is termed ChAcNLS. ChAcNLS was conjugated to the mAb 7G3 (7G3-ChAcNLS), which has nanomolar affinity for the cell-surface leukemic antigen interleukin-3 receptor-α (IL-3Rα). Our aim was to determine whether 7G3-ChAcNLS increased intracellular accumulation while retaining nanomolar affinity and IL-3Rα-positive cell selectivity. Competition ELISA and cell treatment assays were performed. Cell fractionation, confocal microscopy, flow cytometry, and Western blot techniques were used to determine the level of antibody accumulation inside cells and in corresponding nuclei. In addition, the radioisotope copper-64 ((64)Cu) was also utilized as a surrogate molecular cargo to evaluate nuclear and intracellular accumulation by radioactivity counting. 7G3-ChAcNLS effectively escaped endosome entrapment and degradation resulting in a unique intracellular distribution pattern. mAb modification with ChAcNLS maintained 7G3 nM affinity and produced high

  18. Optical imaging of disseminated leukemia models in mice with near-infrared probe conjugated to a monoclonal antibody.

    Directory of Open Access Journals (Sweden)

    Sabrina Pesnel

    Full Text Available BACKGROUND: The assessment of anticancer agents to treat leukemia needs to have animal models closer to the human pathology such as implantation in immunodeficient mice of leukemic cells from patient samples. A sensitive and early detection of tumor cells in these orthotopic models is a prerequisite for monitoring engraftment of leukemic cells and their dissemination in mice. Therefore, we developed a fluorescent antibody based strategy to detect leukemic foci in mice bearing patient-derived leukemic cells using fluorescence reflectance imaging (FRI to determine when to start treatments with novel antitumor agents. METHODS: Two mAbs against the CD44 human myeloid marker or the CD45 human leukocyte marker were labeled with Alexa Fluor 750 and administered to leukemia-bearing mice after having verified the immunoreactivity in vitro. Bioluminescent leukemic cells (HL60-Luc were used to compare the colocalization of the fluorescent mAb with these cells. The impact of the labeled antibodies on disease progression was further determined. Finally, the fluorescent hCD45 mAb was tested in mice engrafted with human leukemic cells. RESULTS: The probe labeling did not modify the immunoreactivity of the mAbs. There was a satisfactory correlation between bioluminescence imaging (BLI and FRI and low doses of mAb were sufficient to detect leukemic foci. However, anti-hCD44 mAb had a strong impact on the tumor proliferation contrary to anti-hCD45 mAb. The use of anti-hCD45 mAb allowed the detection of leukemic patient cells engrafted onto NOD/SCID mice. CONCLUSIONS: A mAb labeled with a near infrared fluorochrome is useful to detect leukemic foci in disseminated models provided that its potential impact on tumor proliferation has been thoroughly documented.

  19. Optical Imaging of Disseminated Leukemia Models in Mice with Near-Infrared Probe Conjugated to a Monoclonal Antibody

    Science.gov (United States)

    Pesnel, Sabrina; Pillon, Arnaud; Créancier, Laurent; Lerondel, Stéphanie; Le Pape, Alain; Recher, Christian; Demur, Cécile; Guilbaud, Nicolas; Kruczynski, Anna

    2012-01-01

    Background The assessment of anticancer agents to treat leukemia needs to have animal models closer to the human pathology such as implantation in immunodeficient mice of leukemic cells from patient samples. A sensitive and early detection of tumor cells in these orthotopic models is a prerequisite for monitoring engraftment of leukemic cells and their dissemination in mice. Therefore, we developed a fluorescent antibody based strategy to detect leukemic foci in mice bearing patient-derived leukemic cells using fluorescence reflectance imaging (FRI) to determine when to start treatments with novel antitumor agents. Methods Two mAbs against the CD44 human myeloid marker or the CD45 human leukocyte marker were labeled with Alexa Fluor 750 and administered to leukemia-bearing mice after having verified the immunoreactivity in vitro. Bioluminescent leukemic cells (HL60-Luc) were used to compare the colocalization of the fluorescent mAb with these cells. The impact of the labeled antibodies on disease progression was further determined. Finally, the fluorescent hCD45 mAb was tested in mice engrafted with human leukemic cells. Results The probe labeling did not modify the immunoreactivity of the mAbs. There was a satisfactory correlation between bioluminescence imaging (BLI) and FRI and low doses of mAb were sufficient to detect leukemic foci. However, anti-hCD44 mAb had a strong impact on the tumor proliferation contrary to anti-hCD45 mAb. The use of anti-hCD45 mAb allowed the detection of leukemic patient cells engrafted onto NOD/SCID mice. Conclusions A mAb labeled with a near infrared fluorochrome is useful to detect leukemic foci in disseminated models provided that its potential impact on tumor proliferation has been thoroughly documented. PMID:22303450

  20. Interdigitated array microelectrode based impedance biosensor coupled with magnetic nanoparticle-antibody conjugates for detection of Escherichia coli O157:H7 in food samples.

    Science.gov (United States)

    Varshney, Madhukar; Li, Yanbin

    2007-05-15

    An impedance biosensor based on interdigitated array microelectrode (IDAM) coupled with magnetic nanoparticle-antibody conjugates (MNAC) was developed and evaluated for rapid and specific detection of E. coli O157:H7 in ground beef samples. MNAC were prepared by immobilizing biotin-labeled polyclonal goat anti-E. coli antibodies onto streptavidin-coated magnetic nanoparticles, which were used to separate and concentrate E. coli O157:H7 from ground beef samples. Magnitude of impedance and phase angle were measured in a frequency range of 10 Hz to 1 MHz in the presence of 0.1M mannitol solution. The lowest detection limits of this biosensor for detection of E. coli O157:H7 in pure culture and ground beef samples were 7.4 x 10(4) and 8.0 x 10(5)CFU ml(-1), respectively. The regression equation for the normalized impedance change (NIC) versus E. coli O157:H7 concentration (N) in ground beef samples was NIC=15.55 N-71.04 with R(2)=0.95. Sensitivity of the impedance biosensor was improved by 35% by concentrating bacterial cells attached to MNAC in the active layer of IDAM above the surface of electrodes with the help of a magnetic field. Based on equivalent circuit analysis, it was observed that bulk resistance and double layer capacitance were responsible for the impedance change caused by the presence of E. coli O157:H7 on the surface of IDAM. Surface immobilization techniques, redox probes, or sample incubation were not used in this impedance biosensor. The total detection time from sampling to measurement was 35 min. PMID:17045791

  1. Optimization of multi-epitopic HIV-1 recombinant protein expression in prokaryote system and conjugation to mouse DEC-205 monoclonal antibody: implication for in-vivo targeted delivery of dendritic cells

    Directory of Open Access Journals (Sweden)

    Roghayeh Rahimi

    2015-02-01

    Full Text Available Objective(s:Multi-epitopic protein vaccines and direction of vaccine delivery to dendritic cells (DCs are promising approaches for enhancing immune responses against mutable pathogens. Escherichia coli is current host for expression of recombinant proteins, and it is important to optimize expression condition. The aim of this study was the optimization of multi-epitopic HIV-1 tat/pol/gag/env recombinant protein (HIVtop4 expression by E. coli and conjugation of purified protein to anti DEC-205 monoclonal antibody as candidate vaccine. Materials and Methods: In this study, expression was induced in BL21 (DE3 E. coli cells by optimization of induction condition, post induction incubation time, temperature and culture            medium formula. Some culture mediums were used for cell culture, and isopropyl-beta-D-thiogalactopyranoside was used for induction of expression. Protein was purified by Ni-NTA column chromatography and confirmed against anti-His antibody in western-blotting. To exploit DCs properties for immunization purposes, recombinant protein chemically coupled to αDEC-205 monoclonal antibody and confirmed against anti-His antibody in western-blotting. Results: The optimum condition for expression was 1 mM IPTG during 4 hr cultures in 2XYT medium, and final protein produced in soluble form. Conjugation of purified protein to αDEC-205 antibody resulted in smears of protein: antibodies conjugate in different molecular weights. [AGA1] . Conclusion: The best cultivation condition for production of HIVtop4 protein is induction by 1 mM IPTG during 4 hr in 2XYT medium. [AGA2] The final concentration of purified protein was 500 µg/ml.

  2. In vitro perforation of human epithelial carcinoma cell with antibody-conjugated biodegradable microspheres illuminated by a single 80 femtosecond near-infrared laser pulse

    Directory of Open Access Journals (Sweden)

    Terakawa M

    2012-05-01

    Full Text Available Mitsuhiro Terakawa, Yasuyuki Tsunoi, Tatsuki MitsuhashiDepartment of Electronics and Electrical Engineering, Keio University, Yokohama, Kanagawa, JapanAbstract: Pulsed laser interaction with small metallic and dielectric particles has been receiving attention as a method of drug delivery to many cells. However, most of the particles are attended by many risks, which are mainly dependent upon particle size. Unlike other widely used particles, biodegradable particles have advantages of being broken down and eliminated by innate metabolic processes. In this paper, the perforation of cell membrane by a focused spot with transparent biodegradable microspheres excited by a single 800 nm, 80 fs laser pulse is demonstrated. A polylactic acid (PLA sphere, a biodegradable polymer, was used. Fluorescein isothiocyanate (FITC-dextran and short interfering RNA were delivered into many human epithelial carcinoma cells (A431 cells by applying a single 80 fs laser pulse in the presence of antibody-conjugated PLA microspheres. The focused intensity was also simulated by the three-dimensional finite-difference time-domain method. Perforation by biodegradable spheres compared with other particles has the potential to be a much safer phototherapy and drug delivery method for patients. The present method can open a new avenue, which is considered an efficient adherent for the selective perforation of cells which express the specific antigen on the cell membrane.Keywords: femtosecond laser, drug delivery, transfection, biodegradable polymer

  3. CD44v6 Monoclonal Antibody-Conjugated Gold Nanostars for Targeted Photoacoustic Imaging and Plasmonic Photothermal Therapy of Gastric Cancer Stem-like Cells

    Science.gov (United States)

    Liang, Shujing; Li, Chao; Zhang, Chunlei; Chen, Yunsheng; Xu, Liang; Bao, Chenchen; Wang, Xiaoyong; liu, Gang; zhang, Fengchun; Cui, Daxiang

    2015-01-01

    Developing safe and effective nanoprobes for targeted imaging and selective therapy of gastric cancer stem cells (GCSCs) has become one of the most promising anticancer strategies. Herein, gold nanostars-based PEGylated multifunctional nanoprobes were prepared with conjugated CD44v6 monoclonal antibodies (CD44v6-GNS) as the targeting ligands. It was observed that the prepared nanoprobes had high affinity towards GCSC spheroid colonies and destroyed them completely with a low power density upon near-infrared (NIR) laser treatment (790 nm, 1.5 W/cm2, 5 min) in vitro experiment. Orthotopic and subcutaneous xenografted nude mice models of human gastric cancer were established. Subsequently, biodistribution and photothermal therapeutic effects after being intravenously injected with the prepared nanoprobes were assessed. Photoacoustic imaging revealed that CD44v6-GNS nanoprobes could target the gastric cancer vascular system actively at 4 h post-injection, while the probes inhibited tumor growth remarkably upon NIR laser irradiation, and even extended survivability of the gastric cancer-bearing mice. The CD44v6-GNS nanoprobes exhibited great potential for applications of gastric cancer targeted imaging and photothermal therapy in the near future. PMID:26155313

  4. The biology of MDR1-P-glycoprotein (MDR1-Pgp in designing functional antibody drug conjugates (ADCs: the experience of gemtuzumab ozogamicin

    Directory of Open Access Journals (Sweden)

    Maurizio Cianfriglia

    2013-06-01

    Full Text Available BACKGROUND: The treatment of cancer remains a formidable challenge owing to the difficulties in differentiating tumor cells from healthy cells to ameliorate the disease without causing intolerable toxicity to patients. In addition, the emergence of MDR1-Pgp mediated multi-drug resistance (MDR it is a biological phenomenon that inhibits the curative potential of chemotherapeutic treatments. One way to improve the selectivity of therapeutic molecules in tumors would be to target them on the tumor site, thereby sparing normal tissues. AIMS: In this overview, we will discuss the biological factors influencing the safety and efficacy of the humanized mAb hP67.6 linked to the potent cytotoxic drug calicheamicin-gamma1 (gemtuzumab ozogamicin that target CD33 cell surface antigen expressed on AML cells. In addition, we highlight key aspects of MDR1-Pgp biology as a platform to understand its functional role in gemtuzumab ozogamicin immunotherapy which is tightly linked to an accurate assessment of the MDR status of AML cells. DISCUSSION: Several factors may affect the efficacy and safety of immunoconjugates. These include the common issues of chemical and antibody therapeutics such as specificity, heterogeneous target antigen expression and the complex pharmacokinetics profile of conveyed antibody. Further, the delivered drug may not be sufficient for providing therapeutic benefit, since the curative cytotoxic compound may be affected by intrinsic or acquired resistance of target cells. These and other potential problems, as well as the possible ways to overcome them will be discussed in this review by examining the biological factors involved in safety and efficacy of the first in class antibody drug conjugate (ADC gentuzumab ozogamicin. Despite this set-back, the extensive recorded data and the lessons learned from gentuzumab ozogamicin recently withdrawn from the market for safety concerns helped to pave the way for next generations of clinically

  5. Polyethylene glycol (PEG) linked to near infrared (NIR) dyes conjugated to chimeric anti-carcinoembryonic antigen (CEA) antibody enhances imaging of liver metastases in a nude-mouse model of human colon cancer.

    Science.gov (United States)

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

    2014-01-01

    We report here that polyethylene glycol (PEG) linked to near infrared dyes conjugated to chimeric mouse-human anti-carcinoembryonic antigen (CEA) antibody greatly improves imaging of liver metastases in a nude mouse model of colon-cancer experimental metastases. PEGylated and non-PEGylated DyLight 650 and 750 dyes were conjugated to the chimeric anti-CEA antibody. The dyes were initially injected intravenously into nude mice without tumors. Tissue biodistribution was determined by tissue sonication and analyzing tissue dye concentration profiles over time. PEGylated dyes had significantly lower accumulation in the liver (p = 0.03 for the 650 dyes; p = 0.002 for the 750 dyes) compared to non-PEGylated dyes. In an experimental liver metastasis model of HT-29 colon cancer, PEGylated dyes conjugated to the anti-CEA antibody showed good labeling of metastatic tumors with high contrast between normal and malignant tissue which was not possible with the non-PEGylated dyes since there was so much non-specific accumulation in the liver. PEGylation of the DyLight 650 and 750 NIR dyes significantly altered tissue biodistribution, allowing brighter tissue labeling, decreased accumulation in normal organs, particularly the liver. This enabled high fidelity and high contrast imaging of liver metastases.

  6. Polyethylene glycol (PEG linked to near infrared (NIR dyes conjugated to chimeric anti-carcinoembryonic antigen (CEA antibody enhances imaging of liver metastases in a nude-mouse model of human colon cancer.

    Directory of Open Access Journals (Sweden)

    Ali A Maawy

    Full Text Available We report here that polyethylene glycol (PEG linked to near infrared dyes conjugated to chimeric mouse-human anti-carcinoembryonic antigen (CEA antibody greatly improves imaging of liver metastases in a nude mouse model of colon-cancer experimental metastases. PEGylated and non-PEGylated DyLight 650 and 750 dyes were conjugated to the chimeric anti-CEA antibody. The dyes were initially injected intravenously into nude mice without tumors. Tissue biodistribution was determined by tissue sonication and analyzing tissue dye concentration profiles over time. PEGylated dyes had significantly lower accumulation in the liver (p = 0.03 for the 650 dyes; p = 0.002 for the 750 dyes compared to non-PEGylated dyes. In an experimental liver metastasis model of HT-29 colon cancer, PEGylated dyes conjugated to the anti-CEA antibody showed good labeling of metastatic tumors with high contrast between normal and malignant tissue which was not possible with the non-PEGylated dyes since there was so much non-specific accumulation in the liver. PEGylation of the DyLight 650 and 750 NIR dyes significantly altered tissue biodistribution, allowing brighter tissue labeling, decreased accumulation in normal organs, particularly the liver. This enabled high fidelity and high contrast imaging of liver metastases.

  7. Imaging small human prostate cancer xenografts after pretargeting with bispecific bombesin-antibody complexes and targeting with high specific radioactivity labeled polymer-drug conjugates

    Energy Technology Data Exchange (ETDEWEB)

    Patil, Vishwesh; Gada, Keyur; Panwar, Rajiv; Ferris, Craig; Khaw, Ban-An [Northeastern University, School of Pharmacy, Department of Pharmaceutical Sciences, Boston, MA (United States); Varvarigou, Alexandra [Institute of Radioisotopes and Radiodiagnostics, National Centre for Scientific Research ' ' Demokritos' ' , Athens (Greece); Majewski, Stan [West Virginia University, Department of Radiology, Nuclear Medicine Imaging Instrumentation Program, Center for Advanced Imaging, Morgantown, WV (United States); Weisenberger, Andrew [Jefferson LA, Thomas Jefferson National Accelerator Facility, Newport News, VA (United States); Tekabe, Yared [Columbia University Medical Center, New York, NY (United States)

    2012-05-15

    Pretargeting with bispecific monoclonal antibodies (bsMAb) for tumor imaging was developed to enhance target to background activity ratios. Visualization of tumors was achieved by the delivery of mono- and divalent radiolabeled haptens. To improve the ability to image tumors with bsMAb, we have combined the pretargeting approach with targeting of high specific activity radiotracer labeled negatively charged polymers. The tumor antigen-specific antibody was replaced with bombesin (Bom), a ligand that binds specifically to the growth receptors that are overexpressed by many tumors including prostate cancer. Bom-anti-diethylenetriaminepentaacetic acid (DTPA) bispecific antibody complexes were used to demonstrate pretargeting and imaging of very small human prostate cancer xenografts targeted with high specific activity {sup 111}In- or {sup 99m}Tc-labeled negatively charged polymers. Bispecific antibody complexes consisting of intact anti-DTPA antibody or Fab' linked to Bom via thioether bonds (Bom-bsCx or Bom-bsFCx, respectively) were used to pretarget PC-3 human prostate cancer xenografts in SCID mice. Negative control mice were pretargeted with Bom or anti-DTPA Ab. {sup 111}In-Labeled DTPA-succinyl polylysine (DSPL) was injected intravenously at 24 h (7.03 {+-} 1.74 or 6.88 {+-} 1.89 MBq {sup 111}In-DSPL) after Bom-bsCx or 50 {+-} 5.34 MBq of {sup 99m}Tc-DSPL after Bom-bsFCx pretargeting, respectively. Planar or single photon emission computed tomography (SPECT)/CT gamma images were obtained for up to 3 h and only planar images at 24 h. After imaging, all mice were killed and biodistribution of {sup 111}In or {sup 99m}Tc activities were determined by scintillation counting. Both planar and SPECT/CT imaging enabled detection of PC-3 prostate cancer lesions less than 1-2 mm in diameter in 1-3 h post {sup 111}In-DSPL injection. No lesions were visualized in Bom or anti-DTPA Ab pretargeted controls. {sup 111}In-DSPL activity in Bom-bsCx pretargeted tumors (1

  8. Antibody-drug conjugates and their application in the treatment of hematological malignancies%抗体药物偶联物及其在恶性血液系统肿瘤治疗中的应用

    Institute of Scientific and Technical Information of China (English)

    林莉; 丁倩; 汤沁; 张珍珍; 代争; 詹金彪

    2012-01-01

    单克隆抗体靶向治疗是目前临床肿瘤治疗的热点.针对抗体分子大而组织穿透性差以及临床使用剂量大、生产成本高的问题,抗体的小型化和高效性设计已成为抗体药物研发的新趋势.近年来,单抗与细胞毒性药物的结合物被称为抗体药物偶联物(antibody-drug conjugates,ADCs),已加入到抗癌药物的行列中,成为新型的抗体药物而受到广泛关注.泛义的ADC通常由抗体、接头(linker)和效应分子等3部分组成.根据效应分子的不同,可将ADC分为化学免疫偶联物、免疫毒素、放射性免疫偶联物等3类.ADC被内化进入细胞后,通过细胞内的化学和酶解作用释放出细胞毒性物质,细胞毒性物质则通过抑制蛋白合成、解聚微管蛋白或断裂双链DNA等作用而对靶细胞产生杀伤作用.近年来,FDA已经批准2种ADC药物上市,有多种处于Ⅱ~Ⅲ期临床试验阶段,取得了显著的临床效果,吸引了越来越多的制药企业争相竞逐.本文介绍ADC的过去和现状,结合临床肿瘤应用中的实际问题,探讨其将来的发展趋势.%Monoclonal antibody-targeted therapy has been a hot spot in current clinical cancer treatment. As current antibody drugs have large molecule sizes leading to poor tissue penetration, and high dosage in clinical application leading to high cost, to overcome the problems, the development of new antibody drugs with miniaturization and high potency has become a new trend. In recent years, the conjugates of monoclonal antibodies and cytotoxins, called antibody-drug conjugates (ADCs), have entered the arsenal of anti-cancer drugs, becoming a new format of antibody drugs and attracting extensive attentions. The ADC molecule usually consists of antibody, linker and effector molecule. According to different effector molecules, ADCs can be divided into three categories as chemo-conjugates, immunotoxins and radio-conjugates. When ADC molecules are internalized into cancer

  9. Anti-EphA10 antibody-conjugated pH-sensitive liposomes for specific intracellular delivery of siRNA

    Directory of Open Access Journals (Sweden)

    Zang X

    2016-08-01

    Full Text Available Xinlong Zang,1 Huaiwei Ding,2 Xiufeng Zhao,3 Xiaowei Li,1 Zhouqi Du,1 Haiyang Hu,1 Mingxi Qiao,1 Dawei Chen,1 Yuihui Deng,1 Xiuli Zhao1 1Department of Pharmaceutics, School of Pharmacy, 2Department of Pharmaceutical Chemistry, School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang, People’s Republic of China; 3Hongqi Hospital affiliated to Mudanjiang Medical University, Mudanjiang, People’s Republic of ChinaAbstract: Therapeutic delivery of small interfering RNA (siRNA is a major challenge that limits its potential clinical application. Here, a pH-sensitive cholesterol–Schiff base–polyethylene glycol (Chol–SIB–PEG-modified cationic liposome–siRNA complex, conjugated with the recombinant humanized anti-EphA10 antibody (Eph, was developed as an efficient nonviral siRNA delivery system. Chol–SIB–PEG was successfully synthesized and confirmed with FTIR and 1H-NMR. An Eph–PEG–SIB–Chol-modified liposome–siRNA complex (EPSLR was prepared and characterized by size, zeta potential, gel retardation, and encapsulation efficiency. Electrophoresis results showed that EPSLR was resistant to heparin replacement and protected siRNA from fetal bovine serum digestion. EPSLR exhibited only minor cytotoxicity in MCF-7/ADR cells. The results of flow cytometry and confocal laser scanning microscopy suggested that EPSLR enhanced siRNA transfection in MCF-7/ADR cells. Intracellular distribution experiment revealed that EPSLR could escape from the endo-lysosomal organelle and release siRNA into cytoplasm at 4 hours posttransfection. Western blot experiment demonstrated that EPSLR was able to significantly reduce the levels of MDR1 protein in MCF-7/ADR cells. The in vivo study of DIR-labeled complexes in mice bearing MCF-7/ADR tumor indicated that EPSLR could reach the tumor site rather than other organs more effectively. All these results demonstrate that EPSLR has much potential for effective siRNA delivery and may

  10. In-vivo imaging of oral squamous cell carcinoma by EGFR monoclonal antibody conjugated near-infrared quantum dots in mice

    Directory of Open Access Journals (Sweden)

    Li YD

    2011-08-01

    Full Text Available Kai Yang, Fu-Jun Zhang, Hong Tang, Cheng Zhao, Yu-An Cao, Xiao-Qiang Lv, Dan Chen, Ya-Dong LiDepartment of Oral and Maxillofacial Surgery, The First Affiliated Hospital, Chongqing Medical University, Chongqing, ChinaObjectives: The purpose of this study was to investigate in-vivo visible imaging of oral squamous cell carcinoma (OSCC by targeting epidermal growth factor receptor (EGFR with near-infrared quantum dots.Materials and methods: Quantum dots with an emission wavelength of 800 nm (QD800 were conjugated to monoclonal antibodies against EGFR, resulting in the probe designated as QD800-EGFR Ab. OSCC cell line (BcaCD885 expressing high levels of EGFR was transplanted subcutaneously into nude mice cheeks to develop an OSCC animal model. QD800-EGFR Ab containing 100 pmol equivalent of QD800 was intravenously injected into the animal model, and in-situ and in-vivo imaging of cheek squamous cell carcinoma was analyzed at 10 different time points.Results and conclusion: In-vivo imaging and immunohistochemical examination of the tumors showed that intravenously injected QD800-EGFR Ab probe could bind EGFR expressed on BcaCD885 cells. Fluorescence signals of BcaCD885 cells labeled with QD800-EGFR Ab probe could be clarly detected, and these fluorescence signals lasted for 24 hours. The most complete tumor images with maximal signal-to-noise ratio were observed from 15 minutes to 6 hours after injection of the probe. To the best of the authors' knowledge, this is the first study that has obtained clear in-situ and in-vivo imaging of head and neck cancer by using QD800-EGFR Ab probe. The authors conclude that the combination of near-infrared quantum dots that are highly penetrating for tissues with EGFR monoclonal antibody has promising prospects in in-vivo imaging of OSCC and development of personalized surgical therapies.Keywords: oral cancer, head and neck cancer, near-infrared fluorescence, visual in-vivo imaging, epidermal growth factor receptor

  11. Anti-EphA10 antibody-conjugated pH-sensitive liposomes for specific intracellular delivery of siRNA.

    Science.gov (United States)

    Zang, Xinlong; Ding, Huaiwei; Zhao, Xiufeng; Li, Xiaowei; Du, Zhouqi; Hu, Haiyang; Qiao, Mingxi; Chen, Dawei; Deng, Yuihui; Zhao, Xiuli

    2016-01-01

    Therapeutic delivery of small interfering RNA (siRNA) is a major challenge that limits its potential clinical application. Here, a pH-sensitive cholesterol-Schiff base-polyethylene glycol (Chol-SIB-PEG)-modified cationic liposome-siRNA complex, conjugated with the recombinant humanized anti-EphA10 antibody (Eph), was developed as an efficient nonviral siRNA delivery system. Chol-SIB-PEG was successfully synthesized and confirmed with FTIR and (1)H-NMR. An Eph-PEG-SIB-Chol-modified liposome-siRNA complex (EPSLR) was prepared and characterized by size, zeta potential, gel retardation, and encapsulation efficiency. Electrophoresis results showed that EPSLR was resistant to heparin replacement and protected siRNA from fetal bovine serum digestion. EPSLR exhibited only minor cytotoxicity in MCF-7/ADR cells. The results of flow cytometry and confocal laser scanning microscopy suggested that EPSLR enhanced siRNA transfection in MCF-7/ADR cells. Intracellular distribution experiment revealed that EPSLR could escape from the endo-lysosomal organelle and release siRNA into cytoplasm at 4 hours posttransfection. Western blot experiment demonstrated that EPSLR was able to significantly reduce the levels of MDR1 protein in MCF-7/ADR cells. The in vivo study of DIR-labeled complexes in mice bearing MCF-7/ADR tumor indicated that EPSLR could reach the tumor site rather than other organs more effectively. All these results demonstrate that EPSLR has much potential for effective siRNA delivery and may facilitate its therapeutic application.

  12. Mortalin antibody-conjugated quantum dot transfer from human mesenchymal stromal cells to breast cancer cells requires cell–cell interaction

    International Nuclear Information System (INIS)

    The role of tumor stroma in regulation of breast cancer growth has been widely studied. However, the details on the type of heterocellular cross-talk between stromal and breast cancer cells (BCCs) are still poorly known. In the present study, in order to investigate the intercellular communication between human mesenchymal stromal cells (hMSCs) and breast cancer cells (BCCs, MDA-MB-231), we recruited cell-internalizing quantum dots (i-QD) generated by conjugation of cell-internalizing anti-mortalin antibody and quantum dots (QD). Co-culture of illuminated and color-coded hMSCs (QD655) and BCCs (QD585) revealed the intercellular transfer of QD655 signal from hMSCs to BCCs. The amount of QD double positive BCCs increased gradually within 48 h of co-culture. We found prominent intercellular transfer of QD655 in hanging drop co-culture system and it was non-existent when hMSCs and BBCs cells were co-cultured in trans-well system lacking imminent cell–cell contact. Fluorescent and electron microscope analyses also supported that the direct cell-to-cell interactions may be required for the intercellular transfer of QD655 from hMSCs to BCCs. To the best of our knowledge, the study provides a first demonstration of transcellular crosstalk between stromal cells and BCCs that involve direct contact and may also include a transfer of mortalin, an anti-apoptotic and growth-promoting factor enriched in cancer cells

  13. Anti-EphA10 antibody-conjugated pH-sensitive liposomes for specific intracellular delivery of siRNA

    Science.gov (United States)

    Zang, Xinlong; Ding, Huaiwei; Zhao, Xiufeng; Li, Xiaowei; Du, Zhouqi; Hu, Haiyang; Qiao, Mingxi; Chen, Dawei; Deng, Yuihui; Zhao, Xiuli

    2016-01-01

    Therapeutic delivery of small interfering RNA (siRNA) is a major challenge that limits its potential clinical application. Here, a pH-sensitive cholesterol–Schiff base–polyethylene glycol (Chol–SIB–PEG)-modified cationic liposome–siRNA complex, conjugated with the recombinant humanized anti-EphA10 antibody (Eph), was developed as an efficient nonviral siRNA delivery system. Chol–SIB–PEG was successfully synthesized and confirmed with FTIR and 1H-NMR. An Eph–PEG–SIB–Chol-modified liposome–siRNA complex (EPSLR) was prepared and characterized by size, zeta potential, gel retardation, and encapsulation efficiency. Electrophoresis results showed that EPSLR was resistant to heparin replacement and protected siRNA from fetal bovine serum digestion. EPSLR exhibited only minor cytotoxicity in MCF-7/ADR cells. The results of flow cytometry and confocal laser scanning microscopy suggested that EPSLR enhanced siRNA transfection in MCF-7/ADR cells. Intracellular distribution experiment revealed that EPSLR could escape from the endo-lysosomal organelle and release siRNA into cytoplasm at 4 hours posttransfection. Western blot experiment demonstrated that EPSLR was able to significantly reduce the levels of MDR1 protein in MCF-7/ADR cells. The in vivo study of DIR-labeled complexes in mice bearing MCF-7/ADR tumor indicated that EPSLR could reach the tumor site rather than other organs more effectively. All these results demonstrate that EPSLR has much potential for effective siRNA delivery and may facilitate its therapeutic application. PMID:27574425

  14. Mortalin antibody-conjugated quantum dot transfer from human mesenchymal stromal cells to breast cancer cells requires cell–cell interaction

    Energy Technology Data Exchange (ETDEWEB)

    Pietilä, Mika [National Institute of Advanced industrial Sciences and Technology, Tsukuba, Ibaraki 305 8562 (Japan); Lehenkari, Petri [Institute of Biomedicine, Department of Anatomy and Cell Biology, University of Oulu, Aapistie 7, P.O. Box 5000, FIN-90014 (Finland); Institute of Clinical Medicine, Division of Surgery, University of Oulu and Clinical Research Centre, Department of Surgery and Intensive Care, Oulu University Hospital, Aapistie 5a, P.O. Box 5000, FIN-90014 (Finland); Kuvaja, Paula [Institute of Biomedicine, Department of Anatomy and Cell Biology, University of Oulu, Aapistie 7, P.O. Box 5000, FIN-90014 (Finland); Department of Pathology, Oulu University Hospital, P.O. Box 50, FIN-90029 OYS, Oulu (Finland); Kaakinen, Mika [Biocenter Oulu, University of Oulu, P.O. Box 5000, FI-90014 (Finland); Kaul, Sunil C.; Wadhwa, Renu [National Institute of Advanced industrial Sciences and Technology, Tsukuba, Ibaraki 305 8562 (Japan); Uemura, Toshimasa, E-mail: t.uemura@aist.go.jp [National Institute of Advanced industrial Sciences and Technology, Tsukuba, Ibaraki 305 8562 (Japan)

    2013-11-01

    The role of tumor stroma in regulation of breast cancer growth has been widely studied. However, the details on the type of heterocellular cross-talk between stromal and breast cancer cells (BCCs) are still poorly known. In the present study, in order to investigate the intercellular communication between human mesenchymal stromal cells (hMSCs) and breast cancer cells (BCCs, MDA-MB-231), we recruited cell-internalizing quantum dots (i-QD) generated by conjugation of cell-internalizing anti-mortalin antibody and quantum dots (QD). Co-culture of illuminated and color-coded hMSCs (QD655) and BCCs (QD585) revealed the intercellular transfer of QD655 signal from hMSCs to BCCs. The amount of QD double positive BCCs increased gradually within 48 h of co-culture. We found prominent intercellular transfer of QD655 in hanging drop co-culture system and it was non-existent when hMSCs and BBCs cells were co-cultured in trans-well system lacking imminent cell–cell contact. Fluorescent and electron microscope analyses also supported that the direct cell-to-cell interactions may be required for the intercellular transfer of QD655 from hMSCs to BCCs. To the best of our knowledge, the study provides a first demonstration of transcellular crosstalk between stromal cells and BCCs that involve direct contact and may also include a transfer of mortalin, an anti-apoptotic and growth-promoting factor enriched in cancer cells.

  15. Ionizing radiation improves glioma-specific targeting of superparamagnetic iron oxide nanoparticles conjugated with cmHsp70.1 monoclonal antibodies (SPION-cmHsp70.1)

    Science.gov (United States)

    Shevtsov, Maxim A.; Nikolaev, Boris P.; Ryzhov, Vyacheslav A.; Yakovleva, Ludmila Y.; Marchenko, Yaroslav Y.; Parr, Marina A.; Rolich, Valerij I.; Mikhrina, Anastasiya L.; Dobrodumov, Anatolii V.; Pitkin, Emil; Multhoff, Gabriele

    2015-12-01

    The stress-inducible 72 kDa heat shock protein Hsp70 is known to be expressed on the membrane of highly aggressive tumor cells including high-grade gliomas, but not on the corresponding normal cells. Membrane Hsp70 (mHsp70) is rapidly internalized into tumor cells and thus targeting of mHsp70 might provide a promising strategy for theranostics. Superparamagnetic iron oxide nanoparticles (SPIONs) are contrast negative agents that are used for the detection of tumors with MRI. Herein, we conjugated the Hsp70-specific antibody (cmHsp70.1) which is known to recognize mHsp70 to superparamagnetic iron nanoparticles to assess tumor-specific targeting before and after ionizing irradiation. In vitro experiments demonstrated the selectivity of SPION-cmHsp70.1 conjugates to free and mHsp70 in different tumor cell types (C6 glioblastoma, K562 leukemia, HeLa cervix carcinoma) in a dose-dependent manner. High-resolution MRI (11 T) on T2-weighted images showed the retention of the conjugates in the C6 glioma model. Accumulation of SPION-cmHsp70.1 nanoparticles in the glioma resulted in a nearly 2-fold drop of values in comparison to non-conjugated SPIONs. Biodistribution analysis using NLR-M2 measurements showed a 7-fold increase in the tumor-to-background (normal brain) uptake ratio of SPION-cmHsp70.1 conjugates in glioma-bearing rats in comparison to SPIONs. This accumulation within Hsp70-positive glioma was further enhanced after a single dose (10 Gy) of ionizing radiation. Elevated accumulation of the magnetic conjugates in the tumor due to radiosensitization proves the combination of radiotherapy and application of Hsp70-targeted agents in brain tumors.The stress-inducible 72 kDa heat shock protein Hsp70 is known to be expressed on the membrane of highly aggressive tumor cells including high-grade gliomas, but not on the corresponding normal cells. Membrane Hsp70 (mHsp70) is rapidly internalized into tumor cells and thus targeting of mHsp70 might provide a promising strategy

  16. Cancer Cell Targeting Using Folic Acid/Anti-HER2 Antibody Conjugated Fluorescent CdSe/CdS/ZnS-Mercaptopropionic Acid and CdTe-Mercaptosuccinic Acid Quantum Dots.

    Science.gov (United States)

    Singh, Gurpal; Kumar, Manoj; Soni, Udit; Arora, Vikas; Bansal, Vivek; Gupta, Dikshi; Bhat, Madhusudan; Dinda, Amit K; Sapra, Sameer; Singh, Harpal

    2016-01-01

    CdSe/CdS/ZnS and CdTe quantum dots (QDs) were synthesized by successive ion layer adsorption and reaction (SILAR) technique and direct aqueous synthesis respectively using thiol stabilizers. Synthesized CdSe/CdS/ZnS and CdTe QDs stabilized with 3-mercaptopropionic acid (MPA) and mercaptosuccinic acid (MSA) were used as fluorescent labels after conjugation with folic acid (FA) and anti-HER2 antibodies. Photoluminescence quantum yield of folated CdSe/CdS/ZnS-MPA and CdTe-MSA QDs was 59% and 77% than that of non-folated hydrophilic QDs. The folate receptor-mediated delivery of folic acid-conjugated CdTe-MSA and CdSe/CdS/ZnS-MPA QDs showed higher cellular internalization as observed by confocal laser scanning microscopic studies. Folated and non-folated CdTe-MSA QDs were highly toxic and exhibited only 10% cell viability as compared to > 80% cell viability with CdSe/CdS/ZnS-MPA QDs over the concentration ranging from 3.38 to 50 pmoles. Immunohistochemistry (IHC) results of human breast cancer tissue samples showed positive results with anti-HER2 antibody conjugated CdSe/CdS/ZnS-MPA QDs with better sensitivity and specificity as compared to conventional IHC analysis using diaminobenzedene staining. PMID:27398438

  17. Peptide-Carrier Conjugation

    DEFF Research Database (Denmark)

    Hansen, Paul Robert

    2015-01-01

    To produce antibodies against synthetic peptides it is necessary to couple them to a protein carrier. This chapter provides a nonspecialist overview of peptide-carrier conjugation. Furthermore, a protocol for coupling cysteine-containing peptides to bovine serum albumin is outlined....

  18. A novel HER2-targeted drug:T-DM1 antibody-drug conjugate%新型抗Her-2药物T-DM1

    Institute of Scientific and Technical Information of China (English)

    岳健; 徐兵河

    2013-01-01

    T-DM1是新型抗体-药物偶联物,具有曲妥珠单抗类似的生物活性,可特异性的将强效抗微管药物DM1释放至Her-2过表达的肿瘤细胞内。T-DM1单药疗效优于拉帕替尼联合卡培他滨,有望成为Her-2阳性晚期乳腺癌的标准二线治疗药物。比较T-DM1与曲妥珠单抗联合紫杉类药物一线治疗晚期乳腺癌的试验正在进行中。该药是继曲妥珠单抗之后又一种全新的抗Her-2药物。美国FDA正式批准T-DM1作为治疗Her-2阳性晚期乳腺癌患者的药物。%T-DM1 is a novel antibody-drug conjugate that has similar biological activity with that of trastuzumab. T-DM1 specifically delivers DM1, the effective anti-microtubule drug, into the cytoplasm of tumor cells with HER2 overexpression. The efficacy of T-DM1 monotherapy is better than lapatinib in combination with capecitabine and T-DM and is expected to become the standard second-line treatment for HER2-positive advanced breast cancer drugs. Clinical trials that compare T-DM1 with trastuzumab joint taxane as the first-line of treatment for advanced breast cancer trials are currently being performed. T-DM1 is a brand new anti-HER2 drug after trastuzumab. U.S. FDA already approved T-DM1 as a drug for the treatment of HER2-positive advanced breast cancer patients.

  19. Bispecific Antibody Conjugated Manganese-Based Magnetic Engineered Iron Oxide for Imaging of HER2/neu- and EGFR-Expressing Tumors

    Science.gov (United States)

    Wu, Shou-Cheng; Chen, Yu-Jen; Wang, Hsiang-Ching; Chou, Min-Yuan; Chang, Teng-Yuan; Yuan, Shyng-Shiou; Chen, Chiao-Yun; Hou, Ming-Feng; Hsu, John Tsu-An; Wang, Yun-Ming

    2016-01-01

    The overexpression of HER2/neu and EGFR receptors plays important roles in tumorigenesis and tumor progression. Targeting these two receptors simultaneously can have a more widespread application in early diagnosis of cancers. In this study, a new multifunctional nanoparticles (MnMEIO-CyTE777-(Bis)-mPEG NPs) comprising a manganese-doped iron oxide nanoparticle core (MnMEIO), a silane-amino functionalized poly(ethylene glycol) copolymer shell, a near infrared fluorescence dye (CyTE777), and a covalently conjugated anti-HER2/neu and anti-EGFR receptors bispecific antibody (Bis) were successfully developed. In vitro T2-weighted MR imaging studies in SKBR-3 and A431 tumor cells incubated with MnMEIO-CyTE777-(Bis)-mPEG NPs showed - 94.8 ± 3.8 and - 84.1 ± 2.8% negative contrast enhancement, respectively. Pharmacokinetics study showed that MnMEIO-CyTE777-(Bis)-mPEG NPs were eliminated from serum with the half-life of 21.3 mins. In vivo MR imaging showed that MnMEIO-CyTE777-(Bis)-mPEG NPs could specifically and effectively target to HER2/neu- and EGFR-expressing tumors in mice; the relative contrast enhancements were 11.8 (at 2 hrs post-injection) and 61.5 (at 24 hrs post-injection) fold higher in SKBR-3 tumors as compared to Colo-205 tumors. T2-weighted MR and optical imaging studies revealed that the new contrast agent (MnMEIO-CyTE777-(Bis)-mPEG NPs) could specifically and effectively target to HER2/neu- and/or EGFR-expressing tumors. Our results demonstrate that MnMEIO-CyTE777-(Bis)-mPEG NPs are able to recognize the tumors expressing both HER2/neu and/or EGFR, and may provide a novel molecular imaging tool for early diagnosis of cancers expressing HER2/neu and/or EGFR. PMID:26722378

  20. In vivo tumor targeting and imaging with anti-vascular endothelial growth factor antibody-conjugated dextran-coated iron oxide nanoparticles

    Directory of Open Access Journals (Sweden)

    Hsieh WJ

    2012-06-01

    Full Text Available Wan-Ju Hsieh,1 Chan-Jung Liang,1 Jen-Jie Chieh,4 Shu-Huei Wang,1 I-Rue Lai,1 Jyh-Horng Chen,2 Fu-Hsiung Chang,3 Wei-Kung Tseng,4–6 Shieh-Yueh Yang,4 Chau-Chung Wu,7 Yuh-Lien Chen11Institute of Anatomy and Cell Biology, College of Medicine, 2Department of Electrical Engineering, 3Institute of Biochemistry and Molecular Biology, National Taiwan University, Taipei, Taiwan; 4Institute of Electro-Optical Science and Technology, National Taiwan Normal University, Taipei, Taiwan; 5Division of Cardiology, Department of Internal Medicine, E-Da Hospital, Taipei, Taiwan; 6Department of Medical Imaging and Radiological Sciences, I-Shou University, Taipei, Taiwan; 7Department of Internal Medicine and Primary Care Medicine, National Taiwan University Hospital, Taipei, TaiwanBackground: Active targeting by specific antibodies combined with nanoparticles is a promising technology for cancer imaging and detection by magnetic resonance imaging (MRI. The aim of the present study is to investigate whether the systemic delivery of antivascular endothelial growth factor antibodies conjugating to the surface of functionalized supermagnetic iron oxide nanoparticles (anti-VEGF-NPs led to target-specific accumulation in the tumor.Methods: The VEGF expression in human colon cancer and in Balb/c mice bearing colon cancers was examined by immunohistochemistry. The distribution of these anti-VEGF-NPs particles or NPs particles were evaluated by MRI at days 1, 2, or 9 after the injection into the jugular vein of Balb/c mice bearing colon cancers. Tumor and normal tissues (liver, spleen, lung, and kidney were collected and were examined by Prussian blue staining to determine the presence and distribution of NPs in the tissue sections.Results: VEGF is highly expressed in human and mouse colon cancer tissues. MRI showed significant changes in the T*2 signal and T2 relaxation in the anti-VEGF-NP- injected-mice, but not in mice injected with NP alone. Examination of paraffin

  1. Preparation of Anti-malaria Antibodies with a Way of Peptide-protein Conjugation%用多肽-蛋白偶联方法制备抗恶性疟原虫抗体

    Institute of Scientific and Technical Information of China (English)

    钱锋

    2012-01-01

    目的 介绍一种多肽-蛋白偶联的流程,并用多肽-蛋白偶联产物制备抗疟原虫抗体. 方法 用化学连接剂Sulfo-EMCS在作为载体蛋白的铜绿假单胞菌重组去毒外毒素(rEPA)上加马来酰亚胺基团,用间接Ellman反应测定载体蛋白上所加的马来酰亚胺基团数量.用马来酰亚胺修饰的载体蛋白滴定Pfs48/45-158多肽[含恶性疟原虫表面蛋白48/45(Pfs48/45)第158~~173氨基酸序列,其N末端带有一个半胱氨酸残基],绘制滴定曲线并用线性回归进行曲线拟合,根据滴定曲线确定理论滴定终点,计算多肽与载体蛋白的偶联比(每摩尔载体蛋白所能结合的多肽的摩尔数).用过量的Pfs48/45-158多肽与马来酰亚胺修饰的rEPA进行反应,规模制备Pfs48/45-158-rEPA多肽-蛋白偶联物,偶联物用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)进行鉴定.用所制备的Pfs48/45-158-rEPA偶联物免疫BALA/c小鼠,制备免疫血清.用ELISA测定免疫小鼠血清抗Pfs48/45-158多肽的抗体效价,用免疫荧光试验(IFA)测定免疫血清识别疟原虫的能力. 结果 通过化学连接剂在每摩尔rEPA上添加约6.94摩尔的马来酰亚胺基团.制备了偶联比约为7.33的Pfs48/45-158-rEPA多肽-蛋白偶联物.偶联物免疫小鼠激发出抗Pfs48/45-158多肽的高抗体应答,免疫血清抗Pfs48/45-158多肽的效价为12 500 ELISA单位(即吸光度A405值为1时的血清稀释度倒数),同时免疫血清可识别疟原虫. 结论 多肽-蛋白偶联是一种可用于制备抗疟原虫抗体的便捷方法,间接Ellman检测、滴定反应和SDS-PAGE分析构成了多肽-蛋白偶联物制备的质控方法,可更好地保证多肽-蛋白偶联物的质量和稳定性.%Objective To introduce a procedure of peptide-protein conjugation and prepare anti-malaria antibodies using a peptide-protein conjugate. Methods The recombinant atoxic form of Pseudomonas aeruginosa exotoxin A (rEPA) was used as carrier

  2. Tandem Native Mass-Spectrometry on Antibody-Drug Conjugates and Submillion da Antibody-Antigen Protein Assemblies on an Orbitrap EMR Equipped with a High-Mass Quadrupole Mass Selector

    NARCIS (Netherlands)

    Dyachenko, Andrey; Wang, Guanbo; Belov, Mike; Makarov, Alexander; De Jong, Rob N.; Van Den Bremer, Ewald T J; Parren, Paul W H I; Heck, Albert J R

    2015-01-01

    Native mass spectrometry is emerging as a powerful tool for the characterization of intact antibodies and antibody-based therapeutics. Here, we demonstrate new possibilities provided by the implementation of a high mass quadrupole mass selector on the recently introduced Orbitrap Exactive EMR mass s

  3. Determination of Conjugation Efficiency of Antibodies and Proteins to the Superparamagnetic Iron Oxide Nanoparticles by Capillary Electrophoresis with Laser-Induced Fluorescence Detection

    International Nuclear Information System (INIS)

    The method based on capillary electrophoresis with laser-induced fluorescence detection (CE/LIF) was developed for determination of magnetic iron oxide nanoparticles (hydrodynamic diameters of 100 nm) functionalized with molecules containing primary amino groups. The magnetic nanoparticles with carboxylic or aminopropyl-trimethoxysilane groups at their surface were conjugated to the model proteins (bovine serum albumin, BSA; streptavidin or goat anti-rabbit immunoglobulin G, IgG) using carbodiimide as a zero-length cross-linker.The nanoparticle-protein conjugates (hydrodynamic diameter 163-194 nm) were derivatized with naphthalene-2,3-dicarboxaldehyde reagent and separated by CE/LIF with a helium-cadmium laser (excitation at 442 nm, emission at 488 nm). The separations were carried out by using a fused-silica capillary (effective length 48 cm, inner diameter 75 um) and 100 mM sodium borate buffer (pH 9.2), the potential was 30 kV. The detection limit for BSA-conjugate was 1.3 pg/10 nl, i.e. about 20 amol. The present method provides an efficient and fast tool for sensitive determination of the efficacy of biomolecular functionalization of magnetic nanoparticles. The CE/LIF technique requires only negligible sample volumes for analysis, which is especially suitable for controlling the process of preparation of functionalized nanoparticles with unique properties aimed to be used for diagnostic or therapeutic purposes

  4. A breakthrough in antibody-drug conjugates in anti-immune rejection%抗免疫排斥反应中抗体药物偶联物的突破性进展

    Institute of Scientific and Technical Information of China (English)

    郭彩龙; 莽源祎; 张雷

    2014-01-01

    BACKGROUND:The immunological rejection between host and graft is the leading cause of organ transplantation failure. The traditional immunosuppressive agents have been unable to meet the needs of clinical treatment. Antibody-drug conjugate, as a type of new drugs, may be hope for the treatment of immune rejection. OBJECTIVE:To comprehensively analyze the composition of antibody-drug conjugates, mechanism of action, clinical research progress as wel as the development trend. METHODS:A computer-based online retrieval was performed to search papers in CNKI and PubMed database using the key words of ADCs, immunosuppressive agents, immunotoxins, organ transplantation, graft rejection in Chinese and English. Recently published or published in the prestigious journals were selected in the same field. After excluding objective-independent papers and repeated studies, 42 papers were included for further analysis. RESULTS AND CONCLUSION:Antibody-drug conjugates, as highly effective and lowly toxic immunosuppressant, have achieved a breakthrough in treatment of targeting tumor, while the role of it in anti-immune rejection is stil at the exploratory stage. For islet transplantation, novel antibody-drug conjugates are required to block CD8+T effector by CD103/E-Cadherin pathway, and wil probably serve as a potential drug intervention for al ograft rejection.%背景:宿主与移植物间的免疫排斥反应是器官移植失败的主要原因之一,传统的免疫抑制剂已无法满足临床治疗的需求,抗体药物偶联物作为一类新型药物,可能为免疫排斥反应的治疗带来希望。  目的:综合分析抗体药物偶联物的构成、作用机制、临床研究进展以及未来的发展趋势。  方法:以“抗体药物偶联物、免疫抑制剂、免疫毒素、器官移植、移植物排斥”,“ADCs,immunosuppressive agents,immunotoxins,organ transplantation,graft rejection”为检索

  5. Preparation and near-quantitative Y-90 labelings of monoclonal antibody-macrocylic chelator (MAb-DOTA) conjugates for radioimmunotherapy (RAIT)

    Energy Technology Data Exchange (ETDEWEB)

    Govindan, S.V.; Griffiths, G.L.; Losman, M.J. [Immunomedics, Inc., Morris Plains, NJ (United States)] [and others

    1996-05-01

    The importance of kinetically inert Y-90-DOTA chelate for RAIT is well recognized. The aim of this study was to adapt a published procedure to overcome long-standing problems related to practical preparation of DOTA-MAb conjugates and poor Y-90 incorporation, and further improve upon labeling efficiencies. Using monoactivated DOTA, we have prepared conjugates of a CEA MAb (MN-14), a lymphoma MAb (LL2), their humanized versions (hMN-14 and hLL2), and the respective divalent fragments, and systematically examined the Y-90-labeling parameters. The DOTA/Mab ratios in conjugates were in the 3.0-6.8 range. In competitive CEA-binding assays, unmodified MN-14, MN-14-DOTA (6.8 chelators) and hMN-14-DOTA (4.4 chelators) exhibited very similar binding patterns. Y-90 labeling yields of >90% were obtained using a labeling time of 2 h at 40-45{degrees}C, pH {approximately}5.5, and DOTA/MAb ratio >3. Incorporations (specific activities):>99%for hMN-14-DOTA (103-170 MBa/mg), 94-97% for hLL2-DOTA (107-185 MBa/mg), 93-98%for hLL2F(ab`){sub 2}-DOTA (111 MBa/mg), and >90% for MN14-DOTA (37-185 MBq/mg). Aggregate content was usually less than 5%. Observed high incorporations are due both to the higher temperature used and DOTA/Y-90 molar ratios which readily exceeded a threshold requirement of three. Labeling efficiencies also depended upon the level of trace metal contaminants in the Y-90 shipment. Incubations of Y-90 labeled DOTA conjugates of hMN-14 and hLL2 in serum and in 1 mM DTPA at 37{degrees}C showed no detectable loss of metal over a 10-day period. These results should allow us to routinely use Y-90-DOTA-MAb conjugates in future preclinical and clinical RAIT studies.

  6. Antibodies conjugated with new highly luminescent Eu3+ and Tb3+ chelates as markers for time resolved immunoassays. Application to simultaneous determination of clenbuterol and free cortisol in horse urine.

    Science.gov (United States)

    Bacigalupo, M A; Meroni, G; Secundo, F; Scalera, C; Quici, S

    2009-12-15

    Highly luminescent Eu(3+) and Tb(3+) complexes of 10-[4-(3-isothiocyanatopropoxy)benzoylmethyl]-1,4,7,10-tetraazacyclododecane-1,4,7 triacetic acid Eu(3+) is a subset of 1 and Tb(3+) is a subset of 1 were conjugated with a goat anti-rabbit IgG and a rabbit anti-mouse IgG, respectively, and applied as markers in a time resolved immunoassay for simultaneous quantitative determination of anabolic compounds clenbuterol (CL) and hydrocortisone (HC). The assay was performed in horse urine, using a monoclonal antibody specific to CL and a rabbit polyclonal antibody specific to the free HC. These lanthanide chelates are very stable and highly luminescent in aqueous solution and allowed to reach 10 microg L(-1) and 40 microg L(-1) sensitivities for CL and for HC, respectively. Application to the horse urine, that is a very complex matrix, has a considerable interest in the control of illegal use of these compounds. PMID:19836578

  7. Radiohalogenated half-antibodies and maleimide intermediate therefor

    Science.gov (United States)

    Kassis, A.I.; Khawli, L.A.

    1991-02-19

    N-(m-radiohalophenyl) maleimide can be conjugated with a reduced antibody having a mercapto group to provide a radiolabeled half-antibody having immunological specific binding characteristics of whole antibody. No Drawings

  8. Intranasal Immunization of Guinea Pigs with an Immunodominant Foot-and-Mouth Disease Virus Peptide Conjugate Induces Mucosal and Humoral Antibodies and Protection against Challenge†

    OpenAIRE

    Fischer, D.; Rood, D.; Barrette, R. W.; Zuwallack, A.; Kramer, E.; Brown, F.; Silbart, L. K.

    2003-01-01

    Guinea pigs immunized intranasally with a keyhole limpet hemocyanin-linked peptide, corresponding to the prominent G-H loop of the VP1 protein of foot-and-mouth disease virus, raised substantial levels of antipeptide and virus-neutralizing antibodies in sera and of peptide-specific secretory immunoglobulin A in nasal secretions. In groups of animals immunized intranasally without adjuvant, 86 percent were fully protected upon challenge with homotypic virus. Surprisingly, animals given the pep...

  9. Trop-2 is a novel target for solid cancer therapy with sacituzumab govitecan (IMMU-132), an antibody-drug conjugate (ADC)*

    Science.gov (United States)

    Goldenberg, David M.; Cardillo, Thomas M.; Govindan, Serengulam V.; Rossi, Edmund A.; Sharkey, Robert M.

    2015-01-01

    Trop-2 is a novel target for ADC therapy because of its high expression by many solid cancers. The rational development of IMMU-132 represents a paradigm shift as an ADC that binds a well-known moderately-cytotoxic drug, SN-38, to the anti-Trop-2 antibody. In vitro and in vivo studies show enhanced efficacy, while there is a gradual release of SN-38 that contributes to the overall effect. IMMU-132 is most efficacious at a high drug:antibody ratio (DAR) of 7.6:1, which does not affect binding and pharmacokinetics. It targets up to 136-fold more SN-38 to a human cancer xenograft than irinotecan, SN-38′s prodrug. IMMU-132 delivers SN-38 in its most active, non-glucuronidated form, which may explain the lower frequency of severe diarrhea than with irinotecan. Thus, this ADC, carrying a moderately-toxic drug targeting Trop-2 represents a novel cancer therapeutic that is showing promising activity in patients with several metastatic cancer types, including triple-negative breast cancer, non-small-cell and small-cell lung cancers. PMID:26101915

  10. Preparation of bioconjugates by solid-phase conjugation to ion exchange matrix-adsorbed carrier proteins

    DEFF Research Database (Denmark)

    Houen, G.; Olsen, D.T.; Hansen, P.R.;

    2003-01-01

    protein was conjugated with glutathione, the conjugation ratio determined by acid hydrolysis, and amino acid analysis performed with quantification of carboxymethyl cysteine. Elution of conjugates from the resin by a salt gradient revealed considerable heterogeneity in the degree of derivatization......, and immunization experiments with the eluted conjugates showed that the more substituted conjugates gave rise to the highest titers of glutathione antibodies. Direct immunization with the conjugates adsorbed to the ion exchange matrix was possible and gave rise to high titers of glutathione antibodies. Conjugates...

  11. Validation of analytical method to calculate the concentration of conjugated monoclonal antibody; Validacao de metodo analitico para calculo de concentracao de anticorpo monoclonal conjugado

    Energy Technology Data Exchange (ETDEWEB)

    Alcarde, Lais F.; Massicano, Adriana V.F.; Oliveira, Ricardo S.; Araujo, Elaine B. de, E-mail: lais_alcarde@hotmail.com [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil)

    2013-07-01

    The objective of this study was to develop a quantitative analytical method using high performance liquid chromatography (HPLC) to determine the antibody concentration in conjunction with bifunctional chelator. Assays were performed using a high performance liquid chromatograph, and the following conditions were used: flow rate of 1 mL / min, 15 min run time, 0.2 M sodium phosphate buffer pH 7.0 as the mobile phase and column of molecular exclusion BioSep SEC S-3000 (300 x 7.8 mm, 5 μM - Phenomenex). The calibration curve was obtained with AcM diluted in 0.2 M sodium phosphate buffer pH 7.0 by serial dilution, yielding the concentrations: 400 μg/mL, 200 μg/mL, 100 μg/mL, 50 μg/mL, 25 μg/mL and 12.5 μg/mL. From the calibration curve calculated the equation of the line and with it the concentration of the immunoconjugate. To ensure the validity of the method accuracy and precision studies were conducted. The accuracy test consisted in the evaluation of 3 samples of known concentration, being this test performed with low concentrations (50 μg/mL), medium (100 μg/mL) and high (200 μg/mL). The precision test consisted of 3 consecutive measurements of one sample of known concentration, subject to the conditions set forth above for the other tests. The correlation coefficient of the standard curve was greater than 97%, the accuracy was satisfactory at low concentrations as well as accuracy. The method was validated by showing it for the accurate and precise determination of the concentration of the immunoconjugate. Furthermore, this assay was found to be extremely important, because using the correct mass of the protein, the radiochemical purity of the radioimmunoconjugate was above 95% in all studies.

  12. Chemically optimized antimyosin Fab conjugates with chelating polymers: importance of the nature of the protein-polymer single site covalent bond for biodistribution and infarction localization.

    Science.gov (United States)

    Trubetskoy, V S; Narula, J; Khaw, B A; Torchilin, V P

    1993-01-01

    Murine antimyosin Fab fragment was conjugated with 111In-labeled N-terminal-modified DTPA-polylysine using three bifunctional reagents: N-hydroxysuccinimide esters of 3-(2-pyridyldithio)propionic acid (SPDP conjugate), 4-(maleimidomethyl)cyclohexanecarboxylic acid (SMCC conjugate) and bromoacetic acid (BrAc conjugate) for potential localization of experimental myocardial infarction. Using various antibody preparations and a rabbit acute myocardial infarction model the following parameters were observed: (1) an in vitro antigen binding activity of SPDP conjugate = SMCC conjugate > BrAc conjugate, (2) a blood clearance rate of SPDP conjugate > BrAc conjugate > SMCC conjugate, (3) a liver and splenic accumulation of SPDP conjugate > BrAc conjugate > SMCC conjugate, and (4) the infarcted tissue activity showed an accumulation of SMCC conjugate > SPDP conjugate > BrAc conjugate. This study exemplifies the importance of rational chemical design of antimyosin Fab-chelating polymer conjugate for improved target tissue localization in vivo.

  13. 不同剂量伤寒Vi 多糖蛋白结合疫苗在小鼠体内诱导的抗体水平分析%Anal ysis of antibody lve el in mou se model induced by diff erent immunization doses of typhoid Vi conjugate vaccine

    Institute of Scientific and Technical Information of China (English)

    刘月萍; 罗树权; 冯宜扬; 冯琪蓉; 金鑫; 周晖国; 周海飞; 谢贵林; 谭小梅

    2015-01-01

    目的:研究伤寒Vi多糖蛋白结合疫苗免疫效果及不同剂量伤寒Vi多糖蛋白结合疫苗在小鼠体内诱导的抗体水平,以确定合适的免疫剂量。方法将150只清洁级NIH雌性小鼠随机分为5组,分别为A组(06.25μg 结合疫苗组)、B组(1.250μg结合疫苗组)、C组(2.500μg结合疫苗组)、D组(2.500μg多糖组)及阴性组(10 mmol/L PBS),每组30只;另领取10只为空白对照(不接种)。 A、B、C、D 组及阴性组小鼠经腹股沟皮下注射,剂量0.1 mL/只,每隔2周免疫1次,共免疫3次,每次免疫后第7天采血。采用ELISA检测小鼠血清抗体效价,同时对不同剂量伤寒Vi多糖蛋白结合疫苗在小鼠体内诱导的抗体效价进行分析比较。结果与D组相比,A 、B、C 三组诱导的抗体水平与之均有统计学意义(P<0.05);A组与B组、A组与C组之间的抗体水平也具有统计学意义(P<0.05),而B组与C组之间的抗体水平无统计学意义(P>0.05)。说明与多糖疫苗相比,伤寒Vi多糖蛋白结合疫苗能够诱导更高的抗体水平,且具有明显的剂次加强效应。同时证明1.250μg的伤寒Vi多糖蛋白结合疫苗可诱导与25.00μg伤寒Vi多糖蛋白结合疫苗相同的抗体水平。结论伤寒Vi多糖蛋白结合疫苗的两种免疫剂量在小鼠体内可诱导相同的抗体水平,在选择接种剂量时,可致免疫应答的无统计学意义的低剂量可能是较为经济和安全的选择。%Objective To study immunological potency of typhoid Vi conjugate vaccine and analyze antibody level in the mouse model induced by different immunization doses of typhoid Vi conjugate vaccine, on which a suitable immunization dose could be set up.Methods A total of 150 NIH female mice ( in clean level) were randomly devided into five groups including--group A (0.625μg conjugate vaccine), group B (1.250μg conjugate vaccine), group C

  14. Interaction of CdTe/CdS quantum dots with antibodies

    Institute of Scientific and Technical Information of China (English)

    Chen Shi; Xiang Yi Huang; Chao Qing Dong; Hong Jin Chen; Ji Cun Ren

    2009-01-01

    In the study,we observed the strong adsorption of CdTe/CdS QDs to antibodies and the formation of QDs-antibodies conjugates.Capillary electrophoresis with laser-induced fluorescence detection(CE-LIF),fluorescence spectrometry and fluorescence correlation spectroscopy(FCS)were used to characterize the QDs conjugates with antibody.We found that the QDs-antibody conjugates possessed high fluorescence,small hydrodynamic radii and good stability in aqueous solution.

  15. Highly sensitive biosensors based on water-soluble conjugated polymers

    Institute of Scientific and Technical Information of China (English)

    XU Hui; WU Haiping; FAN Chunhai; LI Wenxin; ZHANG Zhizhou; HE Lin

    2004-01-01

    Conjugated, conductive polymers are a kind of important organic macromolecules, which has found applications in a variety of areas. The application of conjugated polymers in developing fluorescent biosensors represents the merge of polymer sciences and biological sciences. Conjugated polymers are very good light harvesters as well as fluorescent polymers, and they are also "molecular wires". Through elaborate designs, these important features, i.e. efficient light harvesting and electron/energy transfer, can be used as signal amplification in fluorescent biosensors. This might significantly improve the sensitivity of conjugated polymer-based biosensors. In this article, we reviewed the application of conjugated polymers, via either electron transfer or energy transfer, to detections of gene targets, antibodies or enzymes. We also reviewed recent efforts in conjugated polymer-based solid-state sensor designs as well as chip-based multiple target detection. Possible directions in this conjugated polymer-based biosensor area are also discussed.

  16. Preparation of self-assembled core-shell nano structure of conjugated generation 4.5 poly (amidoamine) dendrimer and monoclonal Anti-IL-6 antibody as bioimaging probe.

    Science.gov (United States)

    Mekuria, Shewaye Lakew; Tsai, Hsieh-Chih

    2015-11-01

    In this article, interleukin-6 (IL-6)-conjugated anionic generation 4.5 (G4.5) poly(amidoamine) (PAMAM) was synthesized through EDC/NHS coupling chemistry and evaluated for its optical properties in vitro. Conjugation was confirmed using Fourier-transformed infrared spectroscopy (FT-IR) and 2-dimensional nuclear magnetic resonance (2D NMR). After IL-6 conjugation, nanoparticle size increased to approximately 70 nm and zeta potential increased from -56.5 ± 0.2 to -19.1 ± 2.4 mV due to neutralization of negatively charged G4.5. Wide-angle X-ray scattering (WAXS) suggested that a layered nanoparticle structure was formed by the G4.5/IL-6 conjugate. Most interestingly, the intrinsic fluorescence of G4.5 significantly increased after IL-6 conjugation and underwent a blue shift as a result of H-aggregation. Furthermore, the cellular uptake of the conjugates by HeLa cells was significantly enhanced in comparison to free G4.5, as demonstrated by confocal microscopy and flow cytometry. These results indicated that the described system may be a potential bioimaging probe in vitro. PMID:26263213

  17. HER2 specific delivery of methotrexate by dendrimer conjugated anti-HER2 mAb

    Science.gov (United States)

    Shukla, Rameshwer; Thomas, Thommey P.; Desai, Ankur M.; Kotlyar, Alina; Park, Steve J.; Baker, James R., Jr.

    2008-07-01

    Herceptin, a humanized monoclonal antibody that binds to human growth factor receptor-2 (HER2), was covalently attached to a fifth-generation (G5) polyamidoamine dendrimer containing the cytotoxic drug methotrexate. The specific binding and internalization of this conjugate labeled with FITC was clearly demonstrated in cell lines overexpressing HER2 by flow cytometry as well as confocal microscopic analysis. In addition, binding and uptake of antibody conjugated dendrimers was completely blocked by excess non-conjugated herceptin. The dendrimer conjugate was also shown to inhibit the dihydrofolate reductase with similar activity to methotrexate. Co-localization experiments with lysotracker red indicate that antibody conjugate, although internalized efficiently into cells, has an unusually long residence time in the lysosome. Somewhat lower cytotoxicity of the conjugate in comparison to free methotrexate was attributed to the slow release of methotrexate from the conjugate and its long retention in the lysosomal pocket.

  18. HER2 specific delivery of methotrexate by dendrimer conjugated anti-HER2 mAb

    Energy Technology Data Exchange (ETDEWEB)

    Shukla, Rameshwer; Thomas, Thommey P; Desai, Ankur M; Kotlyar, Alina; Park, Steve J; Baker, James R Jr [Michigan Nanotechnology Institute for Medicine and Biological Sciences, University of Michigan, 9220 MSRB III, Box 0648, Ann Arbor, MI 48109 (United States)], E-mail: rameshwe@umich.edu, E-mail: jbakerjr@med.umich.edu

    2008-07-23

    Herceptin, a humanized monoclonal antibody that binds to human growth factor receptor-2 (HER2), was covalently attached to a fifth-generation (G5) polyamidoamine dendrimer containing the cytotoxic drug methotrexate. The specific binding and internalization of this conjugate labeled with FITC was clearly demonstrated in cell lines overexpressing HER2 by flow cytometry as well as confocal microscopic analysis. In addition, binding and uptake of antibody conjugated dendrimers was completely blocked by excess non-conjugated herceptin. The dendrimer conjugate was also shown to inhibit the dihydrofolate reductase with similar activity to methotrexate. Co-localization experiments with lysotracker red indicate that antibody conjugate, although internalized efficiently into cells, has an unusually long residence time in the lysosome. Somewhat lower cytotoxicity of the conjugate in comparison to free methotrexate was attributed to the slow release of methotrexate from the conjugate and its long retention in the lysosomal pocket.

  19. HER2 specific delivery of methotrexate by dendrimer conjugated anti-HER2 mAb

    International Nuclear Information System (INIS)

    Herceptin, a humanized monoclonal antibody that binds to human growth factor receptor-2 (HER2), was covalently attached to a fifth-generation (G5) polyamidoamine dendrimer containing the cytotoxic drug methotrexate. The specific binding and internalization of this conjugate labeled with FITC was clearly demonstrated in cell lines overexpressing HER2 by flow cytometry as well as confocal microscopic analysis. In addition, binding and uptake of antibody conjugated dendrimers was completely blocked by excess non-conjugated herceptin. The dendrimer conjugate was also shown to inhibit the dihydrofolate reductase with similar activity to methotrexate. Co-localization experiments with lysotracker red indicate that antibody conjugate, although internalized efficiently into cells, has an unusually long residence time in the lysosome. Somewhat lower cytotoxicity of the conjugate in comparison to free methotrexate was attributed to the slow release of methotrexate from the conjugate and its long retention in the lysosomal pocket

  20. Thyroid Antibodies

    Science.gov (United States)

    ... be limited. Home Visit Global Sites Search Help? Thyroid Antibodies Share this page: Was this page helpful? Also known as: Thyroid Autoantibodies; Antithyroid Antibodies; Antimicrosomal Antibody; Thyroid Microsomal Antibody; ...

  1. Monoclonal antibodies.

    Science.gov (United States)

    2009-01-01

    The ability to produce and exploit monoclonal antibodies (mAbs) has revolutionized many areas of biological sciences. The unique property of an mAb is that it is a single species of immunoglobulin (IG) molecule. This means that the specificity of the interaction of the paratopes on the IG, with the epitopes on an antigenic target, is the same on every molecule. This property can be used to great benefit in immunoassays to provide tests of defined specificity and sensitivity, which improve the possibilities of standardization. The performance of assays can often be determined relating the actual weight of antibody (hence the number of molecules) to the activity. Often the production of an mAb against a specific epitope is the only way that biological entities can be differentiated. This chapter outlines the areas involving the development of assays based on mAbs. The problems involved address include the physical aspects of mAbs and how they may affect assay design and also the implications of results based on monospecific reagents. Often these are not fully understood, leading to assays that are less than satisfactory, which does not justify the relatively high cost of preparing and screening of mAbs. There are many textbooks and reviews dealing with the preparation of mAbs, the principles involved, and various purification and manipulative methods for the preparation of fragments and conjugation. There has been little general information attempting to summarize the best approaches to assay design using mAbs. Much time can be wasted through bad planning, and this is particularly relevant to mAbs. A proper understanding of some basic principles is essential. It is beyond the scope of this chapter to discuss all aspects, but major areas are highlighted. PMID:19219589

  2. A Quantitative Method for Comparing the Brightness of Antibody-dye Reagents and Estimating Antibodies Bound per Cell.

    Science.gov (United States)

    Kantor, Aaron B; Moore, Wayne A; Meehan, Stephen; Parks, David R

    2016-01-01

    We present a quantitative method for comparing the brightness of antibody-dye reagents and estimating antibodies bound per cell. The method is based on complementary binding of test and fill reagents to antibody capture microspheres. Several aliquots of antibody capture beads are stained with varying amounts of the test conjugate. The remaining binding sites on the beads are then filled with a second conjugate containing a different fluorophore. Finally, the fluorescence of the test conjugate compared to the fill conjugate is used to measure the relative brightness of the test conjugate. The fundamental assumption of the test-fill method is that if it takes X molecules of one test antibody to lower the fill signal by Y units, it will take the same X molecules of any other test antibody to give the same effect. We apply a quadratic fit to evaluate the test-fill signal relationship across different amounts of test reagent. If the fit is close to linear, we consider the test reagent to be suitable for quantitative evaluation of antibody binding. To calibrate the antibodies bound per bead, a PE conjugate with 1 PE molecule per antibody is used as a test reagent and the fluorescence scale is calibrated with Quantibrite PE beads. When the fluorescence per antibody molecule has been determined for a particular conjugate, that conjugate can be used for measurement of antibodies bound per cell. This provides comparisons of the brightness of different conjugates when conducted on an instrument whose statistical photoelectron (Spe) scales are known. © 2016 by John Wiley & Sons, Inc.

  3. Synthesis and Antibody Recognition of Cyclic Epitope Peptides, Together with Their Dimer and Conjugated Derivatives Based on Residues 9-22 of Herpes Simplex Virus Type 1 Glycoprotein D

    NARCIS (Netherlands)

    Jakab, Annamaria; Schlosser, Gitta; Feijlbrief, Matty; Welling-Wester, Sytske; Manea, Marilena; Vila-Perello, Miquel; Andreu, David; Ferenc Hudecz, [No Value; Mezo, Gabor

    2009-01-01

    The synthesis of new cyclic peptides comprising the 9-22 epitope (9)LKMADPNRFRGKDL(22) sequence derived from HSV gD-1 is reported. In addition, we describe procedures for the preparation of cyclic peptide dimers and conjugates with an oligotuftsin derivative carrier. The binding of a monoclonal anti

  4. A monoclonal antibody against leptin.

    Science.gov (United States)

    Mahmoudian, Jafar; Jeddi-Tehrani, Mahmood; Bayat, Ali Ahmad; Mahmoudi, Ahmad Reza; Vojgani, Yasaman; Tavangar, Banafsheh; Hadavi, Reza; Zarei, Saeed

    2012-10-01

    Leptin is an important protein that regulates energy storage and homeostasis in humans and animals. Leptin deficiency results in various abnormalities such as diabetes, obesity, and infertility. Producing a high affinity monoclonal antibody against human leptin provides an important tool to monitor and trace leptin function in different biological fluids. In this study, recombinant human leptin was conjugated to KLH and injected into mice. After immunization, mouse myeloma SP2/0 cells were fused with murine splenocytes followed by selection of antibody-producing hybridoma cells. After screening of different hybridoma colonies by ELISA, a high affinity antibody was selected and purified by affinity chromatography. The affinity constant of the antibody was measured by ELISA. Western blot, immunocytochemistry, and flow cytometry experiments were used to characterize the antibody. The anti-leptin antibody had a high affinity (around 1.13 × 10(-9) M) for its antigen. The saturation of the antibody with leptin (20 moles leptin per 1 mole antibody) in Western blot analysis proved that the antibody had specific binding to its antigen. Immunocytochemistry and flow cytometry on JEG-3 (human placental choriocarcinoma cell) cells revealed that the anti-leptin antibody recognized intracellular leptin. In conclusion, we report here the production and characterization of a murine anti-leptin antibody with high affinity for human leptin. PMID:23098305

  5. Antibody Based Surgical Imaging and Photodynamic Therapy for Cancer

    NARCIS (Netherlands)

    de Boer, Esther

    2016-01-01

    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. Revisiting conjugate schedules.

    Science.gov (United States)

    MacAleese, Kenneth R; Ghezzi, Patrick M; Rapp, John T

    2015-07-01

    The effects of conjugate reinforcement on the responding of 13 college students were examined in three experiments. Conjugate reinforcement was provided via key presses that changed the clarity of pictures displayed on a computer monitor in a manner proportional to the rate of responding. Experiment 1, which included seven parameters of clarity change per response, revealed that responding decreased as the percentage clarity per response increased for all five participants. These results indicate that each participant's responding was sensitive to intensity change, which is a parameter of conjugate reinforcement schedules. Experiment 2 showed that responding increased during conjugate reinforcement phases and decreased during extinction phases for all four participants. Experiment 3 also showed that responding increased during conjugate reinforcement and further showed that responding decreased during a conjugate negative punishment condition for another four participants. Directions for future research with conjugate schedules are briefly discussed.

  7. Revisiting conjugate schedules.

    Science.gov (United States)

    MacAleese, Kenneth R; Ghezzi, Patrick M; Rapp, John T

    2015-07-01

    The effects of conjugate reinforcement on the responding of 13 college students were examined in three experiments. Conjugate reinforcement was provided via key presses that changed the clarity of pictures displayed on a computer monitor in a manner proportional to the rate of responding. Experiment 1, which included seven parameters of clarity change per response, revealed that responding decreased as the percentage clarity per response increased for all five participants. These results indicate that each participant's responding was sensitive to intensity change, which is a parameter of conjugate reinforcement schedules. Experiment 2 showed that responding increased during conjugate reinforcement phases and decreased during extinction phases for all four participants. Experiment 3 also showed that responding increased during conjugate reinforcement and further showed that responding decreased during a conjugate negative punishment condition for another four participants. Directions for future research with conjugate schedules are briefly discussed. PMID:26150349

  8. Optimization of multi-epitopic HIV-1 recombinant protein expression in prokaryote system and conjugation to mouse DEC-205 monoclonal antibody: implication for in-vivo targeted delivery of dendritic cells

    OpenAIRE

    Roghayeh Rahimi; Massoumeh Ebtekar; Seyed Mohammad Moazzeni; Ali Mostafaie; Mehdi Mahdavi

    2015-01-01

    Objective(s):Multi-epitopic protein vaccines and direction of vaccine delivery to dendritic cells (DCs) are promising approaches for enhancing immune responses against mutable pathogens. Escherichia coli is current host for expression of recombinant proteins, and it is important to optimize expression condition. The aim of this study was the optimization of multi-epitopic HIV-1 tat/pol/gag/env recombinant protein (HIVtop4) expression by E. coli and conjugation of purified protein to anti DEC-...

  9. A MONOCLONAL-ANTIBODY AGAINST HUMAN BETA-GLUCURONIDASE FOR APPLICATION IN ANTIBODY-DIRECTED ENZYME PRODRUG THERAPY

    NARCIS (Netherlands)

    Haisma, Hidde; VANMUIJEN, M; SCHEFFER, G; SCHEPER, RJ; PINEDO, HM; BOVEN, E

    1995-01-01

    The selectivity of anticancer agents may be improved by antibody-directed enzyme prodrug therapy (ADEPT), The immunogenicity of antibody-enzyme conjugates and the low tumor to normal tissue ratio calls for the use of a human enzyme and the development of a monoclonal antibody (MAb) against that enzy

  10. Pneumococcal Conjugate Vaccine for Adults: A New Paradigm

    OpenAIRE

    Paradiso, Peter R

    2012-01-01

    A 13-valent pneumococcal conjugate vaccine has been studied in adults aged ≥50 years to compare the immune response to that induced by the 23-valent pneumococcal polysaccharide vaccine, which has been the standard of care over the past 30 years. The results demonstrate that adults, regardless of whether they are naive or previously vaccinated with the polysaccharide vaccine, have an overall superior antibody response when vaccinated with the conjugate vaccine compared with the pneumococcal po...

  11. Pneumococcal vaccination of older adults: Conjugate or polysaccharide?

    OpenAIRE

    Fedson, David S; Guppy, Martin J.

    2013-01-01

    Invasive pneumococcal disease continues to be important problem for older adults. Pneumococcal polysaccharide vaccine (PPV23) has a clinical effectiveness of 43–81%, and following primary vaccination and revaccination, antibody responses last 5–10 y. Hyporesponsiveness to a second dose of vaccine has not been shown to be a significant problem. The use of pneumococcal conjugate vaccines (initially PCV7; more recently PCV13) has led to a dramatic fall in the incidence of conjugate vaccine-type ...

  12. Optimization of biotin labeling of antibodies using mouse IgG and goat anti-mouse IgG-conjugated fluorescent beads and their application as capture probes on protein chip.

    Science.gov (United States)

    Lee, Jin Hyung; Choi, Hong Kyung; Chang, Jeong Ho

    2010-10-31

    This study shows the optimization of biotin labeling to antibodies using mouse IgG. Several parameters of the biotin labeling, including the molar ratio of biotin to antibody, the coupling time and the dialysis time, were studied to optimum conditions. The biotin-tagged mouse IgGs were immobilized on avidin-coated PMMA (Polymethyl Methacrylate) plates via a biotin-avidin linkage. The immobilization of the IgG to the chip was quantified using goat anti-mouse IgG bound fluorescent beads. It was found that the binding of the fluorescent beads saturated when a 10-fold or higher molar ratio of biotin to antibody was used. In biotin coupling time tests, sixty minutes was sufficient for the capture probes to bind to the surface. However, the results from the dialysis experiments showed no difference, indicating that 2 hours was sufficient to remove any unbound biotin. Finally, to prove the universality of this protocol using mouse antibodies, the optimum conditions were successfully applied in sandwich immunoassays designed to detect troponin I (TnI) and N-terminal probrain natriuretic peptide (NT-proBNP). PMID:20804762

  13. Antibody-Directed Phototherapy (ADP

    Directory of Open Access Journals (Sweden)

    M. Adil Butt

    2013-04-01

    Full Text Available Photodynamic therapy (PDT is a clinically-approved but rather under-exploited treatment modality for cancer and pre-cancerous superficial lesions. It utilises a cold laser or LED to activate a photochemical reaction between a light activated drug (photosensitiser-drug and oxygen to generate cytotoxic oxygen species. These free radical species damage cellular components leading to cell death. Despite its benefits, the complexity, limited potency and side effects of PDT have led to poor general usage. However, the research area is very active with an increasing understanding of PDT-related cell biology, photophysics and significant progress in molecular targeting of disease. Monoclonal antibody therapy is maturing and the next wave of antibody therapies includes antibody-drug conjugates (ADCs, which promise to be more potent and curable. These developments could lift antibody-directed phototherapy (ADP to success. ADP promises to increase specificity and potency and improve drug pharmacokinetics, thus delivering better PDT drugs whilst retaining its other benefits. Whole antibody conjugates with first generation ADP-drugs displayed problems with aggregation, poor pharmacokinetics and loss of immuno-reactivity. However, these early ADP-drugs still showed improved selectivity and potency. Improved PS-drug chemistry and a variety of conjugation strategies have led to improved ADP-drugs with retained antibody and PS-drug function. More recently, recombinant antibody fragments have been used to deliver ADP-drugs with superior drug loading, more favourable pharmacokinetics, enhanced potency and target cell selectivity. These improvements offer a promise of better quality PDT drugs.

  14. Specific Imaging of Colorectal Cancer Cells using Quantum Dots Probes Conjugated with ND-1 Monoclonal Antibodies%单克隆抗体ND-1-量子点荧光探针对大肠癌细胞的特异成像研究

    Institute of Scientific and Technical Information of China (English)

    王蒴; 方瑾

    2013-01-01

    Objective To prepare the immuno-conjugate of ND-1 monoclonal antibody and quantum dots for targeted fluorescent imaging of colorectal cancer cells. Methods ND-1 , a monoclonal antibody a-gainst human colorectal cancer cells, was covalently coupled to QD605 at differnet ratios using EDC and NHS to optimize the conjugating condition. The obtained QD-linked conjugate was analyzed by fluorespec-trophotometry to characterise its optical properties and anti-photobleach ability. Immunofluorescence assay was utilized to observe the targeted imaging of the probe. Results A high coupling efficiency was a-chieved at 1:40 molar ratio of QD605 to ND-1 monoclonal antibodies. The fluorescence spectrum showed that ND-1-QD605 had similar fluorescent property to free QD, and the fluorescence intensity-time curves showed no significant intensity change during 1h of continuous illumination at 488nm. The fluorescence images demonstrated that ND-1-QD605 was able to specifically bind and image colorectal cancer cells which expressed large external antigens (LEA). Conclusion The results indicate that the QD-linked conjugate can be used to detect cancer cells owing to its ability of targeted binding and imaging. This is a novel approach for studying targeted imaging of colorectal cancer in vivo and clinical diagnosis.%目的 制备抗人大肠癌单克隆抗体ND-1的量子点荧光探针,实现对大肠癌细胞的靶向成像.方法 采用共价偶联方法,以1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(EDC)和N-羟基硫代琥珀酰亚胺(NHS)为缩合剂,通过在反应体系中加入不同摩尔比例的单克隆抗体ND-1和游离量子点QD605进行条件优化,制备偶联产物ND-1-QD605荧光探针;利用荧光光谱扫描技术对ND-1-QD605进行光学特性表征,并检测其抗光漂白能力;利用免疫荧光方法检测ND-1-QD605对大肠癌细胞的靶向结合能力.结果 在量子点QD605与单克隆抗体ND-1摩尔比1:40条件下,可实现二者的高

  15. Quantitative Assessment of Antibody Internalization with Novel Monoclonal Antibodies against Alexa Fluorophores

    OpenAIRE

    Liao-Chan, Sindy; Daine-Matsuoka, Barbara; Heald, Nathan; Wong, Tiffany; Lin, Tracey; Cai, Allen G.; Lai, Michelle; D’Alessio, Joseph A.; Theunissen, Jan-Willem

    2015-01-01

    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 inte...

  16. Production and characterization of antibody against aflatoxin Q1.

    OpenAIRE

    Fan, T. S.; Zhang, G S; Chu, F. S.

    1984-01-01

    Antibodies against aflatoxin Q1 (AFQ1) were obtained from rabbits after immunization of either AFQ1-hemisuccinate or AFQ2a conjugated to bovine serum albumin. Both radioimmunoassay and enzyme-linked immunosorbent assaY (ELISA) were used for the determination of antibody titers and specificities. Antibodies obtained from rabbits after immunization with AFQ1-hemisuccinate-bovine serum albumin had the highest affinity to aflatoxin B1, whereas antibodies obtained from rabbits after immunization w...

  17. Antibody binding to p-Si using LANL SAM chemistry

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, Aaron S [Los Alamos National Laboratory

    2010-12-06

    This NMSBA-sponsored project involves the attachment of antibodies to polymeric silicon (p-Si) surfaces, with the ultimate goal of attaching antibodies to nanowires for Vista Therapeutics, Inc. (Santa Fe, NM). This presentation describes the functionalization of p-Si surfaces. the activation of terminal carboxylates on these surfaces, the conjugation of antibodies, and the analyses undertaken at each step. The results of this work show that antibody conjugation is possible on p-Si coatings using the well-known EDC/NHS activation chemistry.

  18. Highly sensitive electroluminescence immunoassay for Hg(II) ions based on the use of CdSe quantum dots, the methylmercury-6-mercaptonicotinic acid-ovalbumin conjugate, and a specific monoclonal antibody

    International Nuclear Information System (INIS)

    We have designed a rapid and ultrasensitive electrochemiluminescent (ECL) competitive immunoassay for the determination of mercury(II) ion. It is based on the use of CdSe quantum dots (QDs), methylmercury-6-mercaptonicotinic acid-ovalbumin as coating antigen and specific monoclonal antibodies (mAbs) against Hg(II). The latter is quite selective for Hg(II). The coating antigen was immobilized on the surface of a gold electrode via reaction between the functional groups of cysteamine and glutaraldehyde. The mercury(II) ions in a sample and the coating antigen compete for binding sites of QD-labeled monoclonal antibody which binds specifically to Hg(II) ions. The ECL of the system decreases with increasing concentration of Hg(II) because less QD-labeled mAbs are present on the surface of the electrode. Under optimal conditions, the decrease of ECL intensity is linearly related to the logarithm of the Hg(II) concentration in the range from 0.02 to 100 ng mL−1, with a detection limit of 6.2 pg mL−1. As far as we know, this is the first report on an ECL immunoassay for Hg(II) based on a specific monoclonal antibody. The favorable results obtained when this method was applied to real samples indicate that this detection scheme can widely enlarge the applicability of detecting heavy metal ions by exploiting the ECL of QDs for immunoassays. (author)

  19. Preparation and testing of a Haemophilus influenzae Type b/Hepatitis B surface antigen conjugate vaccine.

    Science.gov (United States)

    An, So Jung; Woo, Joo Sung; Chae, Myung Hwa; Kothari, Sudeep; Carbis, Rodney

    2015-03-24

    The majority of conjugate vaccines focus on inducing an antibody response to the polysaccharide antigen and the carrier protein is present primarily to induce a T-cell dependent response. In this study conjugates consisting of poly(ribosylribitolphosphate) (PRP) purified from Haemophilus influenzae Type b bound to Hepatitis B virus surface antigen (HBsAg) virus like particles were prepared with the aim of inducing an antibody response to not only the PRP but also the HBsAg. A conjugate consisting of PRP bound to HBsAg via an adipic acid dihydrazide (ADH) spacer induced strong IgG antibodies to both the PRP and HBsAg. When conjugation was performed without the ADH spacer the induction of an anti-PRP response was equivalent to that seen by conjugate with the ADH spacer, however, a negligible anti-HBsAg response was induced. For comparison, PRP was conjugated to diphtheria toxoid (DT) and Vi polysaccharide purified from Salmonella Typhi conjugated to HBsAg both using an ADH spacer. The PRPAH-DT conjugate induced strong anti-PRP and anti-DT responses, the Vi-AHHBsAg conjugate induced a good anti-HBsAg response but not as strong as that induced by the PRPAH-HBsAg conjugate. This study demonstrated that in mice it was possible to induce robust antibody responses to both polysaccharide and carrier protein provided the conjugate has certain physico-chemical properties. A PRPAH-HBsAg conjugate with the capacity to induce anti-PRP and anti-HBsAg responses could be incorporated into a multivalent pediatric vaccine and simplify formulation of such a vaccine.

  20. ANTITUMOR EFFECTS OF MONOCLONAL ANTIBODY FAB′ FRAGMENT CONTAINING IMMUNOCONJUGATES

    Institute of Scientific and Technical Information of China (English)

    刘小云; 甄永苏

    2002-01-01

    Objective.Using monoclonal antibody (mAb) Fab′ fragment to develop mAb immunoconjugates for cancer. Methods.Fab′ fragment of mAb 3A5 was prepared by digestion of the antibody with pepsin and then reduced by dithiothreitol (DTT),while Fab′ fragment of mAb 3D6 was obtained by digestion of the antibody with ficin and subsequently reduced by β mercaptoethanol.The conjugation between Fab′ fragment and pingyangmycin (PYM),an antitumor antibiotic,was mediated by dextran T 40.Immunoreactivity of Fab′ PYM conjugates with cancer cells was determined by ELISA,and the cytotoxicity of those conjugates to cancer cells was determined by clonogenic assay.Antitumor effects of the Fab′ PYM conjugates were evaluated by subcutaneously transplanted tumors in mice. Results.The molecular weight of Fab′ fragment was approximately 53 kD,while the average molecular weight of Fab′ PYM conjugate was 170 kD.The Fab′ PYM conjugates showed immunoreactivity with antigen relevant cancer cells and selective cytotoxicity against target cells.Administered intravenously,Fab′ PYM conjugates were more effective against the growth of tumors in mice than free PYM and PYM conjugated with intact mAb. Conclusion.Fab′ PYM conjugate may be capable of targeting cancer cells and effectively inhibiting tumor growth,suggesting its therapeutic potential in cancer treatment.

  1. Radioimmunotherapy with engineered antibody fragments

    International Nuclear Information System (INIS)

    Authors have developed and begun evaluating radiometal-chelated (213Bi) engineered antibody fragments as radioimmunotherapy agents that target the HER2/neu (c-erbB-2) antigen. The diabody format was found to have 40-fold greater affinity for HER2/neu and to be associated with significantly greater tumor localization than is achieved with scFv molecule. It is shown that short-lived isotopes like 213Bi would be most effective when used in conjunction with antibodies that targeted diffuse malignancies (leukemia or lymphoma) or when used for very rapid pretargeted radioimmunotherapy application in which the radioisotope is conjugated to a very small ligand

  2. Segmented conjugated polymers

    Indian Academy of Sciences (India)

    G Padmanaban; S Ramakrishnan

    2003-08-01

    Segmented conjugated polymers, wherein the conjugation is randomly truncated by varying lengths of non-conjugated segments, form an interesting class of polymers as they not only represent systems of varying stiffness, but also ones where the backbone can be construed as being made up of chromophores of varying excitation energies. The latter feature, especially when the chromophores are fluorescent, like in MEHPPV, makes these systems particularly interesting from the photophysics point of view. Segmented MEHPPV- samples, where x represents the mole fraction of conjugated segments, were prepared by a novel approach that utilizes a suitable precursor wherein selective elimination of one of the two eliminatable groups is affected; the uneliminated units serve as conjugation truncations. Control of the composition x of the precursor therefore permits one to prepare segmented MEHPPV- samples with varying levels of conjugation (elimination). Using fluorescence spectroscopy, we have seen that even in single isolated polymer chains, energy migration from the shorter (higher energy) chromophores to longer (lower energy) ones occurs – the extent of which depends on the level of conjugation. Further, by varying the solvent composition, it is seen that the extent of energy transfer and the formation of poorly emissive inter-chromophore excitons are greatly enhanced with increasing amounts of non-solvent. A typical S-shaped curve represents the variation of emission yields as a function of composition suggestive of a cooperative collapse of the polymer coil, reminiscent of conformational transitions seen in biological macromolecules.

  3. Antibody Engineering & Therapeutics, the annual meeting of The Antibody Society December 7-10, 2015, San Diego, CA, USA.

    Science.gov (United States)

    Pauthner, Matthias; Yeung, Jenny; Ullman, Chris; Bakker, Joost; Wurch, Thierry; Reichert, Janice M; Lund-Johansen, Fridtjof; Bradbury, Andrew R M; Carter, Paul J; Melis, Joost P M

    2016-01-01

    The 26th Antibody Engineering & Therapeutics meeting, the annual meeting of The Antibody Society united over 800 participants from all over the world in San Diego from 6-10 December 2015. The latest innovations and advances in antibody research and development were discussed, covering a myriad of antibody-related topics by more than 100 speakers, who were carefully selected by The Antibody Society. As a prelude, attendees could join the pre-conference training course focusing, among others, on the engineering and enhancement of antibodies and antibody-like scaffolds, bispecific antibody engineering and adaptation to generate chimeric antigen receptor constructs. The main event covered 4 d of scientific sessions that included antibody effector functions, reproducibility of research and diagnostic antibodies, new developments in antibody-drug conjugates (ADCs), preclinical and clinical ADC data, new technologies and applications for bispecific antibodies, antibody therapeutics for non-cancer and orphan indications, antibodies to harness the cellular immune system, building comprehensive IgVH-gene repertoires through discovering, confirming and cataloging new germline IgVH genes, and overcoming resistance to clinical immunotherapy. The Antibody Society's special session focused on "Antibodies to watch" in 2016. Another special session put the spotlight on the limitations of the new definitions for the assignment of antibody international nonproprietary names introduced by the World Health Organization. The convention concluded with workshops on computational antibody design and on the promise and challenges of using next-generation sequencing for antibody discovery and engineering from synthetic and in vivo libraries. PMID:26909869

  4. Nanobody-photosensitizer conjugates for targeted photodynamic therapy

    NARCIS (Netherlands)

    Heukers, Raimond; van Bergen en Henegouwen, P; Santos Oliveira, Sabrina

    2014-01-01

    Photodynamic therapy (PDT) induces cell death through light activation of a photosensitizer (PS). Targeted delivery of PS via monoclonal antibodies has improved tumor selectivity. However, these conjugates have long half-lives, leading to relatively long photosensitivity in patients. In an attempt t

  5. Meningococcal conjugate vaccines: optimizing global impact

    Directory of Open Access Journals (Sweden)

    Terranella A

    2011-09-01

    Full Text Available Andrew Terranella1,2, Amanda Cohn2, Thomas Clark2 1Epidemic Intelligence Service, Division of Applied Sciences, Scientific Education and Professional Development Program Office, 2Meningitis and Vaccine Preventable Diseases Branch, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA Abstract: Meningococcal conjugate vaccines have several advantages over polysaccharide vaccines, including the ability to induce greater antibody persistence, avidity, immunologic memory, and herd immunity. Since 1999, meningococcal conjugate vaccine programs have been established across the globe. Many of these vaccination programs have resulted in significant decline in meningococcal disease in several countries. Recent introduction of serogroup A conjugate vaccine in Africa offers the potential to eliminate meningococcal disease as a public health problem in Africa. However, the duration of immune response and the development of widespread herd immunity in the population remain important questions for meningococcal vaccine programs. Because of the unique epidemiology of meningococcal disease around the world, the optimal vaccination strategy for long-term disease prevention will vary by country. Keywords: conjugate vaccine, meningitis, meningococcal vaccine, meningococcal disease

  6. Polymers for Protein Conjugation

    Directory of Open Access Journals (Sweden)

    Gianfranco Pasut

    2014-01-01

    Full Text Available Polyethylene glycol (PEG at the moment is considered the leading polymer for protein conjugation in view of its unique properties, as well as to its low toxicity in humans, qualities which have been confirmed by its extensive use in clinical practice. Other polymers that are safe, biodegradable and custom-designed have, nevertheless, also been investigated as potential candidates for protein conjugation. This review will focus on natural polymers and synthetic linear polymers that have been used for protein delivery and the results associated with their use. Genetic fusion approaches for the preparation of protein-polypeptide conjugates will be also reviewed and compared with the best known chemical conjugation ones.

  7. π-Clamp-mediated cysteine conjugation

    Science.gov (United States)

    Zhang, Chi; Welborn, Matthew; Zhu, Tianyu; Yang, Nicole J.; Santos, Michael S.; van Voorhis, Troy; Pentelute, Bradley L.

    2016-02-01

    Site-selective functionalization of complex molecules is one of the most significant challenges in chemistry. Typically, protecting groups or catalysts must be used to enable the selective modification of one site among many that are similarly reactive, and general strategies that selectively tune the local chemical environment around a target site are rare. Here, we show a four-amino-acid sequence (Phe-Cys-Pro-Phe), which we call the ‘π-clamp’, that tunes the reactivity of its cysteine thiol for site-selective conjugation with perfluoroaromatic reagents. We use the π-clamp to selectively modify one cysteine site in proteins containing multiple endogenous cysteine residues. These examples include antibodies and cysteine-based enzymes that would be difficult to modify selectively using standard cysteine-based methods. Antibodies modified using the π-clamp retained binding affinity to their targets, enabling the synthesis of site-specific antibody-drug conjugates for selective killing of HER2-positive breast cancer cells. The π-clamp is an unexpected approach to mediate site-selective chemistry and provides new avenues to modify biomolecules for research and therapeutics.

  8. Evaluation of a competitive enzyme immunoassay for detection of Coxiella burnetii antibody in animal sera.

    OpenAIRE

    Soliman, A.K.; Botros, B A; Watts, D M

    1992-01-01

    A competitive enzyme immunoassay (CEIA) was established and compared with other serological techniques for detecting Coxiella burnetii antibody in camels, goats, and sheep. This technique was evaluated because a conjugated anti-camel immunoglobulin was not available to serve as a direct signal for the demonstration of antigen-antibody reaction. A C. burnetii antibody-positive human serum and a peroxidase-conjugated anti-human immunoglobulin G were used as an indicator system competing against...

  9. Inhibition of the growth of hepatoma and hepatic metastasis by pingyangmycin conjugated with Fab′fragment of monoclonal antibody%应用平阳霉素与单克隆抗体Fab′片段偶联物抑制肝癌生长与肿瘤肝转移

    Institute of Scientific and Technical Information of China (English)

    刘小云; 尚伯扬; 刘秀均; 甄永苏

    2001-01-01

    目的 研制一种具有抑制肝癌生长和肿瘤肝转移作用的单克隆抗体Fab′片段与平阳霉素(PYM)偶联物。方法 以胃蛋白酶酶解结合二巯基苏糖醇(DTT)还原法制备单抗Fab′片段;以葡聚糖T-40(dextran T-40)为中介体制备Fab′片段和PYM偶联物Fab′-PYM;用间接ELISA法、2,3,5-氯化三苯基四氮唑 (TTC)抑菌效价检测法,克隆形成法及动物移植性肿瘤模型测定Fab′-PYM偶联物在体外的生物学活性与在小鼠体内的抗肿瘤作用。结果 Fab′-PYM与BEL-7402、肝癌22(H22)及HT-29细胞呈免疫学阳性反应, 但与KB细胞无反应;偶联物中PYM的抑菌活性为游离PYM的15%;Fab′-PYM对体外培养的BEL-7402细胞、HT-29细胞和KB细胞的50%抑制浓度(IC50)分别为0.02、0.08及0.50 μmol/L;静脉注射10 mg/kg Fab′-PYM偶联物和PYM皮下接种对小鼠肝癌22的抑瘤率分别为86%和62%;腹腔注射,10 mg/kg Fab′-PYM偶联物和PYM对小鼠脾内接种的结肠癌26肝转移的抑制率分别为91%和62%。结论 Fab′-PYM偶联物具有比游离平阳霉素更强的抑制肝癌生长与肿瘤肝转移作用。%Objective To develop an immunoconjugate with inhibitory effect on the growth of hepatoma and hepatic metastasis of tumor by linking Fab′fragment of monoclona antibody (McAb) to pingyangmycin (PYM). Methods Monoclonal antibody Fab′ fragment was obtained by enzymolysis with pepsin and DTT reduction. Linking of the Fab′ fragment and PYM was mediated by dextran T40. Indirect ELISA, TTC bacteriostatic titer test, clonogenic assay and animal models transplanted with tumor were used to assess the biological activities and antitumor effects of Fab′PYM conjugate within and without the bodies of 10 mice. Results The Fab′PYM conjugate showed immunoreactivity to BEL7402, hepatoma 22 (H22) and HT29 cells, but no reaction to KB cells. The bacteriostatic activity of PYM in the conjugate was 15

  10. Synthesis and antimicrobial activity of gold nanoparticle conjugates with cefotaxime

    Science.gov (United States)

    Titanova, Elena O.; Burygin, Gennady L.

    2016-04-01

    Gold nanoparticles (GNPs) have attracted significant interest as a novel platform for various applications to nanobiotechnology and biomedicine. The conjugates of GNPs with antibiotics and antibodies were also used for selective photothermal killing of protozoa and bacteria. Also the conjugates of some antibiotics with GNPs decreased the number of bacterial growing cells. In this work was made the procedure optimization for conjugation of cefotaxime (a third-generation cephalosporin antibiotic) with GNPs (15 nm) and we examined the antimicrobial properties of this conjugate to bacteria culture of E. coli K-12. Addition of cefotaxime solution to colloidal gold does not change their color and extinction spectrum. For physiologically active concentration of cefotaxime (3 μg/mL), it was shown that the optimum pH for the conjugation was more than 9.5. A partial aggregation of the GNPs in saline medium was observed at pH 6.5-7.5. The optimum concentration of K2CO3 for conjugation cefotaxime with GNPs-15 was 5 mM. The optimum concentration of cefotaxime was at 0.36 μg/mL. We found the inhibition of the growth of E. coli K12 upon application cefotaxime-GNP conjugates.

  11. Design and Synthesis of 6α-Corticosteroid Haptens and Their Bovine Serum Albumin (BSA) Conjugates

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    The site of attachment of protein carrier to corticosteroids has great influence on the specificity of produced antibody. In order to obtain highly specific and accurate antibodies for bioimmunoassay determination of cortisol, different tether lengths of 6α-corticosteroid haptens and their BSA conjugates were designed and synthesized.

  12. Qualidade conjugal: mapeando conceitos

    OpenAIRE

    Clarisse Mosmann; Adriana Wagner; Terezinha Féres-Carneiro

    2006-01-01

    Apesar da ampla utilização do conceito de qualidade conjugal, identifica-se falta de clareza conceitual acerca das variáveis que o compõem. Esse artigo apresenta revisão da literatura na área com o objetivo de mapear o conceito de qualidade conjugal. Foram analisadas sete principais teorias sobre o tema: Troca Social, Comportamental, Apego, Teoria da Crise, Interacionismo Simbólico. Pelos postulados propostos nas diferentes teorias, podem-se identificar três grupos de variáveis fundamentais n...

  13. High-conjugation-efficiency aqueous CdSe quantum dots.

    Science.gov (United States)

    Au, Giang H T; Shih, Wan Y; Shih, Wei-Heng

    2013-11-12

    Quantum dots (QDs) are photoluminescent nanoparticles that can be directly or indirectly coupled with a receptor such as an antibody to specifically image a target biomolecule such as an antigen. Recent studies have shown that QDs can be directly made at room temperature and in an aqueous environment (AQDs) with 3-mercaptopropionic acid (MPA) as the capping ligand without solvent and ligand exchange typically required by QDs made by the organic solvent routes (OQDs). In this study, we have synthesized CdSe AQDs and compared their conjugation efficiency and imaging efficacy with commercial carboxylated OQDs in HT29 colon cancer cells using a primary antibody-biotinylated secondary antibody-streptavidin (SA) sandwich. We showed that the best imaging condition for AQDs occurred when one AQD was bound with 3 ± 0.3 SA with a nominal SA/AQD ratio of 4 corresponding to an SA conjugation efficiency of 75 ± 7.5%. In comparison, for commercial CdSe-ZnS OQDs to achieve 2.7 ± 0.4 bound SAs per OQD for comparable imaging efficacy a nominal SA/OQD ratio of 80 was needed corresponding to an SA conjugation efficiency of 3.4 ± 0.5% for CdSe-ZnS OQDs. The more than 10 times better SA conjugation efficiency of the CdSe AQDs as compared to that of the CdSe-ZnS OQDs was attributed to more capping molecules on the AQD surface as a result of the direct aqueous synthesis. More capping molecules on the AQD surface also allowed the SA-AQD conjugate to be stable in cell culture medium for more than three days without losing their staining capability in a flowing cell culture medium. In contrast, SA-OQD conjugates aggregated in cell culture medium and in phosphate buffer saline solution over time. PMID:24151632

  14. Stabilized polyacrylic saccharide protein conjugates

    Science.gov (United States)

    Callstrom, M.R.; Bednarski, M.D.; Gruber, P.R.

    1996-02-20

    This invention is directed to water soluble protein polymer conjugates which are stable in hostile environments. The conjugate comprises a protein which is linked to an acrylic polymer at multiple points through saccharide linker groups. 16 figs.

  15. Comparison of in vitro immunogenicity, tolerogenicity and mitogenicity of dinitrophenyl-levan conjugates with varying epitope density.

    Science.gov (United States)

    Desaymard, C; Ivanyi, L

    1976-01-01

    The mitogenicity, immunogenicity and tolerogenicity of various DNP-levan (DNP-LE) conjugates were compared using in vitro methods. Anti-DNP antibody synthesis induced by DNP-LE conjugates was related to the epitope density of DNP, BUT WAS NOT AFFECTED BY Macrophage dependent and was not influenced by the degree of hapten conjugation. These results imply that mitogenicity of an antigen is not necessarily related to the specific triggering of B cells. PMID:58831

  16. Oligonucleotide conjugates - Candidates for gene silencing therapeutics.

    Science.gov (United States)

    Gooding, Matt; Malhotra, Meenakshi; Evans, James C; Darcy, Raphael; O'Driscoll, Caitriona M

    2016-10-01

    The potential therapeutic and diagnostic applications of oligonucleotides (ONs) have attracted great attention in recent years. The capability of ONs to selectively inhibit target genes through antisense and RNA interference mechanisms, without causing un-intended sideeffects has led them to be investigated for various biomedical applications, especially for the treatment of viral diseases and cancer. In recent years, many researchers have focused on enhancing the stability and target specificity of ONs by encapsulating/complexing them with polymers or lipid chains to formulate nanoparticles/nanocomplexes/micelles. Also, chemical modification of nucleic acids has emerged as an alternative to impart stability to ONs against nucleases and other degrading enzymes and proteins found in blood. In addition to chemically modifying the nucleic acids directly, another strategy that has emerged, involves conjugating polymers/peptide/aptamers/antibodies/proteins, preferably to the sense strand (3'end) of siRNAs. Conjugation to the siRNA not only enhances the stability and targeting specificity of the siRNA, but also allows for the development of self-administering siRNA formulations, with a much smaller size than what is usually observed for nanoparticle (∼200nm). This review concentrates mainly on approaches and studies involving ON-conjugates for biomedical applications. PMID:27521696

  17. DNA-cell conjugates

    Energy Technology Data Exchange (ETDEWEB)

    Hsiao, Shih-Chia; Francis, Matthew B.; Bertozzi, Carolyn; Mathies, Richard; Chandra, Ravi; Douglas, Erik; Twite, Amy; Toriello, Nicholas; Onoe, Hiroaki

    2016-05-03

    The present invention provides conjugates of DNA and cells by linking the DNA to a native functional group on the cell surface. The cells can be without cell walls or can have cell walls. The modified cells can be linked to a substrate surface and used in assay or bioreactors.

  18. Conjugation in "Escherichia coli"

    Science.gov (United States)

    Phornphisutthimas, Somkiat; Thamchaipenet, Arinthip; Panijpan, Bhinyo

    2007-01-01

    Bacterial conjugation is a genetic transfer that involves cell-to-cell between donor and recipient cells. With the current method used to teach students in genetic courses at the undergraduate level, the transconjugants are identified using bacterial physiology and/or antibiotic resistance. Using physiology, however, is difficult for both…

  19. Protein carriers of conjugate vaccines: characteristics, development, and clinical trials.

    Science.gov (United States)

    Pichichero, Michael E

    2013-12-01

    The immunogenicity of polysaccharides as human vaccines was enhanced by coupling to protein carriers. Conjugation transformed the T cell-independent polysaccharide vaccines of the past to T cell-dependent antigenic vaccines that were much more immunogenic and launched a renaissance in vaccinology. This review discusses the conjugate vaccines for prevention of infections caused by Hemophilus influenzae type b, Streptococcus pneumoniae, and Neisseria meningitidis. Specifically, the characteristics of the proteins used in the construction of the vaccines including CRM, tetanus toxoid, diphtheria toxoid, Neisseria meningitidis outer membrane complex, and Hemophilus influenzae protein D are discussed. The studies that established differences among and key features of conjugate vaccines including immunologic memory induction, reduction of nasopharyngeal colonization and herd immunity, and antibody avidity and avidity maturation are presented. Studies of dose, schedule, response to boosters, of single protein carriers with single and multiple polysaccharides, of multiple protein carriers with multiple polysaccharides and conjugate vaccines administered concurrently with other vaccines are discussed along with undesirable consequences of conjugate vaccines. The clear benefits of conjugate vaccines in improving the protective responses of the immature immune systems of young infants and the senescent immune systems of the elderly have been made clear and opened the way to development of additional vaccines using this technology for future vaccine products. PMID:23955057

  20. Specific fluorescein-labeled antibodies to bovine viral diarrhea virus prepared from sera of rabbits immunized with purified virus.

    OpenAIRE

    Hart, R. A.; Rhodes, M B

    1980-01-01

    Specific fluorescein-labeled antibody conjugates to three strains of bovine virus diarrhea virus were prepared from hyperimmune rabbit sera. Viruses used to hyperimmunize the rabbits were purified by four different procedures. Conjugates were comparable in quality and specificity to conjugates prepared from serum of a calf hyperimmunized to bovine virus diarrhea virus in our laboratory. The latter conjugate was tested by Biologics Laboratories, National Veterinary Services, U.S.D.A., Ames, Iowa.

  1. Carrier induced epitopic suppression of antibody responses induced by virus-like particles is a dynamic phenomenon caused by carrier-specific antibodies.

    Science.gov (United States)

    Jegerlehner, Andrea; Wiesel, Melanie; Dietmeier, Klaus; Zabel, Franziska; Gatto, Dominique; Saudan, Philippe; Bachmann, Martin F

    2010-07-26

    Pre-existing immunity against vaccine carrier proteins has been reported to inhibit the immune response against antigens conjugated to the same carrier by a process termed carrier induced epitopic suppression (CIES). Hence understanding the phenomenon of CIES is of major importance for the development of conjugate vaccines. Virus-like particles (VLPs) are a novel class of potent immunological carriers which have been successfully used to enhance the antibody response to virtually any conjugated antigen. In the present study we investigated the impact of a pre-existing VLP-specific immune response on the development of antibody responses against a conjugated model peptide after primary, secondary and tertiary immunization. Although VLP-specific immune responses led to reduced peptide-specific antibody titers, we showed that CIES against peptide-VLP conjugates could be overcome by high coupling densities, repeated injections and/or higher doses of conjugate vaccine. Furthermore we dissected VLP-specific immunity by adoptively transferring VLP-specific antibodies, B-cells or T(helper) cells separately into naïve mice and found that the observed CIES against peptide-VLP conjugates was mainly mediated by carrier-specific antibodies.

  2. Studies on Hypersensitivity—I. Delayed and Arthus-type Skin Reactivity to Protein Conjugates in Guinea Pigs

    Science.gov (United States)

    Benacerraf, B.; Gell, P. G. H.

    1959-01-01

    A study has been made of the immunization of guinea pigs with proteins conjugated with picryl, acetyl and ethoxymethylene-phenyloxazolone groups. Immunization by means of complexes of these substances with anti-protein and anti-hapten antisera have also been studied. Antibody production, anaphylactic and Arthus-type sensitivity and delayed skin sensitivity to the conjugates, to the carrier proteins and to unrelated proteins carrying the same haptenic group have been investigated. Immunization with conjugates is found to be followed by the appearance of delayed hypersensitivity to the protein `carrier' in the absence of detectable antibodies against it, although antibodies are produced at that time against the haptenic group itself. Delayed hypersensitivity to the haptenic group has not been detected at any time: blocking it with specific antibody does not lead to the appearance of delayed sensitivity, but merely suppresses antibody formation against that group. Pure delayed sensitivity has been produced against gelatin, both alone and as a conjugate with picryl. Conjugates with homologous serum proteins are shown to provoke only Arthus-type sensitivity and antibody against the haptenic group. These findings are discussed in view of the light they may throw upon the relation of delayed hypersensitivity to antibody production and upon the process of immunization. PMID:13640680

  3. Preparation and biological evaluation of radiolabelled antibodies with selected carbohydrate modifications

    Energy Technology Data Exchange (ETDEWEB)

    Qi, P. (Alberta Univ., Edmonton, AB (Canada). Faculty of Pharmacy); Sykes, T.R.; Noujaim, A.A. (Alberta Univ., Edmonton, AB (Canada). Faculty of Pharmacy Biomira Inc., Edmonton, AB (Canada)); Koganty, R.R.; Selvaraj, S. (Biomira Inc., Edmonton, AB (Canada))

    1993-05-01

    Two carbohydrates, N-acetylgalactosamine (GalNAc) and galactose-[beta]-1,3-GalNAc have been attached to human IgG (hIgG) by a novel linking reagent, hexafluoroglutaric acid dimethyl ester. Fluorine-19 NMR signals were used for the determination of the conjugation ratio. A third carbohydrate, sialic acid, was conjugated via reductive amination and the conjugation ratio determined by a resorcinol assay. The biological behaviour of these radiodinated antibodies with carbohydrate modification in normal mice indicates an enhanced liver uptake at 15 min post-injection with an associated change in circulating blood levels occurs for the galactose-based hIgG preparations. However, no significant differences in the biodistribution were observed for the sialic acid conjugate. These studies confirm the potential of carbohydrate-antibody conjugation for modifying the behaviour of antibodies in immunoscintigraphy and radioimmunotherapy. (author).

  4. Conjugated polymer nanoparticles

    Science.gov (United States)

    Tuncel, Dönüs; Demir, Hilmi Volkan

    2010-04-01

    Conjugated polymer nanoparticles are highly versatile nano-structured materials that can potentially find applications in various areas such as optoelectronics, photonics, bio-imaging, bio-sensing and nanomedicine. Their straightforward synthesis in desired sizes and properties, biocompatibility and non-toxicity make these materials highly attractive for the aforementioned applications. This feature article reviews the recent developments in the synthesis, characterization, properties and application of these exciting nanostructured materials.

  5. Anti- (conjugate) linearity

    Science.gov (United States)

    Uhlmann, Armin

    2016-03-01

    This is an introduction to antilinear operators. In following Wigner the terminus antilinear is used as it is standard in Physics. Mathematicians prefer to say conjugate linear. By restricting to finite-dimensional complex-linear spaces, the exposition becomes elementary in the functional analytic sense. Nevertheless it shows the amazing differences to the linear case. Basics of antilinearity is explained in sects. 2, 3, 4, 7 and in sect. 1.2: Spectrum, canonical Hermitian form, antilinear rank one and two operators, the Hermitian adjoint, classification of antilinear normal operators, (skew) conjugations, involutions, and acq-lines, the antilinear counterparts of 1-parameter operator groups. Applications include the representation of the Lagrangian Grassmannian by conjugations, its covering by acq-lines. As well as results on equivalence relations. After remembering elementary Tomita-Takesaki theory, antilinear maps, associated to a vector of a two-partite quantum system, are defined. By allowing to write modular objects as twisted products of pairs of them, they open some new ways to express EPR and teleportation tasks. The appendix presents a look onto the rich structure of antilinear operator spaces.

  6. Referencing cross-reactivity of detection antibodies for protein array experiments [version 1; referees: 1 approved, 2 approved with reservations

    OpenAIRE

    Darragh Lemass; Richard O'Kennedy; Kijanka, Gregor S.

    2016-01-01

    Protein arrays are frequently used to profile antibody repertoires in humans and animals. High-throughput protein array characterisation of complex antibody repertoires requires a platform-dependent, lot-to-lot validation of secondary detection antibodies. This article details the validation of an affinity-isolated anti-chicken IgY antibody produced in rabbit and a goat anti-rabbit IgG antibody conjugated with alkaline phosphatase using protein arrays consisting of 7,390 distinct human protei...

  7. Liposomes surface conjugated with human hemoglobin target delivery to macrophages

    OpenAIRE

    Zhang, Ning; Palmer, Andre F.

    2011-01-01

    Current strategies to deliver therapeutic molecules to specific cell and tissue types rely on conjugation of antibodies and other targeting ligands directly to the therapeutic molecule itself or its carrier. This work describes a novel strategy to deliver therapeutic molecules into macrophages that takes advantage of the native hemoglobin (Hb) scavenging activity of plasma haptoglobin (Hp) and the subsequent uptake of the Hb-Hp complex into macrophages via CD163 receptor mediated endocytosis....

  8. Radiolabeled Cetuximab Conjugates for EGFR Targeted Cancer Diagnostics and Therapy

    OpenAIRE

    Wiebke Sihver; Jens Pietzsch; Mechthild Krause; Michael Baumann; Jörg Steinbach; Hans-Jürgen Pietzsch

    2014-01-01

    The epidermal growth factor receptor (EGFR) has evolved over years into a main molecular target for the treatment of different cancer entities. In this regard, the anti-EGFR antibody cetuximab has been approved alone or in combination with: (a) chemotherapy for treatment of colorectal and head and neck squamous cell carcinoma and (b) with external radiotherapy for treatment of head and neck squamous cell carcinoma. The conjugation of radionuclides to cetuximab in combination with the specif...

  9. Theory of Digitized Conjugate Surface and Solution to Conjugate Surface

    Institute of Scientific and Technical Information of China (English)

    Xiao Lai-yuan; Liao Dao-xun; Yi Chuan-yun

    2004-01-01

    In order to meet the needs of designing and processing digitized surfaces, the method to spreading digitized surface has been proposed. The key technique is to solve the problem of digitized conjugate surface. In the paper, the digitized conjugate surface was theoretically investigated, and the solution of conjugate surface based on digitized surface was also studied. The digitized conjugate surface theory was then proposed, and applied to build the model of solving conjugate surface based on digitized surface. A corresponding algorithm was developed. This paper applies the software Conjugater-1.0 that is developed by ourselves to compute the digitized conjugate surfaces of the drum-tooth surface. This study provides theoretical and technical bases for analyzing engagement of digitized surface, simulation and numerical processing technique.

  10. Mouse Monoclonal Antibodies for Liver Cancer Research | NCI Technology Transfer Center | TTC

    Science.gov (United States)

    The National Cancer Institute Laboratory of Molecular Biology seeks parties for collaborative research to co-develop and commercialize antibody drug/toxin conjugates as liver cancer therapy and diagnostics.

  11. Synthetic Antibodies for Reversible Cell Recognition

    Science.gov (United States)

    Zhou, Jing Zhou

    2011-12-01

    Antibody-mediated cell recognition plays a critical role in various biological and biomedical applications. However, strong antibody-cell interactions can lead to the difficulty of separating antibodies from the bound cells in a simple and non-destructive manner, which is often necessary to numerous applications such as cell sorting or separation. Thus, this thesis research is aimed to create an antibody-like nanomaterial with the function of reversible cell recognition It was hypothesized that nucleic acid aptamer and dendrimer could be used as fundamental structural components to develop an antibody-like nanomaterial. The aptamer functions as the binding site of an antibody; the dendrimer is used as a robust, defined nano-scaffold to support the aptamer and to carry small molecules (e.g., fluorophores). To test this hypothesis, a novel method was first developed to discover the essential nucleotides of full-length aptamers to mimic the binding sites of antibodies. The essential nucleotides were further conjugated with a dendrimer to synthesize a monovalent aptamer-dendrimer nanomaterial. The results clearly showed that the essential nucleotides could maintain high affinity and specificity after tethered on dendrimer surface. To further test the hypothesis that antibody-like nanomaterials can be rationally designed to acquire the capability of reversible cell recognition, an aptamer that was selected at 0 °C was used as a model to synthesize a "Y-shaped" nanomaterial by conjugating two aptamers to the same dendrimer. The results showed that the nanomaterial-cell interaction could be affected by the distance between two binding aptamers. In addition, the "Y-shaped" antibody-like nanomaterial could bind target cells more strongly than its monovalent control. Importantly, the strong cell-nanomaterial interaction could be rapidly reversed when the temperature was shifted from 0 °C to 37 °C. In summary, we developed a synthetic antibody that can not only mimic the

  12. Endotoxin contamination of enzyme conjugates used in enzyme-linked immunosorbent assays.

    OpenAIRE

    Bryant, R. E.; Chamovitz, B N; Morse, S A; Apicella, M A; Morthland, V H

    1983-01-01

    The specificity of the enzyme-linked immunosorbent assay(s) is thought to depend on the specificity of the antibody used in the assay system. Therefore, the association of broadly reactive antigens like endotoxin with enzyme conjugates or other enzyme-linked immunosorbent assay reagents has the potential of altering the specificity of reactions in the enzyme-linked immunosorbent assay. Using the Limulus amoebocyte lysate assay, we demonstrated that commercially prepared conjugates of goat ant...

  13. Antibody engineering & therapeutics, the annual meeting of the antibody society December 7–10, 2015, San Diego, CA, USA

    Science.gov (United States)

    Pauthner, Matthias; Yeung, Jenny; Ullman, Chris; Bakker, Joost; Wurch, Thierry; Reichert, Janice M.; Lund-Johansen, Fridtjof; Bradbury, Andrew R.M.; Carter, Paul J.; Melis, Joost P.M.

    2016-01-01

    ABSTRACT The 26th Antibody Engineering & Therapeutics meeting, the annual meeting of The Antibody Society united over 800 participants from all over the world in San Diego from 6–10 December 2015. The latest innovations and advances in antibody research and development were discussed, covering a myriad of antibody-related topics by more than 100 speakers, who were carefully selected by The Antibody Society. As a prelude, attendees could join the pre-conference training course focusing, among others, on the engineering and enhancement of antibodies and antibody-like scaffolds, bispecific antibody engineering and adaptation to generate chimeric antigen receptor constructs. The main event covered 4 d of scientific sessions that included antibody effector functions, reproducibility of research and diagnostic antibodies, new developments in antibody-drug conjugates (ADCs), preclinical and clinical ADC data, new technologies and applications for bispecific antibodies, antibody therapeutics for non-cancer and orphan indications, antibodies to harness the cellular immune system, building comprehensive IgVH-gene repertoires through discovering, confirming and cataloging new germline IgVH genes, and overcoming resistance to clinical immunotherapy. The Antibody Society's special session focused on “Antibodies to watch” in 2016. Another special session put the spotlight on the limitations of the new definitions for the assignment of antibody international nonproprietary names introduced by the World Health Organization. The convention concluded with workshops on computational antibody design and on the promise and challenges of using next-generation sequencing for antibody discovery and engineering from synthetic and in vivo libraries. PMID:26909869

  14. Biological characterization of cetuximab-conjugated gold nanoparticles in a tumor animal model

    Science.gov (United States)

    Kao, Hao-Wen; Lin, Yi-Yu; Chen, Chao-Cheng; Chi, Kwan-Hwa; Tien, Der-Chi; Hsia, Chien-Chung; Lin, Wuu-Jyh; Chen, Fu-Du; Lin, Ming-Hsien; Wang, Hsin-Ell

    2014-07-01

    Gold nanoparticles (AuNPs) are widely applied to the diagnosis and treatment of cancer and can be modified to contain target-specific ligands via gold-thiolate bonding. This study investigated the pharmacokinetics and microdistribution of antibody-mediated active targeting gold nanoparticles in mice with subcutaneous lung carcinoma. We conjugated AuNPs with cetuximab (C225), an antibody-targeting epidermal growth factor receptor (EGFR), and then labeled with In-111, which created EGFR-targeted AuNPs. In vitro studies showed that after a 2 h incubation, the uptake of C225-conjugated AuNPs in high EGFR-expression A549 cells was 14.9-fold higher than that of PEGylated AuNPs; furthermore, uptake was also higher at 3.8-fold when MCF7 cells with lower EGFR-expression were used. MicroSPECT/CT imaging and a biodistribution study conducted by using a A549 tumor xenograft mouse model provided evidence of elevated uptake of the C225-conjugated AuNPs into the tumor cells as a result of active targeting. Moreover, the microdistribution of PEGylated AuNPs revealed that a large portion of AuNPs remained in the tumor interstitium, whereas the C225-conjugated AuNPs displayed enhanced internalization via antibody-mediated endocytosis. Our findings suggest that the anti-EGFR antibody-conjugated AuNPs are likely to be a plausible nano-sized vehicle for drug delivery to EGFR-expressing tumors.

  15. A novel chemistry for conjugating pneumococcal polysaccharides to Luminex microspheres.

    Science.gov (United States)

    Schlottmann, Sonela A; Jain, Neil; Chirmule, Narendra; Esser, Mark T

    2006-02-20

    Here we describe a novel method to conjugate pneumococcal polysaccharides (PnPS) to Luminex microspheres for use in serological assays. 4-(4,6-dimethoxy[1,3,5]triazin-2-yl)-4-methyl-morpholinium (DMTMM) modification of PnPS and conjugation to carboxyl functional groups on Luminex microspheres (COOH-DMTMM method) was shown to be a reproducible chemistry that efficiently conjugated PnPS to Luminex microspheres without affecting the antigenicity of a broad set of PnPS. The COOH-DMTMM method was compared to three other methods for robustness, reproducibility and effect on PnPS antigenicity in a multiplexed assay format. The other methods examined included adsorption of the unmodified PnPS to Luminex microspheres, oxidation of the PnPS to conjugate them to amino-modified microspheres using carbodiimide chemistry and poly-l-lysine modification of the PnPS before conjugating to carboxy Luminex microspheres using carbodiimide chemistry. Of the four methods, the COOH-DMTMM chemistry was shown to be a robust methodology, producing stable PnPS coupled microspheres with a 4-log dynamic range and low cross-reactivity when used in a PnPS-specific IgG serology assay. This novel chemistry should be useful for developing serological assays to measure antibodies to polysaccharides for use in vaccine and epidemiology studies. PMID:16448665

  16. Monoclonal Antibodies to Plant Growth Regulators

    Science.gov (United States)

    Eberle, Joachim; Arnscheidt, Angelika; Klix, Dieter; Weiler, Elmar W.

    1986-01-01

    Four high affinity monoclonal antibodies, which recognize two plant growth regulators from the cytokinin group, namely trans-zeatin riboside and dihydrozeatin riboside and their derivatives are reported. Six hybridomas were produced from three independent fusions of Balb/c spleen cells with P3-NS1-Ag 4-1 (abbreviated NS1) or X63-Ag 8.653 (X63) myeloma cells. The mice had been hyperimmunized with zeatin riboside-bovine serum albumin conjugate or dihydrozeatin riboside-bovine serum albumin conjugate for 3 months. The hybridomas secrete antibodies of the IgG 1 or IgG 2b subclass and allow the detection of femtomole amounts of the free cytokinins, their ribosides, and ribotides in plant extracts. The use of these monoclonals in radio- and enzyme-linked immunosorbent assay is also discussed. PMID:16664848

  17. The influence of bio-conjugation on photoluminescence of CdSe/ZnS quantum dots

    Energy Technology Data Exchange (ETDEWEB)

    Torchynska, Tetyana V. [ESFM Instituto Politécnico Nacional, Av. Instituto Politécnico Nacional, México, D.F. 07738 (Mexico); Vorobiev, Yuri V. [Centro de Investigación y de Estudios Avanzados del IPN (CINVESTAV) Querétaro, Libramiento Norponiente 2000, Fracc. Real de Juriquilla, 76230 Querétaro (Mexico); Makhniy, Victor P. [Yuri Fedkovych Chernivtsi National University, 2 Kotsyubynsky Str., 58012 Chernivtsi (Ukraine); Horley, Paul P., E-mail: paul.horley@cimav.edu.mx [Centro de Investigación en Materiales Avanzados, S.C. (CIMAV), Chihuahua/Monterrey, 120 Avenida Miguel de Cervantes, 31109 Chihuahua (Mexico)

    2014-11-15

    We report a considerable blue shift in the luminescence spectra of CdSe/ZnS quantum dots conjugated to anti-interleukin-10 antibodies. This phenomenon can be explained theoretically by accounting for bio-conjugation as a process causing electrostatic interaction between a quantum dot and an antibody, which reduces effective volume of the dot core. To solve the Schrödinger equation for an exciton confined in the quantum dot, we use mirror boundary conditions that were successfully tested for different geometries of quantum wells.

  18. Immunoglobulin and enzyme-conjugated dextran polymers enhance u-PAR staining intensity of carcinoma cells in peripheral blood smears

    DEFF Research Database (Denmark)

    Werther, K; Normark, M; Hansen, B F;

    1999-01-01

    phenotyping of disseminated carcinoma cells in bone marrow and peripheral blood smears. In the first step, the cells were incubated with antibodies against urokinase plasminogen activator receptor (u-PAR) and subsequently with secondary antibodies conjugated to peroxidase-labeled dextran polymers. A brown...... color reaction was developed with diaminobenzidine as chromogen. In the second step, the cells were incubated with alkaline phosphatase-conjugated murine monoclonal antibodies against a common cytokeratin epitope and a red color reaction was developed with new fuchsin as substrate. This method allows...

  19. Solid-phase fluoroimmunoassay for treponemal antibody.

    OpenAIRE

    Stevens, R W; Schell, R F

    1982-01-01

    An objective, solid-phase fluoroimmunoassay for treponemal antibody was developed with a lysate of virulent Treponema pallidum (Nichols strain) adsorbed on cellulose acetate disks. A probe containing both the antigen and control disks is inserted successively into a serum specimen dilution, a buffer rinse, fluoroscein isothiocyanate-conjugated goat anti-human immunoglobulin G, and a second buffer rinse. Fluorescence signal units are measured with a fluorometer. To establish test calibration c...

  20. Influence of prevaccination immunity on the human B-lymphocyte response to a Haemophilus influenzae type b conjugate vaccine

    DEFF Research Database (Denmark)

    Barington, T; Kristensen, K; Henrichsen, J;

    1991-01-01

    The purpose of this study was to investigate whether preexisting immunity to components of a polysaccharide-protein conjugate influences the B-lymphocyte response to vaccination with the conjugate. Thirty-two healthy adults were vaccinated once or twice with a conjugate (PRP-D) consisting...... of Haemophilus influenzae type b capsular polysaccharide (PRP) and diphtheria toxoid (DT), and the response was related to the prevaccination levels of PRP and DT antibodies. Positive correlations were found between increases in plasma PRP (median, 32.0 micrograms/ml) and DT (1.14 IU/ml) antibodies and numbers...... revaccination with PRP-D, small increases in the level of PRP antibodies (median, 2.9 micrograms/ml; n = 11) were found; no significant increase in the level of DT antibodies was seen. The numbers of PRP AbSC were lower (P = 0.04) and peaked earlier (day 7) than after the first vaccination. The isotype pattern...

  1. Generation and characterization of monoclonal antibodies specific to Coenzyme A

    Directory of Open Access Journals (Sweden)

    Malanchuk O. M.

    2015-06-01

    Full Text Available Aim. Generation of monoclonal antibodies specific to Coenzyme A. Methods. Hybridoma technique. KLH carrier protein conjugated with CoA was used for immunization. Screening of positive clones was performed with BSA conjugated to CoA. Results. Monoclonal antibody that specifically recognizes CoA and CoA derivatives, but not its precursors ATP and cysteine has been generated. Conclusion. In this study, we describe for the first time the production and characterization of monoclonal antibodies against CoA. The monoclonal antibody 1F10 was shown to recognize specifically CoA in Western blotting, ELISA and immunoprecipitation. These properties make this antiboby a particularly valuable reagent for elucidating CoA function in health and disease.

  2. Organometallic B12-DNA conjugate

    DEFF Research Database (Denmark)

    Hunger, Miriam; Mutti, Elena; Rieder, Alexander;

    2014-01-01

    Design, synthesis, and structural characterization of a B12-octadecanucleotide are presented herein, a new organometallic B12-DNA conjugate. In such covalent conjugates, the natural B12 moiety may be a versatile vector for controlled in vivo delivery of oligonucleotides to cellular targets in hum...

  3. Baculovirus display of functional antibody Fab fragments.

    Science.gov (United States)

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

    2015-08-01

    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.

  4. Development of hen antihepatitis B antigen IgY-based conjugate for ELISA assay

    OpenAIRE

    Najat Muayed Nafea; Majeed Arsheed Sabbah; Raghad AL-Suhail; Amir Hossein Mahdavi; Sedigheh Asgary

    2015-01-01

    Background: Chicken antibodies have many advantages to the mammalian antibodies and have several important differences against mammalian IgG with regard to their specificity and large-scale production. In this study, the production, purification, and HRP conjugation of polyclonal IgY against hepatitis virus surface antigen (HBsAg) were carried out. Materials and Methods: Single Comb White Leghorn hens were immunized intramuscularly with hepatitis B vaccine in combination with Freund′s adj...

  5. Dihydroazulene-buckminsterfullerene conjugates

    DEFF Research Database (Denmark)

    Santella, Marco; Mazzanti, Virginia; Jevric, Martyn;

    2012-01-01

    The dihydroazulene (DHA)/vinylheptafulvene (VHF) photo/thermoswitch has recently attracted interest as a molecular switch for molecular electronics. In this field, Buckminsterfullerene, C(60), has been shown to be a useful anchoring group for adhering a molecular wire to an electrode. Here we have...... combined the two units with the overall aim to elucidate how C(60) influences the DHA-VHF switching events. Efficient synthetic protocols for making covalently linked DHA-C(60) conjugates were developed, using Prato, Sonogashira, Hay, and Cadiot-Chodkiewicz reactions. These syntheses provide as well...... a variety of potentially useful DHA and C(60) building blocks for acetylenic scaffolding. The two units were separated by bridges of various lengths, such as oligo(phenyleneethynylene) (OPE2 and OPE3) wires. The distance of separation was found to influence strongly the light-induced ring-opening reaction...

  6. Generalized conjugate gradient squared

    Energy Technology Data Exchange (ETDEWEB)

    Fokkema, D.R.; Sleijpen, G.L.G. [Utrecht Univ. (Netherlands)

    1994-12-31

    In order to solve non-symmetric linear systems of equations, the Conjugate Gradient Squared (CGS) is a well-known and widely used iterative method. In practice the method converges fast, often twice as fast as the Bi-Conjugate Gradient method. This is what you may expect, since CGS uses the square of the BiCG polynomial. However, CGS may suffer from its erratic convergence behavior. The method may diverge or the approximate solution may be inaccurate. BiCGSTAB uses the BiCG polynomial and a product of linear factors in an attempt to smoothen the convergence. In many cases, this has proven to be very effective. Unfortunately, the convergence of BiCGSTAB may stall when a linear factor (nearly) degenerates. BiCGstab({ell}) is designed to overcome this degeneration of linear factors. It generalizes BiCGSTAB and uses both the BiCG polynomial and a product of higher order factors. Still, CGS may converge faster than BiCGSTAB or BiCGstab({ell}). So instead of using a product of linear or higher order factors, it may be worthwhile to look for other polynomials. Since the BiCG polynomial is based on a three term recursion, a natural choice would be a polynomial based on another three term recursion. Possibly, a suitable choice of recursion coefficients would result in method that converges faster or as fast as CGS, but less erratic. It turns out that an algorithm for such a method can easily be formulated. One particular choice for the recursion coefficients leads to CGS. Therefore one could call this algorithm generalized CGS. Another choice for the recursion coefficients leads to BiCGSTAB. It is therefore possible to mix linear factors and some polynomial based on a three term recursion. This way one may get the best of both worlds. The authors will report on their findings.

  7. Human Monoclonal Antibodies Targeting Glypican-2 in Neuroblastoma | NCI Technology Transfer Center | TTC

    Science.gov (United States)

    Researchers at the National Cancer Institute’s Laboratory of Molecular Biology (NCI LMB) have developed and isolated several single domain monoclonal human antibodies against GPC2. NCI seeks parties interested in licensing or co-developing GPC2 antibodies and/or conjugates.

  8. Monoclonal antibodies

    International Nuclear Information System (INIS)

    Monoclonal antibodies (MAbs) are antibodies having single specificity for a given antigen site (epitope). The development of hybridoma technology and the relative ease by which MAbs can be prepared has revolutionized many aspects of serological applications in diagnosis and differentiation of disease producing agents. The property of monospecificity offers advantages in diagnostic applications over polyclonal sera in that tests can be defined exactly with regard to the antigen detected and the affinity of reaction between the given antigenic site and the monoclonal reagent. In addition, MAbs offer better possibilities for test standardization, because the same reagent can be used in different laboratories. Such an MAb can be supplied by a central laboratory or 'grown' from hybridoma cells, ensuring that the resultant product is identical from laboratory to laboratory and that the part of the test involving the MAb reaction is the same. The methodologies for inoculation regimes, mice, cloning methods, selection of fusion partners, etc., have been validated extensively in developed country laboratories. The decision to establish a MAb production facility must be examined on a strict cost-benefit basis, since it is still expensive to produce a product. There are many MAbs available that should be sought to allow exploitation in developing tests. If a production facility is envisaged, it should produce reagents for national needs, i.e. there should be a clear problem oriented approach whereby exact needs are defined. In the field of veterinary applications, MAbs are the central reagent in many immunoassays based on the enzyme linked immunosorbent assay (ELISA). The development of specific tests for diagnosing diseases is dominated by MAbs and has been fuelled by a strong research base, mainly in developed countries allied to developing countries through the study of related diseases. Thus, there are very many assays dependent on MAbs, some of which form the basis of

  9. Pharmacokinetics of internally labeled monoclonal antibodies as a gold standard: comparison of biodistribution of /sup 75/Se-, /sup 111/In-, and /sup 125/I-labeled monoclonal antibodies in osteogenic sarcoma xenografts in nude mice

    Energy Technology Data Exchange (ETDEWEB)

    Koizumi, M.; Endo, K.; Watanabe, Y.; Saga, T.; Sakahara, H.; Konishi, J.; Yamamuro, T.; Toyama, S.

    1989-04-01

    In order to know the true biodistribution of anti-tumor monoclonal antibodies, three monoclonal antibodies (OST6, OST7, and OST15) against human osteosarcoma and control antibody were internally labeled with 75Se by incubating (75Se)methionine and hybridoma cells. 75Se-labeled monoclonal antibodies were evaluated both in vitro and in vivo using the human osteogenic sarcoma cell line KT005, and the results were compared with those of 125I- and 111In-labeled antibodies. 75Se-, 125I- and 111In-labeled monoclonal antibodies had identical binding activities to KT005 cells, and the immunoreactivity was in the decreasing order of OST6, OST7, and OST15. On the contrary, in vivo tumor uptake (% injected dose/g) of 75Se- and 125I-labeled antibodies assessed using nude mice bearing human osteosarcoma KT005 was in the order of OST7, OST6, and OST15. In the case of 111In, the order was OST6, OST7, and OST15. High liver uptake was similarly seen with 75Se- and 111In-labeled antibodies, whereas 125I-labeled antibodies showed the lowest tumor and liver uptake. These data indicate that tumor targeting of antibody conjugates are not always predictable from cell binding studies due to the difference of blood clearance of labeled antibodies. Furthermore, biodistribution of both 111In- and 125I-labeled antibodies are not identical with internally labeled antibody. Admitting that internally labeled antibody is a ''gold standard'' of biodistribution of monoclonal antibody, high liver uptake of 111In-radiolabeled antibodies may be inherent to antibodies. Little, if any, increase in tumor-to-normal tissue ratios of antibody conjugates will be expected compared to those of 111In-labeled antibodies if stably coupled conjugates are administered i.v.

  10. A sulfhydryl-reactive ruthenium (II complex and its conjugation to protein G as a universal reagent for fluorescent immunoassays.

    Directory of Open Access Journals (Sweden)

    Jing-Tang Lin

    Full Text Available To develop a fluorescent ruthenium complex for biosensing, we synthesized a novel sulfhydryl-reactive compound, 4-bromophenanthroline bis-2,2'-dipyridine Ruthenium bis (hexafluorophosphate. The synthesized Ru(II complex was crosslinked with thiol-modified protein G to form a universal reagent for fluorescent immunoassays. The resulting Ru(II-protein G conjugates were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE. The emission peak wavelength of the Ru(II-protein G conjugate was 602 nm at the excitation of 452 nm which is similar to the spectra of the Ru(II complex, indicating that Ru(II-protein G conjugates still remain the same fluorescence after conjugation. To test the usefulness of the conjugate for biosensing, immunoglobulin G (IgG binding assay was conducted. The result showed that Ru(II-protein G conjugates were capable of binding IgG and the more cross-linkers to modify protein G, the higher conjugation efficiency. To demonstrate the feasibility of Ru(II-protein G conjugates for fluorescent immunoassays, the detection of recombinant histidine-tagged protein using the conjugates and anti-histidine antibody was developed. The results showed that the histidine-tagged protein was successfully detected with dose-response, indicating that Ru(II-protein G conjugate is a useful universal fluorescent reagent for quantitative immunoassays.

  11. Specific targeting of tumor cells by lyophilisomes functionalized with antibodies

    NARCIS (Netherlands)

    van Bracht, Etienne; Stolle, Sarah; Hafmans, Theo G.; Boerman, Otto C.; Oosterwijk, Egbert; van Kuppevelt, Toin H.; Daamen, Willeke F.

    2014-01-01

    Lyophilisomes are a novel class of proteinaceous biodegradable nano/micro drug delivery capsules prepared by freezing, annealing and Iyophilization. In the present study, lyophilisomes were functionalized for active targeting by antibody conjugation in order to obtain a selective drug-carrier system

  12. Star-Shaped Conjugated Systems

    Directory of Open Access Journals (Sweden)

    Heiner Detert

    2010-05-01

    Full Text Available The present review deals with the preparation and the properties of star-shaped conjugated compounds. Three, four or six conjugated arms are attached to cross-conjugated cores, which consist of single atoms (B, C+, N, benzene or azine rings or polycyclic ring systems, as for example triphenylene or tristriazolotriazine. Many of these shape-persistent [n]star compounds tend to π-stacking and self-organization, and exhibit interesting properties in materials science: Linear and non-linear optics, electrical conductivity, electroluminescence, formation of liquid crystalline phases, etc.

  13. Cost-effectiveness models of pneumococcal conjugate vaccines : Variability and impact of modeling assumptions

    NARCIS (Netherlands)

    Farkouh, Raymond A; Klok, Rogier M; Postma, Maarten J; Roberts, Craig S; Strutton, David R

    2012-01-01

    Currently, 13-valent pneumococcal conjugate vaccine (PCV); and ten-valent PCV vaccine are marketed. Neither vaccine obtained regulatory approval based on efficacy trials, but instead were approved based on a surrogate end point: immunogenicity data measuring effective antibody levels. Therefore, dir

  14. Effect of increased CRM₁₉₇ carrier protein dose on meningococcal C bactericidal antibody response.

    Science.gov (United States)

    Lee, Lucia H; Blake, Milan S

    2012-04-01

    New multivalent CRM(197)-based conjugate vaccines are available for childhood immunization. Clinical studies were reviewed to assess meningococcal group C (MenC) antibody responses following MenC-CRM(197) coadministration with CRM(197)-based pneumococcal or Haemophilus influenzae type b conjugate vaccines. Infants receiving a total CRM(197) carrier protein dose of ∼50 μg and concomitant diphtheria-tetanus-acellular pertussis (DTaP)-containing vaccine tended to have lower MenC geometric mean antibody titers and continued to have low titers after the toddler dose. Nevertheless, at least 95% of children in the reported studies achieved a MenC serum bactericidal antibody (SBA) titer of ≥ 1:8 after the last infant or toddler dose. SBA was measured using an assay with a baby rabbit or human complement source. Additional studies are needed to assess long-term antibody persistence and MenC CRM(197) conjugate vaccine immunogenicity using alternative dosing schedules.

  15. Development of monoclonal antibody against isoquinoline alkaloid coptisine and its application for the screening of medicinal plants

    OpenAIRE

    Kim, Jun-Sik; Tanaka, Hiroyuki; YUAN, CHUN-SU; Shoyama, Yukihiro

    2004-01-01

    In the development of immunoassay technique, the design of hapten containing a functional group suitable for protein conjugate is the key step for the preparation of antibodies against small molecules. Coptisine (MW 320), a bioactive constituent of Berberis and Coptis species, is small as an immunogen. In addition, coptisine has no reactive group in molecule for conjugating with a protein. To overcome this problem, 9-O-carboxymethyl-berberrubine was designed and conjugated with carrier protei...

  16. Sequential measurements of conjugate observables

    Energy Technology Data Exchange (ETDEWEB)

    Carmeli, Claudio [Dipartimento di Fisica, Universita di Genova, Via Dodecaneso 33, 16146 Genova (Italy); Heinosaari, Teiko [Department of Physics and Astronomy, Turku Centre for Quantum Physics, University of Turku, 20014 Turku (Finland); Toigo, Alessandro, E-mail: claudio.carmeli@gmail.com, E-mail: teiko.heinosaari@utu.fi, E-mail: alessandro.toigo@polimi.it [Dipartimento di Matematica ' Francesco Brioschi' , Politecnico di Milano, Piazza Leonardo da Vinci 32, 20133 Milano (Italy)

    2011-07-15

    We present a unified treatment of sequential measurements of two conjugate observables. Our approach is to derive a mathematical structure theorem for all the relevant covariant instruments. As a consequence of this result, we show that every Weyl-Heisenberg covariant observable can be implemented as a sequential measurement of two conjugate observables. This method is applicable both in finite- and infinite-dimensional Hilbert spaces, therefore covering sequential spin component measurements as well as position-momentum sequential measurements.

  17. Antibody-based biosensor assays for the detection of zilpaterol and markers for prostate cancer

    OpenAIRE

    Dillon, Mary

    2008-01-01

    The research presented in this thesis describes the production and application of antibodies against the drug of abuse zilpaterol, and the application of antibodies against prostate-specific antigen (PSA), a cancer marker. Polyclonal antibodies were used in the development of immunoassays in a competitive ELISA format and on the Biacore (a surface plasmon resonance-based optical biosensor capable of monitoring biomolecular interactions in 'real-time'). A zilpaterol-HSA conjugate was u...

  18. 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)

    2004-07-01

    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.

  19. [Antinuclear antibodies].

    Science.gov (United States)

    Cabiedes, Javier; Núñez-Álvarez, Carlos A

    2010-01-01

    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.

  20. Antibody-directed targeting of lysostaphin adsorbed onto polylactide nanoparticles increases its antimicrobial activity against S. aureus in vitro

    Science.gov (United States)

    Satishkumar, R.; Vertegel, A. A.

    2011-12-01

    The objective of this paper was to study the effect of antibody-directed targeting of S. aureus by comparing the activities of lysostaphin conjugated to biodegradable polylactide nanoparticles (NPs) in the presence and in the absence of co-immobilized anti-S. aureus antibody. Lysostaphin-antibody-NP conjugates were synthesized through physical adsorption at different enzyme:antibody:NP ratios. The synthesized enzyme-NP conjugates were characterized by means of dynamic light scattering and zeta potential analysis, and the total protein binding yield on the NPs was characterized using Alexa Fluor 350 and 594 dyes for the S. aureus antibody and lysostaphin respectively. We observed enhanced antimicrobial activity for both enzyme-coated and enzyme-antibody-coated NPs for lysostaphin coatings corresponding to ~ 40% of the initial monolayer and higher compared to the free enzyme case (p < 0.05). At the highest antibody coating concentration, bacterial lysis rates for antibody-coated samples were significantly higher than for lysostaphin-coated samples lacking the antibody (p < 0.05). Such enzyme-NP conjugates thus have the potential for becoming novel therapeutic agents for treating antibiotic-resistant S. aureus infections.

  1. Flow immunoassay of trinitrophenol based on a surface plasmon resonance sensor using a one-pot immunoreaction with a high molecular weight conjugate.

    Science.gov (United States)

    Kobayashi, Masatoshi; Sato, Masahiro; Li, Yan; Soh, Nobuaki; Nakano, Koji; Toko, Kiyoshi; Miura, Norio; Matsumoto, Kiyoshi; Hemmi, Akihide; Asano, Yasukazu; Imato, Toshihiko

    2005-12-15

    A surface plasmon resonance (SPR) immunosensor based on a competitive immunoreaction for the determination of trinitrophenol (TNP) is described. A goat anti-mouse IgG (1st antibody), which recognizes an Fc moiety of an antibody, was immobilized on a gold film of an SPR sensor chip by physical adsorption. A TNP solution containing a fixed concentration of a mouse anti-TNP monoclonal antibody (2nd antibody) and a TNP-keyhole limpet hemocyanin (KLH) conjugate was incubated in one-pot and introduced into the sensor chip. The TNP-KLH conjugate competes with TNP for binding with the 2nd antibody. The resulting complex of the 2nd antibody with the TNP-KLH conjugate was bound to the 1st antibody, which is immobilized on the sensor chip. The SPR sensor signal based on resonance angle shift is dependent on the concentration of TNP in the incubation solution in the range from 25ppt to 25ppb, and the coefficient of variation of the SPR signals for the 25ppb TNP solution was determined to be 13% (n=4). The experimental results for the adsorption constant of the 1st antibody on the sensor chip and the binding constant of the 1st antibody complex with the 2nd antibody are discussed, together with theoretical considerations. PMID:18970305

  2. Update on the use of meningococcal serogroup C CRM₁₉₇-conjugate vaccine (Meningitec) against meningitis.

    Science.gov (United States)

    Badahdah, Al-Mamoon; Rashid, Harunor; Khatami, Ameneh

    2016-01-01

    Meningitec is a CRM197-conjugated meningococcal serogroup C (MenC) vaccine, first licensed in 1999. It has been used as a primary and booster vaccine in infants, toddlers, older children and adults, and has been shown to be immunogenic and well-tolerated in all age groups, including premature infants. Vaccine effectiveness has been demonstrated using combined data on all three licensed MenC conjugate vaccines. Evidence from clinical trials, however, suggests that the different MenC conjugate vaccines behave differently with respect to the induction and persistence of bactericidal antibody and generation of immune memory. It appears that Meningitec has a less favorable immunologic profile compared particularly to tetanus toxoid (TT) MenC conjugate vaccines. Data from comparative trials have raised interesting questions on priming of the immune system by conjugate vaccines, particularly in infants. The results from these and other studies are reviewed here with specific focus on Meningitec.

  3. Research study of conjugate materials; Conjugate material no chosa kenkyu

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1998-03-01

    The paper reported an introductory research on possibilities of new glass `conjugate materials.` The report took up the structure and synthetic process of conjugate materials to be researched/developed, classified them according to structural elements on molecular, nanometer and cluster levels, and introduced the structures and functions. Further, as glasses with new functions to be proposed, the paper introduced transparent and high-strength glass used for houses and vehicles, light modulation glass which realizes energy saving and optical data processing, and environmentally functional glass which realizes environmental cleaning or high performance biosensor. An initial survey was also conducted on rights of intellectual property to be taken notice of in Japan and abroad in the present situation. Reports were summed up and introduced of Osaka National Research Institute, Electrotechnical Laboratory, and National Industrial Research Institute of Nagoya which are all carrying out leading studies of conjugate materials. 235 refs., 135 figs., 6 tabs.

  4. PREPARATION OF CONJUGATE FOR USE IN AN ELISA FOR HUMORAL IMMUNE RESPONSE AGAINST EGG DROP SYNDROME VIRUS IN LAYER CHICKS

    Directory of Open Access Journals (Sweden)

    M. K. Mansoor, S. U. Rahman, I. Hussain, M. H. Rasool and M. A. Zahoor

    2004-10-01

    Full Text Available An indirect enzyme-linked immunosorbent assay (ELISA was performed for the detection of antibodies against Egg Drop Syndrome (EDS virus. Virus identification was done through haemaggluti- nation inhibition (HI test using known antisera. Antichicken immunoglobulins were raised in goats and purified by ammonium sulphate precipitation technique. These goat-antichicken immunoglobulins were conjugated with horseradish peroxidase. Twenty-seven serum samples were collected from a layers flock vaccinated against EDS and specific antibodies were determined by using a horseradish conjugate.

  5. A Geometric View of Conjugate Priors

    CERN Document Server

    Agarwal, Arvind

    2010-01-01

    In Bayesian machine learning, conjugate priors are popular, mostly due to mathematical convenience. In this paper, we show that there are deeper reasons for choosing a conjugate prior. Specifically, we formulate the conjugate prior in the form of Bregman divergence and show that it is the inherent geometry of conjugate priors that makes them appropriate and intuitive. This geometric interpretation allows one to view the hyperparameters of conjugate priors as the {\\it effective} sample points, thus providing additional intuition. We use this geometric understanding of conjugate priors to derive the hyperparameters and expression of the prior used to couple the generative and discriminative components of a hybrid model for semi-supervised learning.

  6. Persistence Mechanisms of Conjugative Plasmids

    DEFF Research Database (Denmark)

    Bahl, Martin Iain; Hansen, Lars H.; Sørensen, Søren Johannes

    2009-01-01

    maintenance in the host cell. These importantly include the ability to self-mobilize in a process termed conjugative transfer, which may occur across species barriers. Other plasmid stabilizing mechanisms include the multimer resolution system, active partitioning, and post-segregational-killing of plasmid......Are plasmids selfish parasitic DNA molecules or an integrated part of the bacterial genome? This chapter reviews the current understanding of the persistence mechanisms of conjugative plasmids harbored by bacterial cells and populations. The diversity and intricacy of mechanisms affecting the...... successful propagation and long-term continued existence of these extra-chromosomal elements is extensive. Apart from the accessory genetic elements that may provide plasmid-harboring cells a selective advantage, special focus is placed on the mechanisms conjugative plasmids employ to ensure their stable...

  7. Storage Conditions of Conjugated Reagents Can Impact Results of Immunogenicity Assays

    Directory of Open Access Journals (Sweden)

    Robert J. Kubiak

    2016-01-01

    Full Text Available Consistent performance of anti-drug antibody (ADA assays through all stages of clinical development is critical for the assessment of immunogenicity and interpretation of PK, PD, safety, and efficacy. The electrochemiluminescent assays commonly employed for ADA measurement use drug conjugated with ruthenium and biotin to bind ADA in samples. Here we report an association between high nonspecific ADA responses in certain drug-naïve individuals and the storage buffer of the conjugated reagents used in a monoclonal antibody ADA assay. Ruthenylated reagents stored in phosphate-buffered saline (PBS buffer had increased levels of aggregate and produced variable and high baseline responses in some subjects. Reagents stored in a histidine-sucrose buffer (HSB had lower aggregate levels and produced low sample responses. In contrast to PBS, conjugated reagents formulated in HSB remained low in aggregate content and in sample response variability after 5 freeze/thaw cycles. A reagent monitoring control (RMC serum was prepared for the real-time evaluation of conjugated reagent quality. Using appropriate buffers for storage of conjugated reagents together with RMCs capable of monitoring of reagent aggregation status can help ensure consistent, long-term performance of ADA methods.

  8. Macrophages are critical effectors of antibody therapies for cancer.

    Science.gov (United States)

    Weiskopf, Kipp; Weissman, Irving L

    2015-01-01

    Macrophages are innate immune cells that derive from circulating monocytes, reside in all tissues, and participate in many states of pathology. Macrophages play a dichotomous role in cancer, where they promote tumor growth but also serve as critical immune effectors of therapeutic antibodies. Macrophages express all classes of Fcγ receptors, and they have immense potential to destroy tumors via the process of antibody-dependent phagocytosis. A number of studies have demonstrated that macrophage phagocytosis is a major mechanism of action of many antibodies approved to treat cancer. Consequently, a number of approaches to augment macrophage responses to therapeutic antibodies are under investigation, including the exploration of new targets and development of antibodies with enhanced functions. For example, the interaction of CD47 with signal-regulatory protein α (SIRPα) serves as a myeloid-specific immune checkpoint that limits the response of macrophages to antibody therapies, and CD47-blocking agents overcome this barrier to augment phagocytosis. The response of macrophages to antibody therapies can also be enhanced with engineered Fc variants, bispecific antibodies, or antibody-drug conjugates. Macrophages have demonstrated success as effectors of cancer immunotherapy, and further investigation will unlock their full potential for the benefit of patients. PMID:25667985

  9. Production and Application of Monoclonal Antibodies Against Methamphetamine (MA) and Morphine (MP)

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Three monoclonal antibodies (MAbs) against metharnphetamine (BD1, BD2, BD10) and four MAbs against morphine (MP6A8, MP6D9, MP7D6, MP8D9) are obtained by using MA-conjugated bovine serum albumin (BSA) and MP-conjugated BSA as immunogens, respectively. The MAbs against MA belonged to the IgG1 subclass with λ chains. The association constants (Ka) of the antibodies are higher than 107 L/mol (RIA). The MAbs against MP also belonged to the IgG1 subclass with λ chains. Ka of these antibodies is higher thanThree monoclonal antibodies (MAbs) against metharnphetamine (BD1, BD2, BD10) and four MAbs against morphine (MP6A8, MP6D9, MP7D6, MP8D9) are obtained by using MA-conjugated bovine serum albumin (BSA) and MP-conjugated BSA as immunogens, respectively. The MAbs against MA belonged to the IgG1 subclass with λ chains. The association constants (Ka) of the antibodies are higher than 107 L/mol (RIA). The MAbs against MP also belonged to the IgG1 subclass with λ chains. Ka of these antibodies is higher than 108

  10. DNA-Directed Antibody Immobilization for Enhanced Detection of Single Viral Pathogens.

    Science.gov (United States)

    Seymour, Elif; Daaboul, George G; Zhang, Xirui; Scherr, Steven M; Ünlü, Nese Lortlar; Connor, John H; Ünlü, M Selim

    2015-10-20

    Here, we describe the use of DNA-conjugated antibodies for rapid and sensitive detection of whole viruses using a single-particle interferometric reflectance imaging sensor (SP-IRIS), a simple, label-free biosensor capable of imaging individual nanoparticles. First, we characterize the elevation of the antibodies conjugated to a DNA sequence on a three-dimensional (3-D) polymeric surface using a fluorescence axial localization technique, spectral self-interference fluorescence microscopy (SSFM). Our results indicate that using DNA linkers results in significant elevation of the antibodies on the 3-D polymeric surface. We subsequently show the specific detection of pseudotyped vesicular stomatitis virus (VSV) as a model virus on SP-IRIS platform. We demonstrate that DNA-conjugated antibodies improve the capture efficiency by achieving the maximal virus capture for an antibody density as low as 0.72 ng/mm(2), whereas for unmodified antibody, the optimal virus capture requires six times greater antibody density on the sensor surface. We also show that using DNA conjugated anti-EBOV GP (Ebola virus glycoprotein) improves the sensitivity of EBOV-GP carrying VSV detection compared to directly immobilized antibodies. Furthermore, utilizing a DNA surface for conversion to an antibody array offers an easier manufacturing process by replacing the antibody printing step with DNA printing. The DNA-directed immobilization technique also has the added advantages of programmable sensor surface generation based on the need and resistance to high temperatures required for microfluidic device fabrication. These capabilities improve the existing SP-IRIS technology, resulting in a more robust and versatile platform, ideal for point-of-care diagnostics applications. PMID:26378807

  11. Heterologous coproduction of enterocin A and pediocin PA-1 by Lactococcus lactis : Detection by specific peptide-directed antibodies

    NARCIS (Netherlands)

    Martinez, JM; Kok, J; Sanders, JW; Hernandez, PE

    2000-01-01

    Antibodies against enterocin A were obtained by immunization of rabbits with synthetic peptides PH4 and PH5 designed, respectively, on the N- and C-terminal amino acid sequences of enterocin A and conjugated to the carrier protein KLH, Anti-PH4-KLH antibodies not only recognized enterocin A but also

  12. Development of Anti-Isoproturon Polyclonal Antibody

    Institute of Scientific and Technical Information of China (English)

    LI Fang-shi; SUN Feng; LIU Xian-jin; CUI Heng-hua

    2007-01-01

    A competitive enzyme-linked immunosorbent assay (ELISA) suitable for the determination of the urea herbicide isoproturon,3-(4-isopropylphenyl)-1,1-dimethylurea, in food and environmental samples was developed. Two haptens named 1-(3-carboxypropyl)-3-(4-isopropylphenyl)-1-methylurca (hapten 4C) and 1-(5-carboxypentyl)-3-(4-isopropylphenyl)-1-methylurea (hapten 6C) were synthesized. The haptens were coupled to bovine serum albumin (BSA) and ovalbumin(OVA), respectively, using the N-hydroxysuccinimide reaction. The hapten 6C-BSA conjugate was used as the immunogen,with which a high-titer anti-isoproturon polyclonal antibody (pAb) was successfully obtained by immunization of New Zealand white rabbits. The hapten 4C-OVA conjugate was used as coating antigen and a method of the indirect competitive ELISA for isoproturon was established. The haptens were confirmed with TLC, IR, and 1H NMR. The conjugation molar ratios of hapten 4C to OVA and hapten 6C to BSA were 36:1 and 46:1, respectively, as calculated by a UV spectrophotometry.The highest titer of the anti-isoproturon sera determined by a non-competitive indirect ELISA procedure was 1.6×105. The optimal concentrations of the coating antigen and the dilution of the anti-isoproturon sera used in the ELISA were 0.1 mg L-1 and 1.0 × 105, respectively. The concentration of isoproturon that inhibits 50% of antibody-antigen binding (IC50) was 0.07 mg mL-1.The cross-reactivities of six urea herbicides including chlorbromuron, fluometuron, monolinuron were lower than 0.1%. Isoproturon is a small molecule without immune activity and active functional group for attaching to carrier protein. To produce an antibody against isoproturon with high titer and high specificity is the most important step in the development of an immunochemical method for the determination of isoproturon in food and environmental samples. The two haptens synthesized in this study have carboxyl groups and accommodate different lengths of spacer arms, and

  13. PE-Cy5.5 conjugates bind to the cells expressing mouse DEC205/CD205

    Science.gov (United States)

    Park, Chae Gyu; Rodriguez, Anthony; Steinman, Ralph M.

    2012-01-01

    DEC205/CD205, an endocytic receptor of C-type multilectin, is expressed highly in dendritic cells (DCs). DEC205 was shown to efficiently deliver vaccine antigens in surrogate ligands to the antigen processing and presentation machinery of DCs, which resulted in the development of DC-targeted vaccines employing anti-DC monoclonal antibodies (mAbs). During our studies to characterize a variety of anti-DC mAbs including anti-DEC205 by flow cytometric analysis, we discovered that a secondary anti-immunoglobulin antibody conjugated with PE-Cy5.5 bound strongly to the cells expressing mouse DEC205 (mDEC205) without incubation of a primary anti-mDEC205 mAb. In the present study we demonstrate that various antibodies and streptavidin conjugated with PE-Cy5.5 bind to the mDEC205-expressing cells including CHO, KIT6, and HEK293 cells. The interaction between the PE-Cy5.5 conjugates and the cells expressing mDEC205 appears distinctive, since none of PE-Cy5.5 conjugates bind to the cells that express human DEC205 on surface. Besides, only PE-Cy5.5 conjugates bind strongly to mDEC205-expressing cells; PerCP-Cy5.5, APC-Cy5.5, and Cy5.5 conjugates bind weakly; PE, PE-Cy5, Cy5, FITC, or Alexa488 conjugates do not bind. Therefore the use of PE-Cy5.5 conjugates, widely utilized in multicolor flow cytometry, requires precaution against nonspecific binding to mDEC205-positive cells. PMID:22841832

  14. On contravariant product conjugate connections

    Directory of Open Access Journals (Sweden)

    A. M. Blaga

    2012-02-01

    Full Text Available Invariance properties for the covariant and contravariant connections on a Riemannian manifold with respect to an almost product structure are stated. Restricting to a distribution of the contravariant connections is also discussed. The particular case of the conjugate connection is investigated and properties of the extended structural and virtual tensors for the contravariant connections are given.

  15. Application of bispecific antibody against antigen and hapten for immunodetection and immunopurification

    OpenAIRE

    Kim, Hyori; Park, Sunyoung; Lee, Hwa Kyoung; Chung, Junho

    2013-01-01

    We present a bispecific antibody that recognizes an antigen and a hapten and can be applied to various biological assays, including immunoblotting and immunoprecipitation. In immunoblot analysis of serum, an anti-C5 × anti-cotinine bispecific tandem single-chain variable fragment (scFv)-Fc fusion protein and cotinine-conjugated horseradish peroxidase (HRP) generated a clean signal without the high background that was observed in a parallel experiment using HRP-conjugated goat anti-rabbit immu...

  16. Glutathione conjugation as a bioactivation reaction

    NARCIS (Netherlands)

    Bladeren, P.J. van

    2000-01-01

    In general, glutathione conjugation is regarded as a detoxication reaction. However, depending on the properties of the substrate, bioactivation is also possible. Four types of activation reaction have been recognized: direct-acting compounds, conjugates that are activated through cysteine conjugate

  17. Development of a monoclonal antibody-based broad-specificity ELISA for fluroquinolone antibiotics in foods and molecular modeling studies of cross-reactive compounds

    Science.gov (United States)

    Development of a competitive indirect enzyme-linked immunosorbent assay (ciELISA) with monoclonal antibodies (Mabs) having broad specificity for fluoroquinolone (FQ) antibiotics is described. Four FQs, ciprofloxacin (CIP), norfloxacin (NOR), enrofloxacin (ENR) and ofloxacin (OFL) were conjugated to...

  18. Interface states and bio-conjugation of CdSe/ZnS core-shell quantum dots

    Science.gov (United States)

    Torchynska, T. V.

    2009-03-01

    The paper presents the results of photoluminescence (PL) and Raman scattering studies of non-conjugated and bio-conjugated CdSe/ZnS core-shell quantum dots (QDs). The commercial CdSe/ZnS QDs used are characterized by color emission with maxima at 605-610 nm (2.03-2.05 eV). PL spectra of non-conjugated QDs are the superposition of PL bands related to exciton emission in the CdSe core (2.03-2.05 eV) and to hot electron-hole emission via defect states at the CdSe/ZnS interface (2.37 and 2.68 eV). QD conjugation was performed with biomolecules—the antihuman interleukin 10 antibody (antihuman IL10). The PL spectra of bio-conjugated QDs have been changed dramatically: only one PL band related to exciton emission in the CdSe core was detected in bio-conjugated QDs. To explain this effect a model has been proposed which assumes that the QD bio-conjugation process is accompanied by the recharging of acceptor-like interface states at the CdSe/ZnS interface. A comparative analysis of normalized PL spectra of non-conjugated CdSe/ZnS QDs with different intensities of interface state PL has confirmed the proposed electron-hole recombination model in QDs.

  19. Site-specific conjugation of bifunctional chelator BAT to mouse IgG1 Fab' fragment

    Institute of Scientific and Technical Information of China (English)

    Jun LI; Xue-hao WANG; Xiao-ming WANG; Zhao-lai CHEN

    2006-01-01

    Aim: To perform a site-specific conjugation of Fab' fragments of a mouse monoclonal antibody(MoAb) B43(of IgG1 subtype) to a bifunctional chelator 6-[p-(bromoacetamido) benzyl]-l,4,8,11-tetraazacyclotetradecane-N,N',N",N'"-tetraacetic acid (BAT) via the thiol groups in the hinge distal to the antigenbinding site of the Fab'. Methods: B43 was cleaved using a simple 2-step method.First, stable F(ab')2 was produced by pepsin treatment. Fab' with free thiol in the hinge region was then obtained by cysteine reduction of F(ab')2. Second, a sitespecific conjugation of Fab' to thiol-specific BAT was performed in a one-step reaction. Results: The Fab' fragment had approximately 1.8 free thiol groups per molecule after cysteine reduction. The conjugation efficiency and the chemical yield were approximately 1.28 moles chelator/Fab' and 74% of the initial concentration of Fab', respectively. The F(ab')2, Fab' and Fab'-BAT all maintained reasonable antigen-binding properties. 67Cu labeling of the conjugate under standard conditions did not impair the immunoreactivity of Fab'-BAT. Conclusion: This is a simple and efficient method for producing immunoreactive conjugates of Fab'-BAT, which can be used to make radiometal-labeled conjugates for further diagnostic and therapeutic applications.

  20. IBC's 23rd Antibody Engineering and 10th Antibody Therapeutics Conferences and the Annual Meeting of The Antibody Society: December 2-6, 2012, San Diego, CA.

    Science.gov (United States)

    Marquardt, John; Begent, Richard H J; Chester, Kerry; Huston, James S; Bradbury, Andrew; Scott, Jamie K; Thorpe, Philip E; Veldman, Trudi; Reichert, Janice M; Weiner, Louis M

    2012-01-01

    Now in its 23rd and 10th years, respectively, the Antibody Engineering and Antibody Therapeutics conferences are the Annual Meeting of The Antibody Society. The scientific program covers the full spectrum of challenges in antibody research and development from basic science through clinical development. In this preview of the conferences, the chairs provide their thoughts on sessions that will allow participants to track emerging trends in (1) the development of next-generation immunomodulatory antibodies; (2) the complexity of the environment in which antibodies must function; (3) antibody-targeted central nervous system (CNS) therapies that cross the blood brain barrier; (4) the extension of antibody half-life for improved efficacy and pharmacokinetics (PK)/pharmacodynamics (PD); and (5) the application of next generation DNA sequencing to accelerate antibody research. A pre-conference workshop on Sunday, December 2, 2012 will update participants on recent intellectual property (IP) law changes that affect antibody research, including biosimilar legislation, the America Invents Act and recent court cases. Keynote presentations will be given by Andreas Plückthun (University of Zürich), who will speak on engineering receptor ligands with powerful cellular responses; Gregory Friberg (Amgen Inc.), who will provide clinical updates of bispecific antibodies; James D. Marks (University of California, San Francisco), who will discuss a systems approach to generating tumor targeting antibodies; Dario Neri (Swiss Federal Institute of Technology Zürich), who will speak about delivering immune modulators at the sites of disease; William M. Pardridge (University of California, Los Angeles), who will discuss delivery across the blood-brain barrier; and Peter Senter (Seattle Genetics, Inc.), who will present his vision for the future of antibody-drug conjugates. For more information on these meetings or to register to attend, please visit www.IBCLifeSciences.com/Antibody

  1. Purification of anti-bromelain antibodies by affinity precipitation using pNIPAm-linked bromelain.

    Science.gov (United States)

    Mahmood, Rubab

    2016-01-01

    Affinity precipitation has emerged as a very useful technique for the purification of proteins. Here it has been employed for the purification of anti-bromelain antibodies from rabbit serum. A system has been developed for reversibly binding and thermoprecipitating antibodies. Anti-bromelain antibodies were raised in rabbit by immunizing it with bromelain. Poly-N-isopropylacrylamide (pNIPAm)-bromelain conjugate was prepared and incubated with rabbit serum. After that the temperature was raised for thermal precipitation of the polymer. Antibodies were then eluted from the complex by incubating it with a small volume of buffer, pH 3.0. This method is very effective in concentrating the antibodies. Purity and specificity of the antibodies were checked by gel electrophoresis and enzyme-linked immunosorbent assay (ELISA), respectively. The study of the effect of pH and temperature on the binding of the antibodies to the conjugate showed that the optimum binding occurred at pH 8.0 and 25°C.The polymer enzyme conjugate was further used for another cycle. PMID:25569629

  2. Humoral and cell-mediated immunity following vaccination with synthetic Candida cell wall mannan derived heptamannoside-protein conjugate: immunomodulatory properties of heptamannoside-BSA conjugate.

    Science.gov (United States)

    Paulovičová, Lucia; Paulovičová, Ema; Karelin, Alexander A; Tsvetkov, Yury E; Nifantiev, Nikolay E; Bystrický, Slavomír

    2012-10-01

    Chemically defined glycoprotein conjugate composed of synthetically prepared mannan-derived heptamannoside with terminal β-1,2-linked mannose residue attached to the α-1,3-linked mannose residues and BSA as carrier protein (M7-BSA conjugate) was analysed for the capacity to induce protective humoral immunity and appropriate alteration cellular immunity. To identify protective antigenic structure of Candida cell wall mannan M7-BSA conjugate was used for BALB/c mice immunization. The obtained results were compared with placebo group and with heat-inactivated C. albicans whole cells immunization. The administration route of M7-BSA conjugate secondary booster injection significantly affected the intensity of humoral immune response and the specificity of produced antibodies. All prepared sera were able to elevate candidacidal activity of polymorphonuclear leukocytes (PMN) in cooperation with complement. Moreover, polyclonal sera obtained after secondary subcutaneous (s.c.) booster injection of M7-BSA conjugate were able to induce candidacidal activity of PMN also in complement independent manner. M7-BSA conjugate immunization induced increases of phagocytic activity and respiratory burst of granulocytes, caused a raise of the proportion of CD3(+) T lymphocytes and increased the CD4(+)/CD8(+) T lymphocyte ratio. We observed also an increasing proportion of CD4(+)CD25(+) T cells compared to immunization with heat inactivated whole C. albicans cells, which in turn promoted an increase of the CD8(+)CD25(+) cell proportion. Immunization with M7-BSA conjugate induced Th1, Th2 and Th17 immune responses as indicated by the elevation of relevant cytokines levels. These data provide some insights on the immunomodulatory properties of oligomannosides and contribute to the development of synthetic oligosaccharide vaccines against fungal diseases.

  3. Conjugative plasmids of Neisseria gonorrhoeae.

    Directory of Open Access Journals (Sweden)

    Emilia Pachulec

    Full Text Available Many clinical isolates of the human pathogen Neisseria gonorrhoeae contain conjugative plasmids. The host range of these plasmids is limited to Neisseria species, but presence of a tetracycline (tetM determinant inserted in several of these plasmids is an important cause of the rapid spread of tetracycline resistance. Previously plasmids with different backbones (Dutch and American type backbones and with and without different tetM determinants (Dutch and American type tetM determinants have been identified. Within the isolates tested, all plasmids with American or Dutch type tetM determinants contained a Dutch type plasmid backbone. This demonstrated that tetM determinants should not be used to differentiate between conjugal plasmid backbones. The nucleotide sequences of conjugative plasmids with Dutch type plasmid backbones either not containing the tetM determinant (pEP5233 or containing Dutch (pEP5289 or American (pEP5050 type tetM determinants were determined. Analysis of the backbone sequences showed that they belong to a novel IncP1 subfamily divergent from the IncP1alpha, beta, gamma, delta and epsilon subfamilies. The tetM determinants were inserted in a genetic load region found in all these plasmids. Insertion was accompanied by the insertion of a gene with an unknown function, and rearrangement of a toxin/antitoxin gene cluster. The genetic load region contains two toxin/antitoxins of the Zeta/Epsilon toxin/antitoxin family previously only found in Gram positive organisms and the virulence associated protein D of the VapD/VapX toxin/antitoxin family. Remarkably, presence of VapX of pJD1, a small cryptic neisserial plasmid, in the acceptor strain strongly increased the conjugation efficiency, suggesting that it functions as an antitoxin for the conjugative plasmid. The presence of the toxin and antitoxin on different plasmids might explain why the host range of this IncP1 plasmid is limited to Neisseria species. The isolated plasmids

  4. Vi capsular polysaccharide-protein conjugates for prevention of typhoid fever. Preparation, characterization, and immunogenicity in laboratory animals.

    Science.gov (United States)

    Szu, S C; Stone, A L; Robbins, J D; Schneerson, R; Robbins, J B

    1987-11-01

    The Vi has proven to be a protective antigen in two double masked, controlled clinical trials in areas with high rates of typhoid fever (approximately 1% per annum). In both studies the protective efficacy of the Vi was approximately 70%. Approximately 75% of subjects in these areas responded with a fourfold or greater rise of serum Vi antibodies. In contrast, the Vi elicited a fourfold or greater rise in 95-100% of young adults in France and the United States. Methods were devised, therefore, to synthesize Vi-protein conjugates in order to both enhance the antibody response and confer T-dependent properties to the Vi (and theoretically increase its protective action in populations at high risk for typhoid fever). We settled on a method that used the heterobifunctional crosslinking reagent, N-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP), to bind thiol derivatives of the Vi to proteins. This synthetic scheme was reproducible, provided high yields of Vi-protein conjugates, and was applicable to several medically relevant proteins such as diphtheria and tetanus toxoids. The resultant conjugates were more immunogenic in mice and juvenile Rhesus monkeys than the Vi alone. In contrast to the T-independent properties of the Vi, conjugates of this polysaccharide with several medically relevant proteins induced booster responses in mice and in juvenile Rhesus monkeys. Clinical studies with Vi-protein conjugates are planned. This scheme is also applicable to synthesize protein conjugates with other polysaccharides that have carboxyl functions.

  5. Comparison of bifunctional chelates for {sup 64}Cu antibody imaging

    Energy Technology Data Exchange (ETDEWEB)

    Ferreira, Cara L.; Crisp, Sarah; Bensimon, Corinne [MDS Nordion, Vancouver, BC (Canada); Yapp, Donald T.T.; Ng, Sylvia S.W. [British Columbia Cancer Agency Research Centre, Vancouver, BC (Canada); University of British Columba, The Faculty of Pharmaceutical Sciences, Vancouver, BC (Canada); Sutherland, Brent W. [British Columbia Cancer Agency Research Centre, Vancouver, BC (Canada); Gleave, Martin [Prostate Centre at Vancouver General Hospital, Vancouver, BC (Canada); Jurek, Paul; Kiefer, Garry E. [Macrocyclics Inc., Dallas, TX (United States)

    2010-11-15

    Improved bifunctional chelates (BFCs) are needed to facilitate efficient {sup 64}Cu radiolabeling of monoclonal antibodies (mAbs) under mild conditions and to yield stable, target-specific agents. The utility of two novel BFCs, 1-Oxa-4,7,10-triazacyclododecane-5-S-(4-isothiocyanatobenzyl)-4,7,10-triacetic acid (p-SCN-Bn-Oxo-DO3A) and 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-4-S-(4-isothiocyanatobenzyl)-3,6,9-triacetic acid (p-SCN-Bn-PCTA), for mAb imaging with {sup 64}Cu were compared to the commonly used S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-tetraacetic acid (p-SCN-Bn-DOTA). The BFCs were conjugated to trastuzumab, which targets the HER2/neu receptor. {sup 64}Cu radiolabeling of the conjugates was optimized. Receptor binding was analyzed using flow cytometry and radioassays. Finally, PET imaging and biodistribution studies were done in mice bearing either HER2/neu-positive or HER2/neu-negative tumors. {sup 64}Cu-Oxo-DO3A- and PCTA-trastuzumab were prepared at room temperature in >95% radiochemical yield (RCY) in <30 min, compared to only 88% RCY after 2 h for the preparation of {sup 64}Cu-DOTA-trastuzumab under the same conditions. Cell studies confirmed that the immunoreactivity of the mAb was retained for each of the bioconjugates. In vivo studies showed that {sup 64}Cu-Oxo-DO3A- and PCTA-trastuzumab had higher uptake than the {sup 64}Cu-DOTA-trastuzumab at 24 h in HER2/neu-positive tumors, resulting in higher tumor to background ratios and better tumor images. By 40 h all three of the {sup 64}Cu-BFC-trastuzumab conjugates allowed for clear visualization of the HER2/neu-positive tumors but not the negative control tumor. The antibody conjugates of PCTA and Oxo-DO3A were shown to have superior {sup 64}Cu radiolabeling efficiency and stability compared to the analogous DOTA conjugate. In addition, {sup 64}Cu-PCTA and Oxo-DO3A antibody conjugates may facilitate earlier imaging with greater target to background ratios than

  6. Superior Immune Response to Protein-Conjugate versus Free Pneumococcal Polysaccharide Vaccine in Chronic Obstructive Pulmonary Disease

    OpenAIRE

    Dransfield, Mark T.; Nahm, Moon H.; Han, MeiLan K.; Harnden, Sarah; Criner, Gerard J.; Fernando J Martinez; Scanlon, Paul D.; Woodruff, Prescott G.; Washko, George R.; Connett, John E.; Anthonisen, Nicholas R.; Bailey, William C.

    2009-01-01

    Rationale: Debate exists about the immunogenicity and protective efficacy of antibodies produced by the 23-valent pneumococcal polysaccharide vaccine (PPSV23) in chronic obstructive pulmonary disease (COPD). The 7-valent diphtheria-conjugated pneumococcal polysaccharide vaccine (PCV7) induces a more robust immune response than PPSV23 in healthy elderly adults.

  7. Pneumococcal conjugate vaccination does not induce a persisting mucosal IgA response in children with recurrent acute otitis media.

    NARCIS (Netherlands)

    Bogaert, D.; Veenhoven, R.H.; Ramdin, R.; Luijendijk, I.H.; Rijkers, G.T.; Sanders, E.A.M.; Groot, R. de; Hermans, P.W.M.

    2005-01-01

    AIM: In a prospective controlled study in young children with a history of recurrent acute otitis media, we analyzed the salivary IgA and IgG antibody titers upon vaccination with a 7-valent pneumococcal conjugate vaccine (PCV) given once or twice, followed by a 23-valent polysaccharide booster vacc

  8. The ISG15 conjugation system.

    Science.gov (United States)

    Durfee, Larissa A; Huibregtse, Jon M

    2012-01-01

    ISG15 is a ubiquitin-like modifier that is expressed in response to type 1 interferon signaling (IFN-α/β) and plays a role in antiviral responses. The core E1, E2, and E3 enzymes for ISG15 are Ube1L, UbcH8, and Herc5, respectively, and these are all also induced at the transcriptional level by IFN-α/β. We recently showed that Herc5 associates with polysomes and modifies target proteins in a cotranslational manner. Here, we describe the expression of the core conjugating enzymes in human cells, the detection of ISG15 conjugates, and the methods for fractionation of Herc5 with polysomes. PMID:22350882

  9. Conjugated Polymers for Energy Production

    DEFF Research Database (Denmark)

    Livi, Francesco

    This dissertation is aimed at developing materials for flexible, large area, ITO-free polymer solar cells (PSCs) fully printed under ambient conditions. A large screening of conjugated polymers, both novel and well-known materials, has been carried out in order to find suitable candidates...... for scalable PSCs fully printed under ambient conditions [Adv. Energy Mater. 2015, 5, 1402186]. PPDTBT resulted to be the conjugated polymer with the best photovoltaic performance within the 104 synthesized macromolecules. Therefore, further studies have been done on such material. The impact of side chain...... arylation (DAr) and direct arylation polymerization (DArP) have been applied to the preparation of PPDTBT, making this polymer readily available in only 4 synthetic steps and thus easily transferable to a large scale-production setup. DArP avoids organometallic species and therefore is an appealing...

  10. 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.

  11. Monoclonal antibodies and cancer

    International Nuclear Information System (INIS)

    The usefulness of radiolabeled monoclonal antibodies for imaging and treatment of human (ovarian) cancer was investigated. A review of tumor imaging with monoclonal antibodies is presented. Special attention is given to factors that influence the localization of the antibodies in tumors, isotope choice and methods of radiolabeling of the monoclonal antibodies. Two monoclonal antibodies, OC125 and OV-TL3, with high specificity for human epithelial ovarian cancer are characterized. A simple radio-iodination technique was developed for clinical application of the monoclonal antibodies. The behavior of monoclonal antibodies in human tumor xenograft systems and in man are described. Imaging of tumors is complicated because of high background levels of radioactivity in other sites than the tumor, especially in the bloodpool. A technique was developed to improve imaging of human tumor xenographs in nude mice, using subtraction of a specific and a non-specific antibody, radiolabeled with 111In, 67Ga and 131I. To investigate the capability of the two monoclonal antibodies, to specifically localize in human ovarian carcinomas, distribution studies in mice bearing human ovarian carcinoma xenografts were performed. One of the antibodies, OC125, was used for distribution studies in ovarian cancer patients. OC125 was used because of availability and approval to use this antibody in patients. The same antibody was used to investigate the usefulness of radioimmunoimaging in ovarian cancer patients. The interaction of injected radiolabeled antibody OC125 with circulating antigen and an assay to measure the antibody response in ovarian cancer patients after injection of the antibody is described. 265 refs.; 30 figs.; 19 tabs

  12. Conjugated polyelectrolytes fundamentals and applications

    CERN Document Server

    Liu, Bin

    2013-01-01

    This is the first monograph to specifically focus on fundamentals and applications of polyelectrolytes, a class of molecules that gained substantial interest due to their unique combination of properties. Combining both features of organic semiconductors and polyelectrolytes, they offer a broad field for fundamental research as well as applications to analytical chemistry, optical imaging, and opto-electronic devices. The initial chapters introduce readers to the synthesis, optical and electrical properties of various conjugated polyelectrolytes. This is followed by chapters on the applica

  13. Production of a highly group-specific monoclonal antibody against zearalenone and its application in an enzyme-linked immunosorbent assay

    OpenAIRE

    Cha, Sang-Ho; Kim, Sung-Hee; Bischoff, Karyn; Kim, Hyun-Jeong; Son, Seong-Wan; Kang, Hwan-Goo

    2012-01-01

    A monoclonal antibody (mAb) against zearalenone (ZEN) was produced using ZEN-carboxymethoxylamine and -BSA conjugates. Antibody produced by one clone showing a very high binding ability was selected and found to have a higher affinity for ZEN compared to a commerciall ZEN antibody. We developed two direct competitive ELISA systems using the selected antibody (ZEN-coated and anti-ZEN antibody-coated ELISA). Quantitative ranges for the anti-ZEN antibody-coated ELISA and ZEN-coated ELISA were fr...

  14. Time-resolvable fluorescent conjugates for the detection of pathogens in environmental samples containing autofluorescent material

    Science.gov (United States)

    Connally, Russell; Veal, Duncan; Piper, James A.

    2003-07-01

    Water is routinely monitored for environmental pathogens such a Cryptosporidium and Giardia using immunofluorescence microscopy (IFM). Autofluorescence can greatly diminish an operators capacity to resolve labeled pathogens from non-specific background. Naturally fluorescing components (autofluorophores) encountered in biological samples typically have fluorescent lifetimes (τ) of less than 100 nanoseconds and their emissions may be excluded through use of time-resolved fluorescence microscopy (TRFM). TRFM relies on the large differences in τ between autofluorescent molecules and long-lived lanthanide chelates. In TRFM, targets labeled with a time-resolvable fluorescent immunoconjugate are excited by an intense (UV) light pulse. A short delay is imposed to permit the decay of autofluorescence before capture of luminescence from the excited chelate using an image intensified CCD camera. In our experience, autofluorescence can be reduced to insignificant levels with a consequent 30-fold increase in target visibility using TRFM techniques. We report conjugation of a novel europium chelate to a monoclonal antibody specific for Giardia lamblia and use of the immunoconjugate for TRFM studies. Initial attempts to conjugate the same chelate to a monoclonal antibody directed against Cryptosporidium parvum led to poorly fluorescent constructs that were prone to denature and precipitate. We successfully conjugated BHHCT to anti-mouse polyvalent immunoglobulin and used this construct to overcome the difficulties in direct labeling of the anti-Cryptosporidium antibody. Both Giardia and Cryptosporidium were labeled using the anti-mouse protocol with a subsequent 20-fold and 6.6-fold suppression of autofluorescence respectively. A rapid protocol for conjugating and purifying the immunoconjugate was found and methods of quantifying the fluorescence to protein ratio determined. Performance of our TRFM was dependent on the quality and brightness of the immunoconjugate and

  15. [VGKC-complex antibodies].

    Science.gov (United States)

    Watanabe, Osamu

    2013-04-01

    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).

  16. Anti-CD20 multivalent HPMA copolymer-Fab′ conjugates for the direct induction of apoptosis

    OpenAIRE

    Chu, Te-Wei; Yang, Jiyuan; Kopeček, Jindřich

    2012-01-01

    A hybrid biomimetic system comprising high-molecular-weight, linear copolymer of N-(2-hydroxypropyl)methacrylamide (HPMA) grafted with multiple Fab′ fragments of anti-CD20 monoclonal antibody (mAb) was synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization followed by attachment of Fab′ fragments via thioether bonds. Exposure of human non-Hodgkin’s lymphoma (NHL) Raji B cells to the multivalent conjugates resulted in crosslinking of CD20 receptors and commenceme...

  17. Magnetosome Expression of Functional Camelid Antibody Fragments (Nanobodies) in Magnetospirillum gryphiswaldense▿†

    OpenAIRE

    Pollithy, Anna; Romer, Tina; Lang, Claus; Müller, Frank D.; Helma, Jonas; Leonhardt, Heinrich; Rothbauer, Ulrich; Schüler, Dirk

    2011-01-01

    Numerous applications of conventional and biogenic magnetic nanoparticles (MNPs), such as in diagnostics, immunomagnetic separations, and magnetic cell labeling, require the immobilization of antibodies. This is usually accomplished by chemical conjugation, which, however, has several disadvantages, such as poor efficiency and the need for coupling chemistry. Here, we describe a novel strategy to display a functional camelid antibody fragment (nanobody) from an alpaca (Lama pacos) on the surf...

  18. SERUM ANTIBODIES TO WHOLE-CELL AND RECOMBINANT ANTIGENS OF BORRELIA BURGDORFERI IN COTTONTAIL RABBITS

    OpenAIRE

    Magnarelli, Louis A.; Norris, Steven J; Fikrig, Erol

    2012-01-01

    Archived serum samples, from 95 eastern cottontail rabbits (Sylvilagus floridanus) captured in New York, New York, USA and Millbrook, New York, USA, during 1985–86, were analyzed in solid-phase enzyme-linked immunosorbent assays (ELISA) for total and class-specific immunoglobulin (Ig) M antibodies to whole-cell or recombinant antigens of Borrelia burgdorferi sensu stricto. Using a polyvalent conjugate, rabbit sera contained antibodies to whole-cell and recombinant antigens (protein [p]35, p37...

  19. Expression of recombinant multi-coloured fluorescent antibodies in gor -/trxB - E. coli cytoplasm

    OpenAIRE

    Markiv Anatoliy; Beatson Richard; Burchell Joy; Durvasula Ravi V; Kang Angray S

    2011-01-01

    Abstract Background Antibody-fluorophore conjugates are invaluable reagents used in contemporary molecular cell biology for imaging, cell sorting and tracking intracellular events. However they suffer in some cases from batch to batch variation, partial loss of binding and susceptibility to photo-bleaching. In theory, these issues can all be addressed by using recombinant antibody fused directly to genetically encoded fluorescent reporters. However, single-chain fragment variable domains link...

  20. A bicomponent Plasmodium falciparum investigational vaccine composed of protein-peptide conjugates.

    Science.gov (United States)

    Kubler-Kielb, Joanna; Majadly, Fathy; Biesova, Zuzana; Mocca, Christopher P; Guo, Chunyan; Nussenzweig, Ruth; Nussenzweig, Victor; Mishra, Satish; Wu, Yimin; Miller, Louis H; Keith, Jerry M; Liu, Teh-Yung; Robbins, John B; Schneerson, Rachel

    2010-01-19

    There is yet no licensed vaccine against malaria, a serious human disease affecting mostly children, with an annual death rate of about one million. Plasmodia, the malaria-causing parasites, have two obligatory hosts: mammals or birds, in which they multiply asexually, and mosquitoes with sexual multiplication. The most common and serious type of malaria is caused by Plasmodium falciparum. The circumsporozoite protein (CSP), a major surface antigen of sporozoites, is a protective antigen. A unique feature of P. falciparum CSP is its large central domain composed of over 30 tetrapeptide repeats of Asn-Ala-Asn-Pro (NANP). Several NANP peptide-protein conjugates were tested clinically but elicited a low level of CSP antibodies for a short duration. To provide a CSP-based candidate vaccine, we investigated recombinant CSP and NANP conjugates of various peptide lengths, with different N-terminal amino acids, bound at different ratios to various carrier proteins. Injected into mice, CSP alone and CSP or NANP conjugates induced antibodies with booster responses and were positive by the sporozoite immunofluorescent assay. The use of the mosquito stage P. falciparum ookinete surface protein, Pfs25, cross-linked onto itself as a carrier for NANP, induced in mice high levels of uniquely long-lasting antibodies to both vaccine components with secondary biological activities, that will provide immunity to liver infection by sporozoites and block transmission by mosquitoes.

  1. A mechanistic compartmental model for total antibody uptake in tumors.

    Science.gov (United States)

    Thurber, Greg M; Dane Wittrup, K

    2012-12-01

    Antibodies are under development to treat a variety of cancers, such as lymphomas, colon, and breast cancer. A major limitation to greater efficacy for this class of drugs is poor distribution in vivo. Localization of antibodies occurs slowly, often in insufficient therapeutic amounts, and distributes heterogeneously throughout the tumor. While the microdistribution around individual vessels is important for many therapies, the total amount of antibody localized in the tumor is paramount for many applications such as imaging, determining the therapeutic index with antibody drug conjugates, and dosing in radioimmunotherapy. With imaging and pretargeted therapeutic strategies, the time course of uptake is critical in determining when to take an image or deliver a secondary reagent. We present here a simple mechanistic model of antibody uptake and retention that captures the major rates that determine the time course of antibody concentration within a tumor including dose, affinity, plasma clearance, target expression, internalization, permeability, and vascularization. Since many of the parameters are known or can be estimated in vitro, this model can approximate the time course of antibody concentration in tumors to aid in experimental design, data interpretation, and strategies to improve localization. PMID:22974563

  2. ON CONJUGATES AND MODULII OF BICOMPLEX NUMBERS

    Directory of Open Access Journals (Sweden)

    JAISHREE

    2012-06-01

    Full Text Available The paper presents extensive use of complex pairs to develop the algebraic properties of bicomplex numbers and contains various aspects of finding the conjugates and modulii of bicomplex numbers.We discuss three types of conjugations and some specific modulii with complex and hyperbolic ranges. We also examine the impact of different conjugations on the principal ideals I1 and I2.

  3. Mass Spectrometry Based Mechanistic Insights into Formation of Tris Conjugates: Implications on Protein Biopharmaceutics

    Science.gov (United States)

    Kabadi, Pradeep G.; Sankaran, Praveen Kallamvalliillam; Palanivelu, Dinesh V.; Adhikary, Laxmi; Khedkar, Anand; Chatterjee, Amarnath

    2016-08-01

    We present here extensive mass spectrometric studies on the formation of a Tris conjugate with a therapeutic monoclonal antibody. The results not only demonstrate the reactive nature of the Tris molecule but also the sequence and reaction conditions that trigger this reactivity. The results corroborate the fact that proteins are, in general, prone to conjugation and/or adduct formation reactions and any modification due to this essentially leads to formation of impurities in a protein sample. Further, the results demonstrate that the conjugation reaction happens via a succinimide intermediate and has sequence specificity. Additionally, the data presented in this study also shows that the Tris formation is produced in-solution and is not an in-source phenomenon. We believe that the facts given here will open further avenues on exploration of Tris as a conjugating agent as well as ensure that the use of Tris or any ionic buffer in the process of producing a biopharmaceutical drug is monitored closely for the presence of such conjugate formation.

  4. Mass Spectrometry Based Mechanistic Insights into Formation of Tris Conjugates: Implications on Protein Biopharmaceutics

    Science.gov (United States)

    Kabadi, Pradeep G.; Sankaran, Praveen Kallamvalliillam; Palanivelu, Dinesh V.; Adhikary, Laxmi; Khedkar, Anand; Chatterjee, Amarnath

    2016-10-01

    We present here extensive mass spectrometric studies on the formation of a Tris conjugate with a therapeutic monoclonal antibody. The results not only demonstrate the reactive nature of the Tris molecule but also the sequence and reaction conditions that trigger this reactivity. The results corroborate the fact that proteins are, in general, prone to conjugation and/or adduct formation reactions and any modification due to this essentially leads to formation of impurities in a protein sample. Further, the results demonstrate that the conjugation reaction happens via a succinimide intermediate and has sequence specificity. Additionally, the data presented in this study also shows that the Tris formation is produced in-solution and is not an in-source phenomenon. We believe that the facts given here will open further avenues on exploration of Tris as a conjugating agent as well as ensure that the use of Tris or any ionic buffer in the process of producing a biopharmaceutical drug is monitored closely for the presence of such conjugate formation.

  5. Cysteine S-conjugate β-lyases

    OpenAIRE

    Arthur J. L. Cooper; Krasnikov, Boris F.; Pinto, John T.; Bruschi, Sam A.

    2010-01-01

    Cysteine S-conjugate β-lyases are pyridoxal 5′-phosphate (PLP)-containing enzymes that catalyze the conversion of cysteine S-conjugates [RSCH2CH(NH3+)CO2−] and selenium Se-conjugates [RSeCH2CH(NH3+)CO2−] that contain a leaving group in the β position to pyruvate, ammonium and a sulfur-containing fragment (RSH) or selenium-containing fragment (RSeH), respectively. At least ten PLP enzymes catalyze β-elimination reactions with such cysteine S-conjugates. All are enzymes involved in amino acid m...

  6. Heavy chain only antibodies

    DEFF Research Database (Denmark)

    Moghimi, Seyed Moein; Rahbarizadeh, Fatemeh; Ahmadvand, Davoud;

    2013-01-01

    Unlike conventional antibodies, heavy chain only antibodies derived from camel contain a single variable domain (VHH) and two constant domains (CH2 and CH3). Cloned and isolated VHHs possess unique properties that enable them to excel conventional therapeutic antibodies and their smaller antigen-...... for combating HER2+ breast cancer. © 2013 by Tabriz University of Medical Sciences.......Unlike conventional antibodies, heavy chain only antibodies derived from camel contain a single variable domain (VHH) and two constant domains (CH2 and CH3). Cloned and isolated VHHs possess unique properties that enable them to excel conventional therapeutic antibodies and their smaller antigen......-binding fragments in cancer targeting and therapy. VHHs express low immunogenicity, are highly robust and easy to manufacture and have the ability to recognize hidden or uncommon epitopes. We highlight the utility of VHH in design of new molecular, multifunctional particulate and immune cell-based systems...

  7. Engineering antibody therapeutics.

    Science.gov (United States)

    Chiu, Mark L; Gilliland, Gary L

    2016-06-01

    The successful introduction of antibody-based protein therapeutics into the arsenal of treatments for patients has within a few decades fostered intense innovation in the production and engineering of antibodies. Reviewed here are the methods currently used to produce antibodies along with how our knowledge of the structural and functional characterization of immunoglobulins has resulted in the engineering of antibodies to produce protein therapeutics with unique properties, both biological and biophysical, that are leading to novel therapeutic approaches. Antibody engineering includes the introduction of the antibody combining site (variable regions) into a host of architectures including bi and multi-specific formats that further impact the therapeutic properties leading to further advantages and successes in patient treatment. PMID:27525816

  8. Prion-Specific Antibodies Produced in Wild-Type Mice.

    Science.gov (United States)

    Heegaard, Peter M H; Bergström, Ann-Louise; Andersen, Heidi Gertz; Cordes, Henriette

    2015-01-01

    Peptide-specific antibodies produced against synthetic peptides are of high value in probing protein structure and function, especially when working with challenging proteins, including not readily available, non-immunogenic, toxic, and/or pathogenic proteins. Here, we present a straightforward method for production of mouse monoclonal antibodies (MAbs) against peptides representing two sites of interest in the bovine prion protein (boPrP), the causative agent of bovine spongiform encephalopathy ("mad cow disease") and new variant Creutzfeldt-Jakob's disease (CJD) in humans, as well as a thorough characterization of their reactivity with a range of normal and pathogenic (misfolded) prion proteins. It is demonstrated that immunization of wild-type mice with ovalbumin-conjugated peptides formulated with Freund's adjuvant induces a good immune response, including high levels of specific anti-peptide antibodies, even against peptides very homologous to murine protein sequences. In general, using the strategies described here for selecting, synthesizing, and conjugating peptides and immunizing 4-5 mice with 2-3 different peptides, high-titered antibodies reacting with the target protein are routinely obtained with at least one of the peptides after three to four immunizations with incomplete Freund's adjuvant. PMID:26424281

  9. In vitro detection of human breast cancer cells (SK-BR3) using herceptin-conjugated liquid crystal microdroplets as a sensing platform.

    Science.gov (United States)

    Ding, Wang; Gupta, Kailash Chandra; Park, Soo-Young; Kim, Young-Kyoo; Kang, Inn-Kyu

    2016-10-20

    The present study utilizes antibody-protein interactions to develop an LC microdroplet based biosensor for naked eye detection of SK-BR3 human breast cancer cells. The herceptin antibody-conjugated LC microdroplets were fabricated using 4-cyano-4'-pentyl biphenyl (5CB) as the liquid crystalline phase and sodium dodecyl sulfate (SDS) as the surfactant. The poly (styrene-b-acrylic acid) amphiphilic block copolymer (PS-b-PA) played a role as a modifier for the liquid crystalline interfaces. The 5CB molecules in the herceptin antibody-conjugated LC microdroplets have shown an orientation transition from radial to bipolar on selective interactions with targeted SK-BR3 breast cancer cells, which are over expressed by the human epidermal growth factor receptor 2-positive (HER2). The herceptin antibody-conjugated LC microdroplets are found to be highly selective in the detection of SK-BR3 cancer cells in the presence of control cells, such as KB cancer cells and fibroblast (FB), and also in the presence of 10% human blood plasma. The interaction forces of the SK-BR3 cancer cells were only effective in causing orientation transitions in 5CB molecules in the LC microdroplets, which clearly suggested that the herceptin antibody-conjugated LC microdroplets could be used as a selective biosensor for a real-time detection of SK-BR3 cancer cells in biological fluids.

  10. Immunotoxins, ligand-toxin conjugates and molecular targeting.

    Science.gov (United States)

    Soria, M

    1989-01-01

    Biotechnology provides tools for therapeutic exploitation following advances in the elucidation of protein-to-cell and cell-to-cell interactions. Molecular targeting of bacterial and plant toxins to the desired district of action can be achieved through effector molecules like monoclonal antibodies or protein ligands. Biochemical conjugation of these effectors to SO-6, a single-chain Ribosome Inactivating Protein from Saponaria officinalis, yielded powerful cytotoxic agents that are attractive candidates for therapeutic evaluation. Cloning of the gene for this plant toxin has been achieved. Technologies for expression of protein ligands, such as apolipoproteins or several growth factors, are available in recombinant microorganisms, providing adequate partners for the assembly of targeted chimaeras. Domain engineering of structural and functional regions in effector proteins is now possible and will be carried out with the available technologies to improve existing therapy. PMID:2698471

  11. Solution assembly of conjugated polymers

    Science.gov (United States)

    Bokel, Felicia A.

    This dissertation focuses on the solution-state polymer assembly of conjugated polymers with specific attention to nano- and molecular-scale morphology. Understanding how to control these structures holds potential for applications in polymer-based electronics. Optimization of conjugated polymer morphology was performed with three objectives: 1) segregation of donor and acceptor materials on the nanometer length-scale, 2) achieving molecular-scale ordering in terms of crystallinity within distinct domains, and 3) maximizing the number and quality of well-defined donor/acceptor interfaces. Chapter 1 introduces the development of a mixed solvent method to create crystalline poly(3-hexyl thiophene) (P3HT) fibrils in solution. Chapter 2 describes fibril purification and approaches to robust and functional fibrils, while chapters 3 and 4 demonstrate the formation of hybrid nanocomposite wires of P3HT and cadmium selenide (CdSe) nanoparticles by two methods: 1) co-crystallization of free and P3HT-grafted CdSe for composite nanowires and 2) direct attachment of CdSe nanoparticles at fibril edges to give superhighway structures. These composite structures show great potential in the application of optoelectronic devices, such as the active layer of solar cells. Finally, ultrafast photophysical characterization of these polymers, using time-resolved photoluminescence and transient absorption, was performed to determine the aggregation types present in suspended fibrils and monitor the formation and decay of charged species in fibrils and donor-acceptor systems.

  12. Subgap Absorption in Conjugated Polymers

    Science.gov (United States)

    Sinclair, M.; Seager, C. H.; McBranch, D.; Heeger, A. J; Baker, G. L.

    1991-01-01

    Along with X{sup (3)}, the magnitude of the optical absorption in the transparent window below the principal absorption edge is an important parameter which will ultimately determine the utility of conjugated polymers in active integrated optical devices. With an absorptance sensitivity of conjugated polymers poly(1,4-phenylene-vinylene) (and derivitives) and polydiacetylene-4BCMU in the spectral region from 0.55 eV to 3 eV. Our spectra show that the shape of the absorption edge varies considerably from polymer to polymer, with polydiacetylene-4BCMU having the steepest absorption edge. The minimum absorption coefficients measured varied somewhat with sample age and quality, but were typically in the range 1 cm{sup {minus}1} to 10 cm{sup {minus}1}. In the region below 1 eV, overtones of C-H stretching modes were observed, indicating that further improvements in transparency in this spectral region might be achieved via deuteration of fluorination.

  13. Purification and iodination of antibody for use in an immunoradiometric assay for serum ferritin

    International Nuclear Information System (INIS)

    Antibodies were purified by affinity chromatography and radioiodinated by a conjugation method for use in the immunoradiometric assay for serum ferritin. These procedures are simpler and more reproducible than those described earlier, and the radiolabeled preparations were usable for at least 13 weeks

  14. On-chip sample preparation by controlled release of antibodies for simple CD4 counting

    NARCIS (Netherlands)

    Beck, M.; Brockhuis, S.; Velde, van der N.; Breukers, C.; Greve, J.; Terstappen, L.W.M.M.

    2012-01-01

    We present a simple system for CD4 and CD8 counting for point-of-care HIV staging in low-resource settings. Automatic sample preparation is achieved through a dried reagent coating inside a thin (26 μm) counting chamber, allowing the delayed release of fluorochrome conjugated monoclonal antibodies a

  15. Characterization and optimization of heroin hapten-BSA conjugates: method development for the synthesis of reproducible hapten-based vaccines.

    Science.gov (United States)

    Torres, Oscar B; Jalah, Rashmi; Rice, Kenner C; Li, Fuying; Antoline, Joshua F G; Iyer, Malliga R; Jacobson, Arthur E; Boutaghou, Mohamed Nazim; Alving, Carl R; Matyas, Gary R

    2014-09-01

    A potential new treatment for drug addiction is immunization with vaccines that induce antibodies that can abrogate the addictive effects of the drug of abuse. One of the challenges in the development of a vaccine against drugs of abuse is the availability of an optimum procedure that gives reproducible and high yielding hapten-protein conjugates. In this study, a heroin/morphine surrogate hapten (MorHap) was coupled to bovine serum albumin (BSA) using maleimide-thiol chemistry. MorHap-BSA conjugates with 3, 5, 10, 15, 22, 28, and 34 haptens were obtained using different linker and hapten ratios. Using this optimized procedure, MorHap-BSA conjugates were synthesized with highly reproducible results and in high yields. The number of haptens attached to BSA was compared by 2,4,6-trinitrobenzenesulfonic acid (TNBS) assay, modified Ellman's test and matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Among the three methods, MALDI-TOF MS discriminated subtle differences in hapten density. The effect of hapten density on enzyme-linked immunosorbent assay (ELISA) performance was evaluated with seven MorHap-BSA conjugates of varying hapten densities, which were used as coating antigens. The highest antibody binding was obtained with MorHap-BSA conjugates containing 3-5 haptens. This is the first report that rigorously analyzes, optimizes and characterizes the conjugation of haptens to proteins that can be used for vaccines against drugs of abuse. The effect of hapten density on the ELISA detection of antibodies against haptens demonstrates the importance of careful characterization of the hapten density by the analytical techniques described.

  16. Targeting the insulin growth factor-1 receptor with fluorescent antibodies enables high resolution imaging of human pancreatic cancer in orthotopic mouse models

    Science.gov (United States)

    Park, Jeong Youp; Lee, Jin Young; Zhang, Yong; Hoffman, Robert M.; Bouvet, Michael

    2016-01-01

    The goal of the present study was to determine whether insulin-like growth factor-1 receptor (IGF-1R) antibodies, conjugated with bright fluorophores, could enable visualization of pancreatic cancer in orthotopic nude mouse models. IGF-1R antibody (clone 24-31) was conjugated with 550 nm or 650 nm fluorophores. Western blotting confirmed the expression of IGF-1R in Panc-1, BxPC3, and MIAPaCa-2 human pancreatic cancer cell lines. Labeling with fluorophore-conjugated IGF-1R antibody demonstrated fluorescent foci on the membrane of the pancreatic cancer cells. Subcutaneous Panc-1, BxPC-3, and MIA PaCa-2 tumors became fluorescent after intravenous administration of fluorescent IGF-1R antibodies. Orthotopically-transplanted BxPC-3 tumors became fluorescent with the conjugated IGF-1R antibodies, and were easily visible with intravital imaging. Gross and microscopic ex vivo imaging of resected pancreatic tumor and normal pancreas confirmed that fluorescence indeed came from the membrane of cancer cells, and it was stronger from the tumor than the normal tissue. The present study demonstrates that fluorophore-conjugated IGF-1R antibodies can visualize pancreatic cancer and it can be used with various imaging devices such as endoscopy and laparoscopy for diagnosis and fluorescence-guided surgery. PMID:26919100

  17. Probing Antigen-Antibody Interaction Using Fluorescence Coupled Capillary Electrophoresis

    Directory of Open Access Journals (Sweden)

    Pengju Jiang

    2013-09-01

    Full Text Available In this report, the use of fluorescence detection coupled capillary electrophoresis (CE-FL allowed us to fully characterize the antigen-antibody interaction. CE-FL allowed separation of unbound quantum dots (QDs and ligand bound QDs and also revealed an ordered assembly of biomolecules on QDs. Further, we observed FRET from QDs donor to DyLight acceptor, which were covalently conjugated with human IgG and goat anti-human IgG, respectively. The immunocomplex was formed and the mutual affinity of the antigen and antibody brought QDs and DyLight close enough to allow FRET to occur. This novel CE-based technique can be easily extended to other FRET systems based on QDs and may have potential application in the detection of antibodies.

  18. Localization at high resolution of antibody-induced mobilization of vaccinia virus hemagglutinin and the major histocompatibility antigens on the plasma membrane of infected cells

    OpenAIRE

    1982-01-01

    We examined the consequence of simultaneous or independent binding of monospecific antibody to the hemagglutinin (HA) of vaccinia virus and the A-, B- and -determinants of HLA on HeLa or Raji cells or KkDk determinants of H-2 on L929 cells. The bound antibodies were marked by goat-anti-mouse (GAM) or goat-anti-rabbit (GAR) fluorochrome conjugates suitable for light microscopy and GAM or GAR gold conjugates, used in electron microscopy. Specificity and amount of antibody adsorbed was ascertain...

  19. Potentiation of NK cytotoxicity by antibody-C3b/iC3b heteroconjugates.

    Science.gov (United States)

    Yefenof, E; Benizri, R; Reiter, Y; Klein, E; Fishelson, Z

    1990-02-15

    The interaction of two Burkitt lymphoma lines, Raji and Rael, with human C and NK cells was analyzed. Raji cells activate the alternative C pathway (ACP) and then bind C3 fragments. Consequently, the cells become more sensitive to lysis by CR3-bearing NK cells but not to C lysis. In contrast, Rael cells are poor ACP activators, do not bind C3 fragments, and are therefore resistant to C-dependent NK lysis. As suggested earlier, the difference between Raji and Rael could be attributed to the presence or absence of CR2, respectively, on their surface. To potentiate C- and NK-dependent lysis of target cells, we generated heteroconjugates composed of a murine antitransferrin receptor mAb and of human C C3b or iC3b. Antibody-C3b conjugates induced C3 deposition on Rael cells and elevated C3 deposition on Raji cells in human serum. Both Raji and Rael cells coated with antibody-C3b conjugates were efficiently lyzed by the cytolytic ACP in human serum. This conjugate had a small enhancing effect on target cell lysis by NK cells which could be markedly increased by combined treatment of the target cell with antibody-C3b conjugate and C5-depleted human serum. On the other hand, antibody-iC3b conjugates efficiently potentiated lysis of target cells by NK cells in the absence of serum. The iC3b-directed cytotoxicity was mediated by CR3-bearing NK effector cells. Anti-C3 but not anti-mouse Ig antibodies abrogated the activity of the antibody-iC3b conjugate. These results further demonstrate that NK cytotoxicity may be potentiated by opsonizing the target cells with C3 fragments and suggest that antibody-C3b/iC3b conjugates could be potent tools for targeting and potentiation of the lytic action of both C and NK cells against tumor cells. PMID:2303717

  20. CONJUGATE-SYMPLECTICITY OF LINEAR MULTISTEP METHODS

    Institute of Scientific and Technical Information of China (English)

    Ernst Hairer

    2008-01-01

    For the numerical treatment of Hamiltonian differential equations, symplectic integra-tors are the most suitable choice, and methods that are conjugate to a symplectic integrator share the same good long-time behavior. This note characterizes linear multistep methods whose underlying one-step method is conjugate to a symplectic integrator. The bounded-hess of parasitic solution components is not addressed.

  1. The Conjugate Acid-Base Chart.

    Science.gov (United States)

    Treptow, Richard S.

    1986-01-01

    Discusses the difficulties that beginning chemistry students have in understanding acid-base chemistry. Describes the use of conjugate acid-base charts in helping students visualize the conjugate relationship. Addresses chart construction, metal ions, buffers and pH titrations, and the organic functional groups and nonaqueous solvents. (TW)

  2. DENDRIMER CONJUGATES FOR SELECTIVE OF PROTEIN AGGREGATES

    DEFF Research Database (Denmark)

    2004-01-01

    Dendrimer conjugates are presented, which are formed between a dendrimer and a protein solubilising substance. Such dendrimer conjugates are effective in the treatment of protein aggregate-related diseases (e.g. prion-related diseases). The protein solubilising substance and the dendrimer together...

  3. Kinetic models of conjugated metabolic cycles

    Science.gov (United States)

    Ershov, Yu. A.

    2016-01-01

    A general method is developed for the quantitative kinetic analysis of conjugated metabolic cycles in the human organism. This method is used as a basis for constructing a kinetic graph and model of the conjugated citric acid and ureapoiesis cycles. The results from a kinetic analysis of the model for these cycles are given.

  4. LEDs based on conjugated PPV block copolymers

    NARCIS (Netherlands)

    Brouwer, H.J.; Hilberer, A.; Krasnikov, V.V.; Werts, M.; Wildeman, J.; Hadziioannou, G.

    1997-01-01

    A way to control the bandgap in semi-conducting polymers is by preparing polymers with a partially conjugated backbone. In our laboratory, three conjugated copolymers containing PPV trimers as light emitting chromophores have been synthesized, which emit in the blue, green and orange wavelength regi

  5. CONJUGATED BLOCK-COPOLYMERS FOR ELECTROLUMINESCENT DIODES

    NARCIS (Netherlands)

    Hilberer, A; Gill, R.E; Herrema, J.K; Malliaras, G.G; Wildeman, J.; Hadziioannou, G

    1995-01-01

    In this article we review results obtained in our laboratory on the design and study of new light-emitting polymers. We are interested in the synthesis and characterisation of block copolymers with regularly alternating conjugated and non conjugated sequences. The blocks giving rise to luminescence

  6. Synthetic teichoic acid conjugate vaccine against nosocomial Gram-positive bacteria.

    Science.gov (United States)

    Laverde, Diana; Wobser, Dominique; Romero-Saavedra, Felipe; Hogendorf, Wouter; van der Marel, Gijsbert; Berthold, Martin; Kropec, Andrea; Codee, Jeroen; Huebner, Johannes

    2014-01-01

    Lipoteichoic acids (LTA) are amphiphilic polymers that are important constituents of the cell wall of many Gram-positive bacteria. The chemical structures of LTA vary among organisms, albeit in the majority of Gram-positive bacteria the LTAs feature a common poly-1,3-(glycerolphosphate) backbone. Previously, the specificity of opsonic antibodies for this backbone present in some Gram-positive bacteria has been demonstrated, suggesting that this minimal structure may be sufficient for vaccine development. In the present work, we studied a well-defined synthetic LTA-fragment, which is able to inhibit opsonic killing of polyclonal rabbit sera raised against native LTA from Enterococcus faecalis 12030. This promising compound was conjugated with BSA and used to raise rabbit polyclonal antibodies. Subsequently, the opsonic activity of this serum was tested in an opsonophagocytic assay and specificity was confirmed by an opsonophagocytic inhibition assay. The conjugated LTA-fragment was able to induce specific opsonic antibodies that mediate killing of the clinical strains E. faecalis 12030, Enterococcus faecium E1162, and community-acquired Staphylococcus aureus strain MW2 (USA400). Prophylactic immunization with the teichoic acid conjugate and with the rabbit serum raised against this compound was evaluated in active and passive immunization studies in mice, and in an enterococcal endocarditis rat model. In all animal models, a statistically significant reduction of colony counts was observed indicating that the novel synthetic LTA-fragment conjugate is a promising vaccine candidate for active or passive immunotherapy against E. faecalis and other Gram-positive bacteria.

  7. RBC Antibody Screen

    Science.gov (United States)

    ... the baby is Rh-positive and the mother's antibody status is negative for anti-D, the mother is given additional RhIG. This test also may be used to help diagnose autoimmune-related hemolytic anemia ... when a person produces antibodies against his or her own RBC antigens. This ...

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

    DEFF Research Database (Denmark)

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

    1993-01-01

    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....

  9. Switching assay as a novel approach for specific antigen- antibody interaction analysis using magnetic nanoparticles

    Science.gov (United States)

    Parr, M.; Illarionov, R.; Marchenko, Y.; Yakovleva, L.; Nikolaev, B.; Ischenko, A.; Shevtsov, M.

    2016-08-01

    Switching assay was applied for the detection of antigen-antibody interaction between 70-kDa heat shock protein (Hsp70) and anti-Hsp70 monoclonal antibodies in water solutions using conjugates with magnetic iron oxide nanoparticles (MNPs). Hsp70 is a ubiquitous intracellular protein that plays a crucial role in cancerogenesis and many other pathologies. Detection of the Hsp70 level in the biological fluids might have a prognostic and diagnostic value in clinic. The developed switch assay for the detection of Hsp70 demonstrated high sensitivity for antigen-antibody interaction analysis thus proving its potential for further preclinical and clinical studies.

  10. Theoretical analysis and simulation of conjugate heights for dual-conjugate AO system in lidar

    Institute of Scientific and Technical Information of China (English)

    Xueke Ding; Jian Rong; Hong Bai; Xiu Wang; Jine Shen; Fang Li

    2008-01-01

    A multi-conjugate adaptive optics (MCAO) can offer a possibility of widening field of view (FOV) characterized by the isoplanatic angle, and the choose of conjugate height becomes a basic problem for MCAO,which influences the size of iosplanatic angle. Considering the application of lidar, the isoplanatic angle's expressions of two deformable mirrors (DMs) MCAO for uplink and downlink are deduced. The effects of conjugate heights for dual-conjugate AO are thoughtfully discussed, and the isoplanatic angles are further analyzed. The results show that the isopanatic angle varies with the conjugate height and reaches the maximum as the conjugate height is at the optimal altitude. Moreover, the optimal conjugate height changes with the propagation distance.

  11. Immunization of newborns with bacterial conjugate vaccines.

    Science.gov (United States)

    van den Biggelaar, Anita H J; Pomat, William S

    2013-05-17

    Bacterial conjugate vaccines are based on the principle of coupling immunogenic bacterial capsular polysaccharides to a carrier protein to facilitate the induction of memory T-cell responses. Following the success of Haemophilus influenzae type b conjugate vaccines in the 1980s, conjugate vaccines for Streptococcus pneumoniae and Neisseria meningitidis infections were developed and proven to be effective in protecting children against invasive disease. In this review, the use of conjugate vaccines in human newborns is discussed. Neonatal Haemophilus influenzae type b and pneumococcal conjugate vaccination schedules have been trialed and proven to be safe, with the majority of studies demonstrating no evidence for the induction of immune tolerance. Whether their neonatal administration also results in an earlier induction of clinical protection in the first 2-3 critical months of life is still to be demonstrated. PMID:22728221

  12. Selection of Recombinant Human Antibodies.

    Science.gov (United States)

    Tomszak, Florian; Weber, Susanne; Zantow, Jonas; Schirrmann, Thomas; Hust, Michael; Frenzel, André

    2016-01-01

    Since the development of therapeutic antibodies the demand of recombinant human antibodies is steadily increasing. Traditionally, therapeutic antibodies were generated by immunization of rat or mice, the generation of hybridoma clones, cloning of the antibody genes and subsequent humanization and engineering of the lead candidates. In the last few years, techniques were developed that use transgenic animals with a human antibody gene repertoire. Here, modern recombinant DNA technologies can be combined with well established immunization and hybridoma technologies to generate already affinity maturated human antibodies. An alternative are in vitro technologies which enabled the generation of fully human antibodies from antibody gene libraries that even exceed the human antibody repertoire. Specific antibodies can be isolated from these libraries in a very short time and therefore reduce the development time of an antibody drug at a very early stage.In this review, we describe different technologies that are currently used for the in vitro and in vivo generation of human antibodies. PMID:27236551

  13. Anti-insulin antibody test

    Science.gov (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

  14. Photoconductive properties of conjugated polymers

    CERN Document Server

    Halls, J J M

    1997-01-01

    The research described in my dissertation has involved the fabrication and characterisation of photovoltaic cells based on conjugated polymers, including the widely studied polymer poly(p-phenylenevinylene). These materials have semiconducting properties which arise from the delocalisation of electrons along the pi-electron systems of the polymer chains. Research into these materials is motivated both by their novel electronic properties, and also their potential for use in a wide range of applications including light-emitting diodes (LEDs), thin-film transistors, and photovoltaic cells (solar cells and light detectors). Light absorbed in a photovoltaic cell generates opposite charges which are collected at two different electrodes, giving rise to an electric current

  15. Distribution of anticancer antibiotic daunomycin in the rat heart and kidney revealed by immunocytochemistry using monoclonal antibodies

    DEFF Research Database (Denmark)

    Fujiwara, Kunio; Shin, Masashi; Hougaard, David M.;

    2007-01-01

    Two monoclonal antibodies (ADM-1-11 and 79-31 mAbs) were raised against daunomycin (DM) conjugated to bovine serum albumin via the cross-linker N-(gamma-maleimidobutyryloxy)succinimide. The monoclonal antibodies (mAbs) specifically detected DM as well as its analogs doxorubicin and epirubicin......, but did not react with other anticancer antibiotics, including pepleomycin, mitomycin C, and actinomycin D. The mAbs reacted strongly with glutaraldehyde-conjugated DM in an enzyme linked immunosorbent assay (ELISA) used as a model system for immunocytochemistry as well as in appropriately pretreated...

  16. Optimization of the DTPA mixed-anhydride reaction with antibodies at low concentration

    International Nuclear Information System (INIS)

    Diethylenetriaminepentaacetic acid (DTPA) was conjugated with antibody to human serum albumin (Ab) at low concentration (300 μg/ml, 2.0 μM/ via the DTPA carboxycarbonyl mixed-anhydride method. To study parameters determining the balance between the degree of conjugation and the antibody-binding activaty of Ab, a known concentration of the anhydride prepared at isobutylchloroformate (IBC)-to-DTPA ratios of 1, 2.1, or 4.2 was reacted with Ab. The percentage yields of the anhydride were determined by spectrophotometric and gravimetric titration. By the former method the percentage yields, based on DTPA concentration, were 18, 24, and 220, respectively, when the IBC-to-DTPA ratios were 1, 2.1, and 4.2. The corresponding percentage yields were 17, 39, and 262 when determined by the latter method. When the anhydride was prepared at an IBC-to-DTPA ratio of 2.1, an optimum conjugation giving three indium atoms per Ab was obtained, wtih 64% retention of antibody-binding activity. For an IBC-to-DTPA ratio of 1, the antibody retained almost 100% binding activity but the number of indium atoms incorporated (0.2) was too small. For an IBC-to-DTPA ratio of 4.2, up to 22 indium atoms were incorporated but antibody-binding activity was completely destroyed

  17. The generation of rhenium-188-labeled antibodies by direct labeling methods

    International Nuclear Information System (INIS)

    Rhenium-188 having similar chemistry to Tc-99m and favorable decay properties, is an attractive agent for radioimmunotherapy, despite the greater difficulties in antibody labeling with this element. The authors have succeeded in generating a reproducible process for the production of 188Re-IgC conjugates in near quantitative yield with highly preserved immunoreactivity. Incubation of perrhenate with a thiol-containing antibody in the presence of a reductant gives rise to radiolabeled antibody in yields approaching > 95% at 1-3 hr time periods, with unreduced perrhenate as the only other species. 188Re from a 188W/188Re generator system has been used to label antibody with a specific activity up to 15 mCi/mg. Animal biodistribution in LS174T tumor bearing nude mice out to 96 hours verified its stability with good tumor/non-tumor ratios being seen, while the strong uptake and retention in the tumor further reinforced this conclusion. Use of this approach, with the readily available 188Re source from the generator, gives a clinically viable procedure for the generation of 188Re antibody conjugates ready for immediate therapeutic use in as simple a manner as the corresponding technetium conjugates are now used for radioimmunodetection

  18. Homogeneous plate based antibody internalization assay using pH sensor fluorescent dye.

    Science.gov (United States)

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

    2016-04-01

    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

  19. Referencing cross-reactivity of detection antibodies for protein array experiments.

    Science.gov (United States)

    Lemass, Darragh; O'Kennedy, Richard; Kijanka, Gregor S

    2016-01-01

    Protein arrays are frequently used to profile antibody repertoires in humans and animals. High-throughput protein array characterisation of complex antibody repertoires requires a platform-dependent, lot-to-lot validation of secondary detection antibodies. This article details the validation of an affinity-isolated anti-chicken IgY antibody produced in rabbit and a goat anti-rabbit IgG antibody conjugated with alkaline phosphatase using protein arrays consisting of 7,390 distinct human proteins. Probing protein arrays with secondary antibodies in absence of chicken serum revealed non-specific binding to 61 distinct human proteins. The cross-reactivity of the tested secondary detection antibodies points towards the necessity of platform-specific antibody characterisation studies for all secondary immunoreagents. Secondary antibody characterisation using protein arrays enables generation of reference lists of cross-reactive proteins, which can be then excluded from analysis in follow-up experiments. Furthermore, making such cross-reactivity lists accessible to the wider research community may help to interpret data generated by the same antibodies in applications not related to protein arrays such as immunoprecipitation, Western blots or other immunoassays. PMID:27335636

  20. Radiolabeled Cetuximab Conjugates for EGFR Targeted Cancer Diagnostics and Therapy

    Directory of Open Access Journals (Sweden)

    Wiebke Sihver

    2014-03-01

    Full Text Available The epidermal growth factor receptor (EGFR has evolved over years into a main molecular target for the treatment of different cancer entities. In this regard, the anti-EGFR antibody cetuximab has been approved alone or in combination with: (a chemotherapy for treatment of colorectal and head and neck squamous cell carcinoma and (b with external radiotherapy for treatment of head and neck squamous cell carcinoma. The conjugation of radionuclides to cetuximab in combination with the specific targeting properties of this antibody might increase its therapeutic efficiency. This review article gives an overview of the preclinical studies that have been performed with radiolabeled cetuximab for imaging and/or treatment of different tumor models. A particularly promising approach seems to be the treatment with therapeutic radionuclide-labeled cetuximab in combination with external radiotherapy. Present data support an important impact of the tumor micromilieu on treatment response that needs to be further validated in patients. Another important challenge is the reduction of nonspecific uptake of the radioactive substance in metabolic organs like liver and radiosensitive organs like bone marrow and kidneys. Overall, the integration of diagnosis, treatment and monitoring as a theranostic approach appears to be a promising strategy for improvement of individualized cancer treatment.

  1. TAT Peptide and Its Conjugates: Proteolytic Stability

    Science.gov (United States)

    Grunwald, Jacob; Rejtar, Tomas; Sawant, Rupa; Wang, Zhouxi; Torchilin, Vladimir P.

    2009-01-01

    The proteolytic cleavage of TATp, TATp-PEG1000-PE conjugate (TATp-conjugate), and TATp as TATp-conjugate in mixed micelles made of TATp-conjugate and PEG5000-PE (2.5% mol of TATp-conjugate, TATp-Mic) were studied by HPLC with fluorescent detection using fluorenylmethyl chloroformate (FMOC) labeling and by MALDI-TOF MS analysis. The cleavage kinetics were analyzed in human blood plasma and in trypsin-containing phosphate buffered saline (PBS), pH 7.4, to simulate the proteolytic activity of human plasma. The trypsinolysis of free TATp, TATp-conjugate, and TATp-Mic revealed that the main initial fragmentation is an endocleavage at the carboxyl terminus resulting in an Arg-Arg (RR) dimer. The trypsinolysis followed pseudo-first-order kinetics. The cleavage of the free TATp was relatively fast with a half-life of a few minutes (t1/2 ∼ 3.5 min). The TATp-conjugate showed more stability with about a 3-fold increase in half-life (t1/2 ∼ 10 min). TATp in TATp-Mic was highly protected against proteolysis with an over 100-fold increase in half-life (t1/2 ∼ 430 min). The shielding of TATp by PEG moieties in the proposed TATp-Mic is of great importance for its potential use as a cell-penetrating moiety for multifunctional “smart” drug delivery systems with detachable PEG. PMID:19601640

  2. Measurement of Anti-Erythropoiesis-Stimulating Agent IgG4 Antibody as an Indicator of Antibody-Mediated Pure Red Cell Aplasia

    Science.gov (United States)

    Weeraratne, Dohan K.; Kuck, Andrew J.; Chirmule, Narendra

    2013-01-01

    Patients treated with erythropoietin-based erythropoiesis-stimulating agents (ESAs) can develop a rare but life-threatening condition called antibody-mediated pure red cell aplasia (amPRCA). The antibody characteristics in a nephrology patient with amPRCA include high antibody concentrations with neutralizing activity and a mixed IgG subclass including anti-ESA IgG4 antibodies. In contrast, anti-ESA IgG4 antibody is generally not detected in baseline samples and antibody-positive non-PRCA patients. Therefore, we validated a highly sensitive immunoassay on the ImmunoCAP 100 instrument to quantitate anti-ESA IgG4 antibodies using a human recombinant anti-epoetin alfa (EPO) IgG4 antibody as a calibrator. The biotinylated ESA was applied to a streptavidin ImmunoCAP, and bound anti-ESA IgG4 antibodies were detected using a β-galactosidase-conjugated mouse anti-human IgG4 antibody. The validated assay was used to detect anti-ESA IgG4 in amPRCA and non-PRCA patients. The immunoassay detected 15 ng/ml of human anti-EPO IgG4 antibody in the presence of a 200 M excess of human anti-ESA IgG1, IgG2, or IgM antibody and tolerated 2 μg/ml of soluble erythropoietin. All patient samples with confirmed amPRCA had measurable anti-ESA IgG4 antibodies. In addition, 94% (17/18) of non-PRCA patient samples were antibody negative or had below 15 ng/ml of anti-ESA IgG4 antibodies. This novel immunoassay can measure low-nanogram quantities of human anti-ESA IgG4 antibodies in the presence of other anti-ESA antibodies. An increased concentration of anti-ESA IgG4 antibody is associated with the development of amPRCA. We propose that the measurement of anti-ESA specific IgG4 antibodies may facilitate early detection of amPRCA in patients receiving all ESAs structurally related to human erythropoietin. PMID:23114696

  3. Mitigation of nonlinearities using conjugate data repetition.

    Science.gov (United States)

    Eliasson, Henrik; Johannisson, Pontus; Karlsson, Magnus; Andrekson, Peter A

    2015-02-01

    We investigate a time-domain implementation of generalized phase-conjugated twin waves which we call conjugate data repetition. A theory based on time-domain perturbation analysis explaining the mitigation of nonlinear effects is provided, and the concept is evaluated using numerical simulations. Compared to PM-QPSK at the same channel bit rate, the single-channel transmission reach in a conventional system with standard single-mode fiber of conjugate data repetition-QPSK is increased by approximately a factor of 2. PMID:25836107

  4. Modelling conjugation with stochastic differential equations

    DEFF Research Database (Denmark)

    Philipsen, Kirsten Riber; Christiansen, Lasse Engbo; Hasman, Henrik;

    2010-01-01

    Enterococcus faecium strains in a rich exhaustible media. The model contains a new expression for a substrate dependent conjugation rate. A maximum likelihood based method is used to estimate the model parameters. Different models including different noise structure for the system and observations are compared...... using a likelihood-ratio test and Akaike's information criterion. Experiments indicating conjugation on the agar plates selecting for transconjugants motivates the introduction of an extended model, for which conjugation on the agar plate is described in the measurement equation. This model is compared...

  5. Bio-Conjugates for Nanoscale Applications

    DEFF Research Database (Denmark)

    Villadsen, Klaus

    Bio-conjugates for Nanoscale Applications is the title of this thesis, which covers three different projects in chemical bio-conjugation research, namely synthesis and applications of: Lipidated fluorescent peptides, carbohydrate oxime-azide linkers and N-aryl O-R2 oxyamine derivatives. Lipidated...... fluorescent peptides were designed and synthesized as part of two collaboration projects utilizing peptide conjugates in nanoscale applications to increase understanding of the role peptides and proteins plays in biological systems. Ras proteins are membrane bound molecular switches involved in many signaling...

  6. HIV Antibody Test

    Science.gov (United States)

    ... be limited. Home Visit Global Sites Search Help? HIV Antibody and HIV Antigen (p24) Share this page: Was this page helpful? Also known as: HIV Screening Tests; AIDS Test; AIDS Screen; HIV Serology; ...

  7. Antinuclear antibody panel

    Science.gov (United States)

    ... blood may be due to: Chronic liver disease Collagen vascular disease Drug-induced lupus erythematosus Myositis (inflammatory muscle disease) ... Saunders; 2011:chap 51. Read More Antibody Arthritis Collagen vascular disease Drug-induced lupus erythematosus Liver disease Scleroderma Systemic ...

  8. Anti-cartilage antibody.

    Science.gov (United States)

    Greenbury, C L; Skingle, J

    1979-08-01

    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.

  9. Pneumococcal Serotype 19F Conjugate Vaccine Induces Cross-Protective Immunity to Serotype 19A in a Murine Pneumococcal Pneumonia Model

    OpenAIRE

    Jakobsen, Håvard; Sigurdsson, Viktor D.; Sigurdardottir, Sigurveig; Schulz, Dominique; Jonsdottir, Ingileif

    2003-01-01

    Immunization with a pneumococcal conjugate vaccine (PNC) containing serotype 19F induces cross-reactive antibodies to 19A in mice and human infants. Active immunization with PNC and passive immunization with serum samples from infants vaccinated with PNC containing serotype 19F, but not serotype 19A, protected against lung infection caused by both serotypes in a murine model.

  10. Antibody tumor penetration

    Science.gov (United States)

    Thurber, Greg M.; Schmidt, Michael M.; Wittrup, K. Dane

    2009-01-01

    Antibodies have proven to be effective agents in cancer imaging and therapy. One of the major challenges still facing the field is the heterogeneous distribution of these agents in tumors when administered systemically. Large regions of untargeted cells can therefore escape therapy and potentially select for more resistant cells. We present here a summary of theoretical and experimental approaches to analyze and improve antibody penetration in tumor tissue. PMID:18541331

  11. Expression of Recombinant Antibodies

    OpenAIRE

    Frenzel, André; Hust, Michael; Schirrmann, Thomas

    2013-01-01

    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 transg...

  12. Detection of Cryptosporidium parvum oocysts in bovine feces by monoclonal antibody capture enzyme-linked immunosorbent assay.

    OpenAIRE

    Anusz, K Z; Mason, P H; Riggs, M W; Perryman, L E

    1990-01-01

    A monoclonal antibody enzyme-linked immunosorbent assay (ELISA) was developed to detect Cryptosporidium parvum oocysts in bovine feces. Fecal oocysts were concentrated by centrifugation through Formalin-ethyl acetate solution and captured with monoclonal antibody 18.280.2 reactive with C. parvum oocysts. Captured oocysts were detected with goat anti-oocyst serum, following the addition of a peroxidase conjugate of rabbit anti-goat immunoglobulin and O-phenylenediamine substrate. The assay was...

  13. Macrophage-specific RAM11 monoclonal antibody cross-reacts with basal cells of stratified squamous epithelia.

    OpenAIRE

    Tadeusz Cichocki; Joanna Zarzecka; Alicja Furgal-Borzych; Jan A. Litwin; Grzegorz J. Lis

    2007-01-01

    RAM11 is a mouse monoclonal anti-rabbit macrophage antibody recognizing connective tissue and vascular (atheromatous tissue) macrophages. This study demonstrates a cross-reaction of RAM11 with an unknown antigen in rabbit normal epithelial cells. Formalin-fixed, paraffin sections of the New Zealand White rabbit normal skin, oral mucosa, esophagus, small intestine and lung were immunostained with RAM11 antibody followed by goat anti-mouse Cy-3-conjugated antiglobulin. RAM11-positive immunofluo...

  14. Antibody informatics for drug discovery

    DEFF Research Database (Denmark)

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

    2014-01-01

    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...

  15. Multiplexed detection of protein cancer markers on Au/Ag-barcoded nanorods using fluorescent-conjugated polymers.

    Science.gov (United States)

    Zheng, Weiming; He, Lin

    2010-07-01

    Integration of fluorescent-conjugated polymers as detection moiety with metallic striped nanorods for multiplexed detection of clinically important cancer marker proteins in an immunoassay format was demonstrated in this report. Specifically, cationic conjugated polymers were introduced to protein complexes through electrostatic binding to negatively charged double-stranded DNA, which was tagged on detection antibodies prior to antigen recognition. The intense fluorescence emission of conjugated polymers resulted in highly sensitive detection of cancer marker proteins wherein an undiluted bovine serum sample as low as approximately 25 target molecules captured on each particle was detectable. Meanwhile, the use of polymer molecules as the detection probe did not obscure the optical pattern of underlying nanorods, i.e., the encoding capability of barcoded nanorods was preserved, which allowed simultaneous detection of three cancer marker proteins with good specificity.

  16. Conjugated amplifying polymers for optical sensing applications.

    Science.gov (United States)

    Rochat, Sébastien; Swager, Timothy M

    2013-06-12

    Thanks to their unique optical and electrochemical properties, conjugated polymers have attracted considerable attention over the last two decades and resulted in numerous technological innovations. In particular, their implementation in sensing schemes and devices was widely investigated and produced a multitude of sensory systems and transduction mechanisms. Conjugated polymers possess numerous attractive features that make them particularly suitable for a broad variety of sensing tasks. They display sensory signal amplification (compared to their small-molecule counterparts) and their structures can easily be tailored to adjust solubility, absorption/emission wavelengths, energy offsets for excited state electron transfer, and/or for use in solution or in the solid state. This versatility has made conjugated polymers a fluorescence sensory platform of choice in the recent years. In this review, we highlight a variety of conjugated polymer-based sensory mechanisms together with selected examples from the recent literature.

  17. Design and Application of Antimicrobial Peptide Conjugates

    Directory of Open Access Journals (Sweden)

    Andre Reinhardt

    2016-05-01

    Full Text Available Antimicrobial peptides (AMPs are an interesting class of antibiotics characterized by their unique antibiotic activity and lower propensity for developing resistance compared to common antibiotics. They belong to the class of membrane-active peptides and usually act selectively against bacteria, fungi and protozoans. AMPs, but also peptide conjugates containing AMPs, have come more and more into the focus of research during the last few years. Within this article, recent work on AMP conjugates is reviewed. Different aspects will be highlighted as a combination of AMPs with antibiotics or organometallic compounds aiming to increase antibacterial activity or target selectivity, conjugation with photosensitizers for improving photodynamic therapy (PDT or the attachment to particles, to name only a few. Owing to the enormous resonance of antimicrobial conjugates in the literature so far, this research topic seems to be very attractive to different scientific fields, like medicine, biology, biochemistry or chemistry.

  18. Engineering antibodies for cancer therapy.

    Science.gov (United States)

    Boder, Eric T; Jiang, Wei

    2011-01-01

    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.

  19. Conjugated microporous polymers: design, synthesis and application.

    Science.gov (United States)

    Xu, Yanhong; Jin, Shangbin; Xu, Hong; Nagai, Atsushi; Jiang, Donglin

    2013-10-21

    Conjugated microporous polymers (CMPs) are a class of organic porous polymers that combine π-conjugated skeletons with permanent nanopores, in sharp contrast to other porous materials that are not π-conjugated and with conventional conjugated polymers that are nonporous. As an emerging material platform, CMPs offer a high flexibility for the molecular design of conjugated skeletons and nanopores. Various chemical reactions, building blocks and synthetic methods have been developed and a broad variety of CMPs with different structures and specific properties have been synthesized, driving the rapid growth of the field. CMPs are unique in that they allow the complementary utilization of π-conjugated skeletons and nanopores for functional exploration; they have shown great potential for challenging energy and environmental issues, as exemplified by their excellent performance in gas adsorption, heterogeneous catalysis, light emitting, light harvesting and electrical energy storage. This review describes the molecular design principles of CMPs, advancements in synthetic and structural studies and the frontiers of functional exploration and potential applications.

  20. Immunogenicity and Tolerance of a 7-Valent Pneumococcal Conjugate Vaccine in Nonresponders to the 23-Valent Pneumococcal Vaccine

    OpenAIRE

    Zielen, S; Bühring, I.; Strnad, N.; Reichenbach, J; Hofmann, D.

    2000-01-01

    There is still a lack of effective vaccination strategies for patients with a deficient antibody response to bacterial polysaccharide antigens. In an open trial, we evaluated the immunogenicity and tolerance of a new 7-valent pneumococcal conjugate vaccine in 22 infection-prone nonresponders to pneumococcal polysaccharide vaccine and 21 controls. In the patient group, nonresponsiveness was confirmed by repeated vaccination with a 23-valent pneumococcal polysaccharide vaccine. The study protoc...

  1. DNA-mediated strand displacement facilitates sensitive electronic detection of antibodies in human serums.

    Science.gov (United States)

    Dou, Baoting; Yang, Jianmei; Shi, Kai; Yuan, Ruo; Xiang, Yun

    2016-09-15

    We describe here the development of a sensitive and convenient electronic sensor for the detection of antibodies in human serums. The sensor is constructed by self-assembly formation of a mixed monolayer containing the small molecule epitope conjugated double stranded DNA probes on gold electrode. The target antibody binds the epitope on the dsDNA probe and lowers the melting temperature of the duplex, which facilitates the displacement of the antibody-linked strand of the duplex probe by an invading methylene blue-tagged single stranded DNA (MB-ssDNA) through the strand displacement reaction and leads to the capture of many MB-ssDNA on the sensor surface. Subsequent electrochemical oxidation of the methylene blue labels results in amplified current response for sensitive monitoring of the antibodies. The antibody assay conditions are optimized and the sensor exhibits a linear range between 1.0 and 25.0nM with a detection limit of 0.67nM for the target antibody. The sensor is also selective and can be employed to detect the target antibodies in human serum samples. With the advantages of using small molecule epitope as the antibody recognition element over traditional antigen, the versatile manipulability of the DNA probes and the unique properties of the electrochemical transduction technique, the developed sensor thus hold great potential for simple and sensitive detection of different antibodies and other proteins in real samples. PMID:27111124

  2. Second generation competitive enzyme immunoassay for detection of bovine antibody to Brucella abortus.

    Science.gov (United States)

    Nielsen, K; Smith, P; Yu, W L; Elmgren, C; Nicoletti, P; Perez, B; Bermudez, R; Renteria, T

    2007-09-20

    A second generation competitive enzyme immunoassay (CELISA) for detection of bovine antibody to Brucella abortus was developed. This assay was different from previously developed CELISAs in that the detection reagent used was a recombinant combination of the receptor portions of protein A and protein G, labelled with horseradish peroxidase. This eliminates the need for polyclonal anti-mouse-enzyme conjugate reagents for detection thus allowing for true standardization. The assay utilized a monoclonal antibody specific for a common epitope of the O-polysaccharide (OPS) of smooth lipopolysaccharide (SLPS) derived from B. abortus S1119.3 but which did not react with protein A/G. This monoclonal antibody was used to compete with antibody in the bovine test serum. Binding of bovine antibody to the smooth lipopolysaccharide antigen was then measured directly with the protein A/G enzyme conjugate. In this case, development of colour in the reaction was indicative of the presence of bovine antibody. The performance characteristics, sensitivity, specificity and exclusion of B. abortus S19 vaccinated animals, of the assay were very similar to those of the classical CELISA. PMID:17467200

  3. Production of antibody labeled gold nanoparticles for influenza virus H5N1 diagnosis kit development

    International Nuclear Information System (INIS)

    Preparation of colloidal gold conjugated antibodies specific for influenza A/H5N1 and its use in developing a virus A/H5N1 rapid diagnostic kit is presented. Colloidal gold nanoparticles (AuNPs) were prepared through citrate reduction. Single chain antibodies specific to H5N1 (scFv7 and scFv24) were produced using pTI2 + vector and E. coli strain HB2151. These antibodies were purified by affinity chromatography technique employing HiTrap Chelating HP columns pre-charged with Ni2 + . The method for preparation of antibody–colloidal gold conjugate was based on electrostatic force binding antibody with colloidal gold. The effect of factors such as pH and concentration of antibody has been quantitatively analyzed using spectroscopic methods after adding 1 wt% NaCl which induced AuNP aggregation. The morphological study by scanning electron microscopy (SEM) showed that the average size of the spherical AuNPs was 23 nm with uniform sizes. The spectroscopic properties of colloidal AuNPs showed the typical surface plasmon resonance band at 523 nm in UV-visible spectrum. The optimal pH of conjugated colloidal gold was found between 8.0 and 10.0. The activity of synthesized antibody labeled AuNPs for detection of H5N1 flu virus was checked by dot blot immunological method. The results confirmed the ability in detection of the A/H5N1 virus of the prepared antibody labeled gold particles and opened up the possibility of using them in manufacturing rapid detection kit for this virus. (paper)

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

    Energy Technology Data Exchange (ETDEWEB)

    Quinn, T.P.

    2003-12-31

    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

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

    International Nuclear Information System (INIS)

    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 99mTc and 188Re 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 nuclear

  6. Evaluation of components of X-ray irradiated 7-valent pneumococcal conjugate vaccine and pneumococcal vaccine polyvalent and X-ray and gamma-ray irradiated acellular pertussis component of DTaP vaccine products

    Science.gov (United States)

    May, J. C.; Rey, L.; Lee, Chi-Jen; Arciniega, Juan

    2004-09-01

    Samples of pneumococcal vaccine polyvalent, 7-valent pneumococcal conjugate vaccine, and two different diphtheria and tetanus toxoids and acellular pertussis vaccines adsorbed were irradiated with X-rays and/or gamma-rays (Co-60). Mouse IgG and IgM antibody responses (ELISA) for types 9V, 14, 18C, and 19F pneumococcal polysaccharides and conjugates indicated that the polysaccharides were more tolerant of the radiation than the conjugates. The mouse antibody response for the detoxified pertussis toxin (PT) antigen, filamentous hemagglutinin antigen (FHA), pertactin (PRN), and fimbriae types 2 and 3 (FIM) antigens for the appropriate vaccine type indicated that the antibody response was not significantly changed in the 25 kGy X-ray irradiated vaccines frozen in liquid nitrogen compared to the control vaccine.

  7. Evaluation of components of X-ray irradiated 7-valent pneumococcal conjugate vaccine and pneumococcal vaccine polyvalent and X-ray and gamma-ray irradiated acellular pertussis component of DTaP vaccine products

    International Nuclear Information System (INIS)

    Samples of pneumococcal vaccine polyvalent, 7-valent pneumococcal conjugate vaccine, and two different diphtheria and tetanus toxoids and acellular pertussis vaccines adsorbed were irradiated with X-rays and/or gamma-rays (Co-60). Mouse IgG and IgM antibody responses (ELISA) for types 9V, 14, 18C, and 19F pneumococcal polysaccharides and conjugates indicated that the polysaccharides were more tolerant of the radiation than the conjugates. The mouse antibody response for the detoxified pertussis toxin (PT) antigen, filamentous hemagglutinin antigen (FHA), pertactin (PRN), and fimbriae types 2 and 3 (FIM) antigens for the appropriate vaccine type indicated that the antibody response was not significantly changed in the 25 kGy X-ray irradiated vaccines frozen in liquid nitrogen compared to the control vaccine

  8. Evaluation of components of X-ray irradiated 7-valent pneumococcal conjugate vaccine and pneumococcal vaccine polyvalent and X-ray and gamma-ray irradiated acellular pertussis component of DTaP vaccine products

    Energy Technology Data Exchange (ETDEWEB)

    May, J.C. E-mail: may@cber.fda.gov; Rey, L. E-mail: louis.rey@bluewin.ch; Lee, C.-J.; Arciniega, Juan

    2004-10-01

    Samples of pneumococcal vaccine polyvalent, 7-valent pneumococcal conjugate vaccine, and two different diphtheria and tetanus toxoids and acellular pertussis vaccines adsorbed were irradiated with X-rays and/or gamma-rays (Co-60). Mouse IgG and IgM antibody responses (ELISA) for types 9V, 14, 18C, and 19F pneumococcal polysaccharides and conjugates indicated that the polysaccharides were more tolerant of the radiation than the conjugates. The mouse antibody response for the detoxified pertussis toxin (PT) antigen, filamentous hemagglutinin antigen (FHA), pertactin (PRN), and fimbriae types 2 and 3 (FIM) antigens for the appropriate vaccine type indicated that the antibody response was not significantly changed in the 25 kGy X-ray irradiated vaccines frozen in liquid nitrogen compared to the control vaccine.

  9. The antibody-linked chelating polymers for nuclear therapy and diagnostics

    Energy Technology Data Exchange (ETDEWEB)

    Torchilin, V.P.; Klibanov, A.L. (USSR Cardiology Research Center, Moscow (USSR))

    1991-01-01

    This review deals with the problem of protein modification with chelating polymers. The main purpose of this approach is the preparation of monoclonal antibodies labeled with heavy metal isotopes ({alpha}-, {beta}-, and {delta}-emitting metals and metals used for NMR-tomography). Traditional binding of metals with proteins via chelating agents directly coupled to protein molecule does not allow binding a high number of metal atoms per single protein molecule and can also alter protein specific properties. At the same time, metal-to-protein binding via intermediate chelating polymer makes possible the binding of several dozen metal atoms per single protein without affecting its specific properties. Moreover, the variations in polymer properties and molecular weight allow controlled modified antibody biodistribution and clearance rate. Modified antibodies can be used successfully for nuclear and NMR diagnostics and for nuclear therapy. The following problems are discussed: the chemistry of the coupling of chelating groups to polymer backbone; the binding of chelating polymers to proteins, including monoclonal antibodies; the ability of chelating polymer-to-protein conjugates to bind heavy metals; the influence of the modification on protein conformation and specific properties; the behavior of metal-containing conjugates in vivo; the practical use of conjugates obtained for radioimmunoimaging, radioimmunotherapy, NMR-tomography, and in vitro immunoassays. Future prospects of the approach are also discussed.101 references.

  10. The antibody-linked chelating polymers for nuclear therapy and diagnostics

    International Nuclear Information System (INIS)

    This review deals with the problem of protein modification with chelating polymers. The main purpose of this approach is the preparation of monoclonal antibodies labeled with heavy metal isotopes (α-, β-, and δ-emitting metals and metals used for NMR-tomography). Traditional binding of metals with proteins via chelating agents directly coupled to protein molecule does not allow binding a high number of metal atoms per single protein molecule and can also alter protein specific properties. At the same time, metal-to-protein binding via intermediate chelating polymer makes possible the binding of several dozen metal atoms per single protein without affecting its specific properties. Moreover, the variations in polymer properties and molecular weight allow controlled modified antibody biodistribution and clearance rate. Modified antibodies can be used successfully for nuclear and NMR diagnostics and for nuclear therapy. The following problems are discussed: the chemistry of the coupling of chelating groups to polymer backbone; the binding of chelating polymers to proteins, including monoclonal antibodies; the ability of chelating polymer-to-protein conjugates to bind heavy metals; the influence of the modification on protein conformation and specific properties; the behavior of metal-containing conjugates in vivo; the practical use of conjugates obtained for radioimmunoimaging, radioimmunotherapy, NMR-tomography, and in vitro immunoassays. Future prospects of the approach are also discussed.101 references

  11. Production and Characterization of Monoclonal Antibodies Against Thytoxine

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Four hybridoma cell lines (T410D11,T415611, T413A4, T409F6) producing MAbs againstthytoxine(T4) are established by using T4-conjugated bovine serum albumin as an immunogen. These monoclonal antibodies have high affinitiess and specific against T4. The association constants of these MAbs are higher than 108 L/mol. Their cross-reactivities with T3, T2 and rT3 are lower than 0.4%, 0.04% and 0.22%, respectively. The clinical application of the T4 ELISA Kit

  12. Prion-Specific Antibodies Produced in Wild-Type Mice

    DEFF Research Database (Denmark)

    Heegaard, Peter M. H.; Bergström, Ann-Louise; Andersen, Heidi Gertz;

    2015-01-01

    method for production of mouse monoclonal antibodies (MAbs) against peptides representing two sites of interest in the bovine prion protein (boPrP), the causative agent of bovine spongiform encephalopathy ("mad cow disease") and new variant Creutzfeldt-Jakob's disease (CJD) in humans, as well...... as a thorough characterization of their reactivity with a range of normal and pathogenic (misfolded) prion proteins. It is demonstrated that immunization of wild-type mice with ovalbumin-conjugated peptides formulated with Freund's adjuvant induces a good immune response, including high levels of specific anti...

  13. Probing Antigen-Antibody Interaction Using Fluorescence Coupled Capillary Electrophoresis

    OpenAIRE

    Pengju Jiang; Jiang Xia; Jingyan Li; Cheli Wang; Yue Zhang; Lin Qiu; Jianhao Wang

    2013-01-01

    In this report, the use of fluorescence detection coupled capillary electrophoresis (CE-FL) allowed us to fully characterize the antigen-antibody interaction. CE-FL allowed separation of unbound quantum dots (QDs) and ligand bound QDs and also revealed an ordered assembly of biomolecules on QDs. Further, we observed FRET from QDs donor to DyLight acceptor, which were covalently conjugated with human IgG and goat anti-human IgG, respectively. The immunocomplex was formed and the mutual affinit...

  14. Surface modification of polyacrylonitrile fiber for immobilization of antibodies and detection of analyte

    Energy Technology Data Exchange (ETDEWEB)

    Jain, Swati, E-mail: swatijain.iitd@gmail.com [Center for Biomedical Engineering, Indian Institute of Technology, New Delhi, 110016 (India); Chattopadhyay, Sruti, E-mail: srutic@hotmail.com [Center for Biomedical Engineering, Indian Institute of Technology, New Delhi, 110016 (India); Jackeray, Richa, E-mail: richajackeray.iitd@gmail.com [Center for Biomedical Engineering, Indian Institute of Technology, New Delhi, 110016 (India); Singh, Harpal, E-mail: harpal2000@yahoo.com [Center for Biomedical Engineering, Indian Institute of Technology, New Delhi, 110016 (India)

    2009-11-10

    Pendent nitrile groups of multifilamentous polyacrylonitrile (PAN) fibers were reduced to amino groups using lithium aluminum hydride for different time of reduction and amine content was estimated by performing acid-base titrations. Attenuated total reflection-fourier transform infrared spectroscopy (ATR-FTIR) and Differential Scanning Calorimetry (DSC) were used for the characterization of the generated amino groups and thermal properties of the reduced fibers, respectively. The surface morphology of the fibers after reduction and immobilization was characterized using Scanning Electron Microscope (SEM). The newly formed amino groups of the fibers were activated by using glutaraldehyde for the covalent linking of Goat anti-Rabbit IgG-HRP (GAR-HRP) antibody enzyme conjugate. Modified PAN fibers were evaluated as a matrix for sandwich ELISA by using Goat anti-Rabbit antibody (GAR-IgG), Rabbit anti-Goat (RAG-IgG) as analyte and enzyme conjugate GAR-HRP. The fibers reduced for 24 h were able to detect the analyte RAG-IgG at a concentration as low as 3.75 ng mL{sup -1} with 12% skimmed milk as blocking reagent for the optimized concentration of primary antibody GAR-IgG 3 {mu}g mL{sup -1} and peroxidase conjugate GAR-HRP dilution of 8000 fold. The sensitivity, specificity and reproducibility of the developed immunoassay was further established with antibodies present in human blood using Rabbit anti-Human (RAH-IgG) antibody and the corresponding HRP enzyme conjugate. As low as 0.1 {mu}L of human blood was sufficient to perform the assay with the modified fibers.

  15. Anti-TNP monoclonal antibodies as reagents for enzyme immunoassay (ELISA).

    Science.gov (United States)

    Léo, P; Ucelli, P; Augusto, E F; Oliveira, M S; Tamashiro, W M

    2000-12-01

    The aim of this study was to produce anti-TNP monoclonal antibodies (MAbs) that could be conjugated and used for the detection of antigen-antibody reactions, in which the antigen specific-antibody had been previously bound to trinitrophenyl (TNP). For hybridoma production, SP2/0-Ag14 cells were fused with spleen cells from mice previously immunized with TNP-ovalbumin (TNP-OVA). After 10 days, enzyme-linked immunoadsorbent assay (ELISA) was used to detect anti-TNP antibodies in the supernatants, and five cultures were found to be strictly positive for TNP. Three of these were subsequently cloned by limiting dilution, and 15 clones were chosen for expansion based on the criterion of high reactivity against TNP. Anti-TNP MAbs produced by those clones were isotyped as IgG1, and purified by Sepharose-protein G affinity cromatography from ascites developed in BALB/c mice. Two purified MAbs (1B2.1B6 and 1B2.1E12) were coupled to horseradish peroxidase (HRPO). The resulting conjugates were evaluated in ELISA tests for interferon-gamma and interleukin-4 detection, in which the secondary anti-cytokine antibodies were coupled either to TNP or biotin. The performance of anti-TNP conjugates in these assays were compared with a biotin-streptavidin/peroxidase system. Both types of conjugates were similarly able to detect cytokines with r2 (linear correlation coefficient) close to unity value. Growth studies of one of those hybridomas (1B2.1B6) yielded a specific growth rate of 0.042 h(-1) and a doubling time of 16.5 h. Data discussed here show that at least two MAbs against TNP raised in this work can be used as a reagent for enzyme immunoassays. PMID:11152399

  16. Surface modification of polyacrylonitrile fiber for immobilization of antibodies and detection of analyte

    International Nuclear Information System (INIS)

    Pendent nitrile groups of multifilamentous polyacrylonitrile (PAN) fibers were reduced to amino groups using lithium aluminum hydride for different time of reduction and amine content was estimated by performing acid-base titrations. Attenuated total reflection-fourier transform infrared spectroscopy (ATR-FTIR) and Differential Scanning Calorimetry (DSC) were used for the characterization of the generated amino groups and thermal properties of the reduced fibers, respectively. The surface morphology of the fibers after reduction and immobilization was characterized using Scanning Electron Microscope (SEM). The newly formed amino groups of the fibers were activated by using glutaraldehyde for the covalent linking of Goat anti-Rabbit IgG-HRP (GAR-HRP) antibody enzyme conjugate. Modified PAN fibers were evaluated as a matrix for sandwich ELISA by using Goat anti-Rabbit antibody (GAR-IgG), Rabbit anti-Goat (RAG-IgG) as analyte and enzyme conjugate GAR-HRP. The fibers reduced for 24 h were able to detect the analyte RAG-IgG at a concentration as low as 3.75 ng mL-1 with 12% skimmed milk as blocking reagent for the optimized concentration of primary antibody GAR-IgG 3 μg mL-1 and peroxidase conjugate GAR-HRP dilution of 8000 fold. The sensitivity, specificity and reproducibility of the developed immunoassay was further established with antibodies present in human blood using Rabbit anti-Human (RAH-IgG) antibody and the corresponding HRP enzyme conjugate. As low as 0.1 μL of human blood was sufficient to perform the assay with the modified fibers.

  17. Antibody affinity maturation

    DEFF Research Database (Denmark)

    Skjødt, Mette Louise

    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......-2. Based on the presented data we suggest that affinity maturation of the model antibody proceeds through multiple incremental steps of subtle improvements. We moreover conclude that the best affinity improved candidates are likely to be obtained through optimization of both the antigen...... 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...

  18. GH and IGF-I induction by passive immunization of rainbow trout Oncorhynchus mykiss Walbaum using a somatostatin 14 antibody

    Science.gov (United States)

    Inhibition of the growth axis by somatostatin was studied in juvenile rainbow trout using passive immunization with a previously isolated somatostatin antibody (antiSS-14). Upon subcutaneously injection of laying hens (Gallus domesticus) with conjugated somatostatin-14 (SS-14), the antiSS-14 was iso...

  19. 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)

    1979-03-15

    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.

  20. Large Scale Generation and Characterization of Anti-Human CD34 Monoclonal Antibody in Ascetic Fluid of Balb/c Mice

    Directory of Open Access Journals (Sweden)

    Koushan Sineh sepehr

    2013-02-01

    Full Text Available Purpose: Monoclonal antibodies or specific antibodies are now an essential tool of biomedical research and are of great commercial and medical value. The purpose of this study was to produce large scale of monoclonal antibody against CD34 in order to diagnostic application in leukemia and purification of human hematopoietic stem/progenitor cells. Methods: For large scale production of monoclonal antibody, hybridoma cells that produce monoclonal antibody against human CD34 were injected into the peritoneum of the Balb/c mice which have previously been primed with 0.5 ml Pristane. 5 ml ascitic fluid was harvested from each mouse in two times. Evaluation of mAb titration was assessed by ELISA method. The ascitic fluid was examined for class and subclasses by ELISA mouse mAb isotyping Kit. mAb was purified from ascitic fluid by affinity chromatography on Protein A-Sepharose. Purity of monoclonal antibody was monitored by SDS -PAGE and the purified monoclonal antibody was conjugated with FITC. Results: Monoclonal antibodies with high specificity and sensitivity against human CD34 by hybridoma technology were prepared. The subclass of antibody was IgG1 and its light chain was kappa. Conclusion: The conjugated monoclonal antibody could be a useful tool for isolation, purification and characterization of human hematopoietic stem cells.

  1. Design and characteristics of cytotoxic fibroblast growth factor 1 conjugate for fibroblast growth factor receptor-targeted cancer therapy

    Directory of Open Access Journals (Sweden)

    Szlachcic A

    2016-08-01

    Full Text Available Anna Szlachcic, Malgorzata Zakrzewska, Michal Lobocki, Piotr Jakimowicz, Jacek Otlewski Department of Protein Engineering, Faculty of Biotechnology, University of Wroclaw, Wroclaw, Poland Abstract: Fibroblast growth factor receptors (FGFRs are attractive candidate cancer therapy targets as they are overexpressed in multiple types of tumors, such as breast, prostate, bladder, and lung cancer. In this study, a natural ligand of FGFR, an engineered variant of fibroblast growth factor 1 (FGF1V, was conjugated to a potent cytotoxic drug, monomethyl auristatin E (MMAE, and used as a targeting agent for cancer cells overexpressing FGFRs, similar to antibodies in antibody–drug conjugates. The FGF1V–valine–citrulline–MMAE conjugate showed a favorable stability profile, bound FGFRs on the cell surface specifically, and efficiently released the drug (MMAE upon cleavage by the lysosomal protease cathepsin B. Importantly, the conjugate showed a prominent cytotoxic effect toward cell lines expressing FGFR. FGF1V–vcMMAE was highly cytotoxic at concentrations even an order of magnitude lower than those found for free MMAE. This effect was FGFR-specific as cells lacking FGFR did not show any increased mortality. Keywords: fibroblast growth factor 1, FGF receptor, targeted cancer therapy, cytotoxic conjugates, FGFR-dependent cancer, MMAE, auristatin

  2. Identification of highly reactive cysteine residues at less exposed positions in the Fab constant region for site-specific conjugation.

    Science.gov (United States)

    Shiraishi, Yasuhisa; Muramoto, Takashige; Nagatomo, Kazutaka; Shinmi, Daisuke; Honma, Emiko; Masuda, Kazuhiro; Yamasaki, Motoo

    2015-06-17

    Engineered cysteine residues are currently used for the site-specific conjugation of antibody-drug conjugates (ADC). In general, positions on the protein surface have been selected for substituting a cysteine as a conjugation site; however, less exposed positions (with less than 20% of accessible surface area [ASA]) have not yet been evaluated. In this study, we engineered original cysteine positional variants of a Fab fragment, with less than 20% of ASA, and evaluated their thiol reactivities through conjugation with various kinds of payloads. As a result, we have identified three original cysteine positional variants (heavy chain: Hc-A140C, light chain: Lc-Q124C and Lc-L201C), which exhibited similar monomer content, thermal stability, and antigen binding affinity in comparison to the wild-type Fab. In addition, the presence of cysteine in these positions made it possible for the Fab variants to react with variable-sized molecules with high efficiency. The favorable physical properties of the cysteine positional variants selected in our study suggest that less exposed positions, with less than 20% of ASA, provide an alternative for creating conjugation sites.

  3. Geometric and Meshing Properties of Conjugate Curves for Gear Transmission

    Directory of Open Access Journals (Sweden)

    Dong Liang

    2014-01-01

    Full Text Available Conjugate curves have been put forward previously by authors for gear transmission. Compared with traditional conjugate surfaces, the conjugate curves have more flexibility and diversity in aspects of gear design and generation. To further extend its application in power transmission, the geometric and meshing properties of conjugate curves are discussed in this paper. Firstly, general principle descriptions of conjugate curves for arbitrary axial position are introduced. Secondly, geometric analysis of conjugate curves is carried out based on differential geometry including tangent and normal in arbitrary contact direction, characteristic point, and curvature relationships. Then, meshing properties of conjugate curves are further revealed. According to a given plane or spatial curve, the uniqueness of conjugated curve under different contact angle conditions is discussed. Meshing commonality of conjugate curves is also demonstrated in terms of a class of spiral curves contacting in the given direction for various gear axes. Finally, a conclusive summary of this study is given.

  4. Preparation and Characteristic Identification of Monoclonal Antibody Against Sulfamethazine

    Institute of Scientific and Technical Information of China (English)

    DING Liangjun; LI Jichang; FU Rui; ZHOU Yanjun; HUO Guicheng

    2006-01-01

    Two artificial antigens were synthesized successfully by diazotizing method, sulfamethazine(SM2)-human serum albumin (HSA) was used for the immunogen, and SM2-ovalbumin(OVA) was used for the coating antigen.The coupled reaction was successful by confirmation of the ultraviolet scanning spectrometer, and the conjugation ratio of SM2 with HSA and OVA was 9:1 and 15:1, respectively. Using cell-fusion and limiting dilution method to reclone 5times to get 3 hybridoma strains, which could stably secret monoclonal antibody (Mab), named CB7, BC4 and BB12. The subtype of BC4 Mab was IgG1 and chain, the molecular weight was 162 ku, the numbers of chromosomal were about 90,the affinity constant was 6.1 × 1012 M-1. No cross reactivity was seen between the Mab and the other 4 sulfonamides, as well as the 2 carries proteins. The Mab antibody had excellent stability.

  5. Comparison of three different conjugation strategies in the construction of herceptin-bearing paclitaxel-loaded nanoparticles.

    Science.gov (United States)

    Yu, Kongtong; Zhou, Yulin; Li, Yuhuan; Sun, Xiangshi; Sun, Fengying; Wang, Xinmei; Mu, Hongyan; Li, Jie; Liu, Xiaoyue; Teng, Lesheng; Li, Youxin

    2016-08-19

    Research on quantitatively controlling the ligand density on the surface of nanocarriers is in the frontier and becomes a technical difficulty for targeted delivery system designing. In this study, we developed an improved pre-conjugation (Imp) strategy, in which herceptin as a ligand was pre-conjugated with DSPE-PEG2000-Mal via chemical cross-linking, followed by conjugation onto the surface of pre-prepared paclitaxel-loaded PLGA/DODMA nanoparticles (PDNs) through hydrophobic interaction and electrostatic attraction for paclitaxel delivery. Compared with the post-conjugation (Pos) strategy, in which the ligand was conjugated onto the nanoparticle surface after the preparation of the nanoparticles, it realized a precise control targeting effect via adjustment of the herceptin density on the surface of the nanoparticles. Within the range of 0-20% of DSPE-PEG2000-herceptin in the blend, it showed a linear relation with the ligand density on the surface of the nanoparticles. The Imp strategy protected the bioactivity of the ligand during the preparation of nanoparticles. At the same time it avoided the waste of an excess amount of herceptin to drive the conjugation reaction in comparison with the post-conjugation (Pos) strategy. The nanoparticles from the Imp strategy showed much better cytotoxicity (p < 0.001), tumor targeting and cellular uptake efficiency (p < 0.001) than that of the other strategies in BT474 cells, in which BT474 cells were HER2 receptor over-expression breast cancer cell lines. A significant reduction in cellular uptake of the nanoparticles from the Imp strategy was observed in the presence of sucrose and cytochalasin D, indicating that clathrin-mediated and caveolae-dependent endocytosis was as a primary mechanism of cellular entry for these antibody-modified nanoparticles. PMID:27367271

  6. Production of Group Specific Monoclonal Antibody to Aflatoxins and its Application to Enzyme-linked Immunosorbent Assay

    OpenAIRE

    Kim, Sung-Hee; Cha, Sang-Ho; Karyn, Bischoff; Park, Sung-Won; Son, Seong-Wan; KANG, HWAN-GOO

    2011-01-01

    Through the present study, we produced a monoclonal antibody against aflatoxin B1 (AFB1) using AFB1- carboxymethoxylamine BSA conjugates. One clone showing high binding ability was selected and it was applied to develop a direct competitive ELISA system. The epitope densities of AFB1-CMO against BSA and KLH were about 1 : 6 and 1 : 545, respectively. The monoclonal antibody (mAb) from cloned hybridoma cell was the IgG1 subclass with λ-type light chains. The IC50s of the monoclonal antibody de...

  7. Detection of antibodies to variant antigens on Plasmodium falciparum-infected erythrocytes by flow cytometry

    DEFF Research Database (Denmark)

    Staalsoe, T; Giha, H A; Dodoo, D;

    1999-01-01

    with ethidium-bromide-labelled mature forms of P. falciparum parasites were sequentially exposed to immune plasma, goat anti-human immunoglobulin (Ig) G, and fluorescein-isothiocyanate-conjugated rabbit anti-goat Ig. Plasma antibodies recognising antigens exposed on the surface of parasitised erythrocytes were......: Our FCM assay predominantly detects antibodies that recognise PfEMP1 and thus constitutes a convenient assay for the analysis of acquisition, maintenance, and diversity of anti-PfEMP1-specific antibodies and for the examination of class and subclass characteristics....

  8. Monoclonal antibodies in myeloma

    DEFF Research Database (Denmark)

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

    2015-01-01

    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...

  9. Prediction of Antibody Epitopes

    DEFF Research Database (Denmark)

    Nielsen, Morten; Marcatili, Paolo

    2015-01-01

    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...

  10. Complex conjugate pairs in stationary Sturmians

    International Nuclear Information System (INIS)

    Sturmian expansions enable a simply accurate separable specification of two nucleon t matrices based upon realistic two nucleon interactions. Sturmian eigenstates specified by stationary scattering boundary conditions are particularly useful in that context, and they can be calculated by solving a generalised eigenvalue equation using real and symmetric matrices. In general, the spectrum of such an equation may contain complex eigenvalues. But to each complex eigenvalues there is a corresponding conjugate partner. In studies using realistic two nucleon potentials, and in certain positive energy intervals, these complex conjugated pairs indeed appear in the Sturmian spectrum. However, it is demonstrated that it is possible to recombine the complex conjugate pairs and corresponding states into a new, (and useful) pair of real eigenvalues and eigenstates with which of effect separable expansions of the (real) two nucleon reactance matrices. 8 refs

  11. Complex conjugate pairs in stationary Sturmian eigenstates

    International Nuclear Information System (INIS)

    Sturmian eigenstates specified by stationary scattering boundary conditions are particularly useful in contexts such as forming simple separable two nucleon t matrices, and are determined via solution of generalized eigenvalue equation using real and symmetric matrices. In general, the spectrum of such an equation may contain complex eigenvalues. But to each complex eigenvalue there is a corresponding conjugate partner. In studies using realistic nucleon-nucleon potentials, and in certain positive energy intervals, these complex conjugated pairs indeed appear in the Sturmian spectrum. However, as we demonstrate herein, it is possible to recombine the complex conjugate pairs and corresponding states into a new, sign-definite pair of real quantities with which to effect separable expansions of the (real) nucleon-nucleon reactance matrices

  12. Triplex glue by synthesizing conjugated flexible intercalators.

    Science.gov (United States)

    Pedersen, Erik B; Osman, Amany M A; Globisch, Daniel; Paramasivam, Manikandan; Cogoi, Susanna; Bomholt, Niels; Jørgensen, Per T; Xodo, Luigi E; Filichev, Vyacheslav V

    2008-01-01

    Bulge insertions of conjugated intercalators into the DNA triplex structure are found to give a dramatic contribution to the triplex stability. On the other hand insertions of conjugated intercalators are found to diminish quadruplex structures and in this way breaking down the self association of G-rich oligonucleotides under physiologically potassium ion conditions. A large number of intercalators are described here and they all result in dramatic increases of thermal stability of the corresponding triplexes. Another interesting aspect of conjugated intercalators is their use for assembling alternate strand triplexes. Targeting of neighbouring purine sequences on each their strand in the duplex DNA is a challenge for the 5'- 5' connectivity of the TFOs because of a large distance between the 5'-ends. The intercalator approach offers a linkage with the proper combination of flexibility and rigidity to produce alternate strand triplexes with higher stability than a similar wild type triplex of the same total length. PMID:18776241

  13. Conjugate Meningococcal Vaccines Development: GSK Biologicals Experience

    Directory of Open Access Journals (Sweden)

    Jacqueline M. Miller

    2011-01-01

    Full Text Available Meningococcal diseases are serious threats to global health, and new vaccines specifically tailored to meet the age-related needs of various geographical areas are required. This paper focuses on the meningococcal conjugate vaccines developed by GSK Biologicals. Two combined conjugate vaccines were developed to help protect infants and young children in countries where the incidence of meningococcal serogroup C or serogroup C and Y disease is important: Hib-MenC-TT vaccine, which offers protection against Haemophilus influenzae type b and Neisseria meningitidis serogroup C diseases, is approved in several countries; and Hib-MenCY-TT vaccine, which adds N. meningitidis serogroup Y antigen, is currently in the final stages of development. Additionally, a tetravalent conjugate vaccine (MenACWY-TT designed to help protect against four meningococcal serogroups is presently being evaluated for global use in all age groups. All of these vaccines were shown to be highly immunogenic and to have clinically acceptable safety profiles.

  14. Tight-binding treatment of conjugated polymers

    DEFF Research Database (Denmark)

    Lynge, Thomas Bastholm

    This PhD thesis concerns conjugated polymers which constitute a constantly growing research area. Today, among other things, conjugated polymers play a role in plastic based solar cells, photodetectors and light emitting diodes, and even today such plastic-based components constitute an alternative...... of tomorrow. This thesis specifically treats the three conjugated polymers trans-polyacetylene (tPA), poly(para-phenylene) (PPP) and poly(para-phe\\-nylene vinylene) (PPV). The present results, which are derived within the tight-binding model, are divided into two parts. In one part, analytic results...... are derived for the optical properties of the polymers expressed in terms of the optical susceptibility both in the presence and in the absence of a static electric field. In the other part, the cumputationally efficient Density Functional-based Tight-Binding (DFTB) model is applied to the description...

  15. Conjugate gradient algorithms using multiple recursions

    Energy Technology Data Exchange (ETDEWEB)

    Barth, T.; Manteuffel, T.

    1996-12-31

    Much is already known about when a conjugate gradient method can be implemented with short recursions for the direction vectors. The work done in 1984 by Faber and Manteuffel gave necessary and sufficient conditions on the iteration matrix A, in order for a conjugate gradient method to be implemented with a single recursion of a certain form. However, this form does not take into account all possible recursions. This became evident when Jagels and Reichel used an algorithm of Gragg for unitary matrices to demonstrate that the class of matrices for which a practical conjugate gradient algorithm exists can be extended to include unitary and shifted unitary matrices. The implementation uses short double recursions for the direction vectors. This motivates the study of multiple recursion algorithms.

  16. Conjugation in hydrogen-bonded systems

    CERN Document Server

    Novakovskaya, Yulia V

    2012-01-01

    Analysis of the electron density distribution in clusters composed of hydrogen fluoride, water, and ammonia molecules, especially within the hydrogen-bond domains, reveals the existence of both \\sigma- and \\pi-binding between molecules. The \\sigma-kind density distribution determines the mutual orientation of molecules. A \\pi-system may be delocalized conjugated, which provides additional stabilization of molecular clusters. In those clusters where the sequence of hydrogen bonds is not planar, a peculiar kind of \\pi-conjugation exists. HF anion and H5O2 cation are characterized by quasi-triple bonds between the electronegative atoms. The most long-lived species stabilized by delocalized \\pi-binding are rings and open or closed hoops composed of fused rings. It is conjugated \\pi-system that determines cooperativity phenomenon.

  17. Antibodies to benzo(a)pyrene and their use in immunoassays

    Energy Technology Data Exchange (ETDEWEB)

    Murchison, C.M.

    1984-12-10

    Antibodies to benzo(a)pyrene were produced by coupling the carcinogen to bovine serum albumin and immunizing New Zealand white rabbits and Fischer rats with the conjugate. Sera was collected from blood samples and assayed to determine antibody titer using the following procedures: Ouchterlony double diffusion, passive hemagglutination, and an enzyme immunoassay (EIA). Results showed significant antibody titer, the highest found in samples taken from our subcutaneously injected rabbit. Sera from this rabbit showed a titer of 1/256 in the hemagglutination assay and a titer of 1/15,625 in the EIA. Isolated specific antibodies will be used in the development of fluoroimmuno sensor. 21 references, 4 figures, 3 tables.

  18. Production and characterization of polyclonal antibody against a synthetic peptide from β-actin protein

    Directory of Open Access Journals (Sweden)

    Nazila Amini

    2014-06-01

    Full Text Available Objective(s:Antibodies against actin, as one of the most widely studied structural and multifunctional housekeeping proteins in eukaryotic cells, are used as internal loading controls in western blot analyses. The aim of this study was to produce polyclonal antibody against a synthetic peptide derived from N-terminal region of β-actin protein to be used as a protein loading control in western blot and other assay systems. Materials and Methods: A synthetic peptide derived from β-actin protein was designed and conjugated to Keyhole limpet hemocyanin (KLH (and used to immunize a white New Zealand rabbit. The antibody was purified from serum by affinity chromatography column. The purity of the antibody was determined by SDS-PAGE and its ability to recognize the immunizing peptide was measured by ELISA. The reactivity of the antibody with β-actin protein in a panel of different cell lysates was then evaluated by western blot. In addition, the reactivity of the antibody with the corresponding protein was also evaluated by Immunocytochemistry and Immunohistochemistry in different samples. Results: The antibody could recognize the immunizing peptide in ELISA. It could also recognize            β-actin protein in western blot as well as in immunocytochemistry and immunohistochemistry. Conclusion: Our data suggest that this antibody may be used as an internal control in western blot analyses as well as in other immunological applications such as ELISA,immunocytochemistry and immunohistochemistry.

  19. Production of anti-idiotype antibodies for deoxynivalenol and their evaluation with three immunoassay platforms.

    Science.gov (United States)

    Maragos, C M

    2014-05-01

    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.

  20. Towards a peptide-based suspension array for the detection of pestivirus antibodies in swine.

    Science.gov (United States)

    van der Wal, Fimme J; Jelsma, Tinka; Fijten, Helmi; Achterberg, René P; Loeffen, Willie L A

    2016-09-01

    Classical swine fever (CSF) is a highly contagious and lethal disease in swine. Serological tests for the diagnosis of CSF need not only to detect antibodies against CSFV, but also need to differentiate these from antibodies against other pestiviruses. To investigate the possibilities of specific peptide-based serology, various synthetic peptides that represent a well-described linear epitope of the CSFV E2 protein (TAVSPTTLR) were used to test the viability of a peptide-based suspension array for the detection of antibodies against pestiviruses in swine. The results show that N-terminally biotinylated peptides can bind to avidin conjugated beads, and function in detection of the corresponding monoclonal antibody WH303. There are indications that the length of the spacer between epitope and biotin affect the efficiency of the peptide-antibody interaction. A protocol was established that enables probing for antibodies in porcine sera, where neutravidin-blocking of serum and the use of empty control beads for normalization was crucial. With a set of porcine sera with antibodies against various pestiviruses, the proof of concept of a peptide-based suspension array for specific detection of antibodies against pestiviruses in porcine sera was demonstrated. PMID:27166561

  1. Lupus anticoagulants and antiphospholipid antibodies

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/000547.htm 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 ...

  2. Conjugation of alginate to a synthetic peptide containing T- and B-cell epitopes as an induction for protective immunity against Pseudomonas aeruginosa.

    Science.gov (United States)

    Farjaha, Ali; Owlia, Parviz; Siadat, Seyed Davar; Mousavi, Seyed Fazlollah; Shafieeardestani, Mehdi

    2014-12-20

    Pseudomonas aeruginosa is a major cause of respiratory tract infections worldwide, particularly in hospitalized patients with immunosuppressed conditions and cystic fibrosis (CF). Excessive use of antibiotics means that there is currently resistance among bacterial infections to many drugs. Vaccination is a strategy that can reduce mortality and morbidity rates in infections such as those caused by P. aeruginosa. Alginate has a critical role in such infections and affects pathogenicity of the bacterium. In this work, the bioinformatics approach was used to design and synthesis a carrier peptide (ERRANAVRDVLVNEY), derived from OMP F P. aeruginosa. This peptide contained both B- and T-cell epitopes based on prediction models. Conjugation of alginate to carrier peptide was performed and then analyzed by Fourier transform infrared spectroscopy (FTIR). Results of this study on mice showed that the conjugate elicited anti-alginate-IgG that were not detected after immunization with naive alginate. The effect of the antibodies to alginate conjugate was evaluated as highly opsonic and showed moderate to high-level killing activity against two mucoid strains. IgG1 was also dominant among IgG subclasses. Mice vaccinated with the conjugate vaccine survived lethal challenges (2 ×LD 50). Furthermore, using an acute pneumonia model of infection in mice, determined that levels of P. aeruginosa in mice were significantly reduced in the vaccinated group. Thus, tests confirmed ability of this conjugate to elicit protective and opsonophagocytic antibodies that candidate our vaccine for further studies. PMID:25449544

  3. Phase conjugation of high energy lasers.

    Energy Technology Data Exchange (ETDEWEB)

    Bliss, David E; Valley, Michael T.; Atherton, Briggs W.; Bigman, Verle Howard; Boye, Lydia Ann; Broyles, Robin Scott; Kimmel, Mark W.; Law, Ryan J.; Yoder, James R.

    2013-01-01

    In this report we explore claims that phase conjugation of high energy lasers by stimulated Brillouin scattering (SBS) can compensate optical aberrations associated with severely distorted laser amplifier media and aberrations induced by the atmosphere. The SBS media tested was a gas cell pressurized up to 300 psi with SF6 or Xe or both. The laser was a 10 Hz, 3J, Q-switched Nd:YAG with 25 ns wide pulses. Atmospheric aberrations were created with space heaters, helium jets and phase plates designed with a Kolmogorov turbulence spectrum characterized by a Fried parameter, ro , ranging from 0.6 6.0 mm. Phase conjugate tests in the laboratory were conducted without amplification. For the strongest aberrations, D/ro ~ 20, created by combining the space heaters with the phase plate, the Strehl ratio was degraded by a factor of ~50. Phase conjugation in SF6 restored the peak focusable intensity to about 30% of the original laser. Phase conjugate tests at the outdoor laser range were conducted with laser amplifiers providing gain in combination with the SBS cell. A large 600,000 BTU kerosene space heater was used to create turbulence along the beam path. An atmospheric structure factor of Cn2 = 5x10-13 m2/3 caused the illumination beam to expand to a diameter 250mm and overfill the receiver. The phase conjugate amplified return could successfully be targeted back onto glints 5mm in diameter. Use of a lenslet arrays to lower the peak focusable intensity in the SBS cell failed to produce a useful phase conjugate beam; The Strehl ratio was degraded with multiple random lobes instead of a single focus. I will review literature results which show how multiple beams can be coherently combined by SBS when a confocal reflecting geometry is used to focus the laser in the SBS cell.

  4. A Cell-Based Internalization and Degradation Assay with an Activatable Fluorescence-Quencher Probe as a Tool for Functional Antibody Screening.

    Science.gov (United States)

    Li, Yan; Liu, Peter Corbett; Shen, Yang; Snavely, Marshall D; Hiraga, Kaori

    2015-08-01

    For the development of therapeutically potent anti-cancer antibody drugs, it is often important to identify antibodies that internalize into cells efficiently, rather than just binding to antigens on the cell surface. Such antibodies can mediate receptor endocytosis, resulting in receptor downregulation on the cell surface and potentially inhibiting receptor function and tumor growth. Also, efficient antibody internalization is a prerequisite for the delivery of cytotoxic drugs into target cells and is critical for the development of antibody-drug conjugates. Here we describe a novel activatable fluorescence-quencher pair to quantify the extent of antibody internalization and degradation in the target cells. In this assay, candidate antibodies were labeled with a fluorescent dye and a quencher. Fluorescence is inhibited outside and on the surface of cells, but activated upon endocytosis and degradation of the antibody. This assay enabled the development of a process for rapid characterization of candidate antibodies potentially in a high-throughput format. By employing an activatable secondary antibody, primary antibodies in purified form or in culture supernatants can be screened for internalization and degradation. Because purification of candidate antibodies is not required, this method represents a direct functional screen to identify antibodies that internalize efficiently early in the discovery process. PMID:26024945

  5. Recombinant antibodies and tumor targeting

    OpenAIRE

    Sheikholvaezin, Ali

    2006-01-01

    Different antibody derived constructs are rapidly advancing as putative tools for treatment of malignant diseases. Antibody engineering has added significant new technologies to modify size, affinities, solubility, stability and biodistribution properties for immunoconjugates. In the present thesis, the aim was to increase our knowledge on how new recombinant antibodies could be tailored to optimize localization to experimental tumors in mice. One hybridoma, producing the monoclonal antibody ...

  6. Half-Antibody Functionalized Lipid-Polymer Hybrid Nanoparticles for Targeted Drug Delivery to Carcinoembryonic Antigen (CEA) Presenting Pancreatic Cancer Cells

    Science.gov (United States)

    Hu, Che-Ming Jack; Kaushal, Sharmeela; Tran Cao, Hop S.; Aryal, Santosh; Sartor, Marta; Esener, Sadik; Bouvet, Michael; Zhang, Liangfang

    2010-01-01

    Current chemotherapy regimens against pancreatic cancer are met with little success as poor tumor vascularization significantly limits the delivery of oncological drugs. High-dose targeted drug delivery, through which a drug delivery vehicle releases a large payload upon tumor localization, is thus a promising alternative strategy against this lethal disease. Herein, we synthesize anti-CEA half-antibody conjugated lipid-polymer hybrid nanoparticles and characterize their ligand conjugation yields, physicochemical properties, and targeting ability against pancreatic cancer cells. Under the same drug loading, the half-antibody targeted nanoparticles show enhanced cancer killing effect compared to the corresponding non-targeted nanoparticles. PMID:20394436

  7. Synthesis of cyanopyridine based conjugated polymer

    Directory of Open Access Journals (Sweden)

    B. Hemavathi

    2016-06-01

    Full Text Available This data file contains the detailed synthetic procedure for the synthesis of two new cyanopyridine based conjugated polymer P1 and P2 along with the synthesis of its monomers. The synthesised polymers can be used for electroluminescence and photovoltaic (PV application. The physical data of the polymers are provided in this data file along with the morphological data of the polymer thin films. The data provided here are in association with the research article entitled ‘Cyanopyridine based conjugated polymer-synthesis and characterisation’ (Hemavathi et al., 2015 [3].

  8. Conjugated polymers as actuators: modes of actuation

    DEFF Research Database (Denmark)

    Skaarup, Steen

    2007-01-01

    The physical and chemical properties of conjugated polymers often depend very strongly on the degree of doping with anions or cations. The movement of ions in and out of the polymer matrix as it is redox cycled is also accompanied by mechanical changes. Both the volume and the stiffness can exhibit...... significant differences between the oxidized and reduced states. These effects form the basis of the use of conjugated polymers as actuators (or “artificial muscles”) controllable by a small (1-10 V) voltage. Three basic modes of actuation (bending, linear extension and stiffness change) have been proposed...

  9. Conjugated Polymers as Actuators: Modes of Actuation

    DEFF Research Database (Denmark)

    Skaarup, Steen

    The physical and chemical properties of conjugated polymers often depend very strongly on the degree of doping with anions or cations. The movement of ions in and out of the polymer matrix as it is redox cycled is also accompanied by mechanical changes. Both the volume and the stiffness can exhibit...... significant differences between the oxidized and reduced states. These effects form the basis of the use of conjugated polymers as actuators (or “artificial muscles”) controllable by a small (1-10 V) voltage. Three basic modes of actuation (bending, linear extension and stiffness change) have been proposed...

  10. Dynamics of Photogenerated Polarons in Conjugated Polymers

    Science.gov (United States)

    An, Z.; Wu, C. Q.; Sun, X.

    2004-11-01

    Within a tight-binding electron-phonon interacting model, we investigate the dynamics of photoexcitations to address the generation mechanism of charged polarons in conjugated polymers by using a nonadiabatic evolution method. Besides the neutral polaron exciton which is well known, we identify a novel product of lattice dynamic relaxation from the photoexcited states in a few hundreds of femtoseconds, which is a mixed state composed of both charged polarons and neutral excitons. Our results show that the charged polarons are generated directly with a yield of about 25%, which is independent of the excitation energies, in good agreement with results from experiments. Effects of the conjugation length are also discussed.

  11. Theoretical study of conjugated porphyrin polymers

    DEFF Research Database (Denmark)

    Pedersen, T.G.; Lynge, T.B.; Kristensen, P.K.;

    2005-01-01

    for these applications. From a theoretical analysis of excitons in long metalloporphyrin chains, we demonstrate that the binding energy is much lower than in usual conjugated polymers. Our calculated absorption spectra are in good agreement with measurements. (c) 2004 Elsevier B.V. All rights reserved.......The optical gap of conjugated triply linked porphyrin chains is exceptionally low (similar to 0.5 eV). Hence, such chains are candidates for organic infrared detectors and solar cells harvesting the infrared part of the solar spectrum. However, a low exciton binding energy is required...

  12. Conjugate metamaterials and the perfect lens

    CERN Document Server

    Xu, Yadong; Xu, Lin; Chen, Huanyang

    2015-01-01

    In this letter, we show how transformation optics makes it possible to design what we call conjugate metamaterials. We show that these materials can also serve as substrates for making a subwavelength-resolution lens. The so-called "perfect lens", which is a lens that could focus all components of light (including propagating and evanescent waves), can be regarded as a limiting case, in which the respective conjugate metamaterials approach the characteristics of left-handed metamaterials, which have a negative refractive index.

  13. Mass-Production and Characterization of Anti-CD20 Monoclonal Antibody in Peritoneum of Balb/c Mice

    Directory of Open Access Journals (Sweden)

    Leili Aghebati

    2013-02-01

    Full Text Available Purpose: Monoclonal antibodies are important tools are used in basic research as well as, in diagnosis, imaging and treatment of immunodeficiency diseases, infections and cancers. The purpose of this study was to produce large scale of monoclonal antibody against CD20 in order to diagnostic application in leukemia and lymphomas disorders. Methods: Hybridoma cells that produce monoclonal antibody against human CD20 were administered into the peritoneum of the Balb/c mice which have previously been primed with 0.5 ml Pristane. After twelve days, approximately 7 ml ascetic fluid was harvested from the peritoneum of each mouse. Evaluation of mAb titration was assessed by ELISA method. In the present study, we describe a protocol for large scale production of MAbs. Results: We prepared monoclonal antibodies (mAbs with high specificity and sensitivity against human CD20 by hybridoma method and characterized them by ELISA. The subclass of antibody was IgG2a and its light chain was kappa. Ascetic fluid was purified by Protein-A Sepharose affinity chromatography and the purified monoclonal antibody was conjugated with FITC and Immunofluorescence was done for confirming the specific binding. Conclusion: The conjugated monoclonal antibody could have application in diagnosis B-cell lymphomas, hairy cell leukemia, B-cell chronic lymphocytic leukemia, and melanoma cancer stem cells.

  14. Preparation of an antibody that recognizes and neutralizes Dictyostelium differentiation-inducing factor-1.

    Science.gov (United States)

    Kubohara, Yuzuru; Kikuchi, Haruhisa; Nakamura, Koji; Matsuo, Yusuke; Oshima, Yoshiteru

    2010-05-28

    In the development of the cellular slime mold Dictyostelium discoideum, the differentiation-inducing factor-1 (DIF-1; 1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one) plays an important role in the regulation of cell differentiation and chemotaxis; however, the cellular signaling systems involving DIF-1 remain to be elucidated. To obtain a probe for DIF-1, we synthesized a DIF derivative (DIF-1-NH(2); 6-amino-1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one), and prepared an anti-DIF-1 antibody using a DIF-1-NH(2)-conjugated macromolecule as the immunogen. A 100-fold dilution of the antibody bound to DIF-1-NH(2)-conjugated resin, and this binding was inhibited by co-addition of 20 microM DIF-1 or DIF-1-NH(2). In a monolayer culture of HM44 cells, a DIF-deficient D. discoideum strain, 0.5 nM exogenous DIF-1 induced stalk cell formation in approximately 60% of the cells; this induction was dose-dependently inhibited by the antibody (diluted 12.5- or 25-fold). Furthermore, this inhibition by the antibody was recovered by co-addition of 2.5 or10 nM DIF-1. The results indicate that the anti-DIF-1 antibody recognizes DIF-1 and neutralizes its function. PMID:20416278

  15. Flow-pattern Guided Fabrication of High-density Barcode Antibody Microarray.

    Science.gov (United States)

    Ramirez, Lisa S; Wang, Jun

    2016-01-01

    Antibody microarray as a well-developed technology is currently challenged by a few other established or emerging high-throughput technologies. In this report, we renovate the antibody microarray technology by using a novel approach for manufacturing and by introducing new features. The fabrication of our high-density antibody microarray is accomplished through perpendicularly oriented flow-patterning of single stranded DNAs and subsequent conversion mediated by DNA-antibody conjugates. This protocol outlines the critical steps in flow-patterning DNA, producing and purifying DNA-antibody conjugates, and assessing the quality of the fabricated microarray. The uniformity and sensitivity are comparable with conventional microarrays, while our microarray fabrication does not require the assistance of an array printer and can be performed in most research laboratories. The other major advantage is that the size of our microarray units is 10 times smaller than that of printed arrays, offering the unique capability of analyzing functional proteins from single cells when interfacing with generic microchip designs. This barcode technology can be widely employed in biomarker detection, cell signaling studies, tissue engineering, and a variety of clinical applications. PMID:26780370

  16. Production and Characterization of Polyclonal Antibody for the N-Methylcarbamate Insecticide Metolcarb

    Institute of Scientific and Technical Information of China (English)

    ZHANG Qi; LI Tie-jun; ZHU Xiao-xia; XU Li-na; LIU Feng-quan; HU Bai-shi; JIANG Ying-hua; CAO Bin

    2007-01-01

    The hapten, 3-{[1-(3-(methyl)phenyloxy)-carbonyl]amino}propanoic acid (HOM), mimicking the analyte metolcarb, was synthesized and verified by mass spectrometry (MS) and 1H-nuclear magnetic resonance spectrometry (1H-NMR). Then,HOM was conjugated with the carrier proteins bovine serum albumin (BSA) and ovalbumin (OVA) with stoichiometric amounts of N-hydroxysuccinimide/dicyclohexylcarbodimide (NHS/DCC) using the activated ester method. Polyclonal antibodies were raised against the conjugate of HOM-BSA in rabbits. Antiserum titres were determined by noncompetitive indirect ELISA procedures and the titer of pAb01 reached 1.28 × 106. The cross-reactivities of the structurally related Nmethylcarbamate insecticides were 0.0% except for dimethacarb. These results indicate that the antibody pAb01 with strong affinity and high specificity can be used to develop a sensitive and rapid detection protocol for metolcarb residue.

  17. Succinylated polylysine as a possible link between an antibody molecule and deferoxamine

    Energy Technology Data Exchange (ETDEWEB)

    Slinkin, M.A.; Klibanov, A.L.; Khaw, B.A.; Torchilin, V.P. (USSR Cardiology Research Center, Academy of Medical Sciences, Moscow (USSR))

    1990-07-01

    Modification of antibodies with chelating polymers may be helpful for radioimmunoimaging, radioimmunotherapy, and NMR tomography. Succinylated polylysine was activated with carbodiimide/N-hydroxysulfosuccinimide in dimethyl sulfoxide and isolated as a dry solid. Sulfosuccinimide-esterified polymer was used for the two-stage coupling of an amino-containing chelating agent (deferoxamine) to monoclonal R11D10 (IgG) or its Fab fragment. Conjugates were separated from free components by using gel-chromatography and anion-exchange chromatography. Antibody-coupling efficiency and the loss of its immunoreactivity upon modification have been studied for polymers with different deferoxamine content. Specific binding of 67Ga to the corresponding antigen via the conjugate has been demonstrated.

  18. Anti-enrofloxacin Antibody Production by Using Enrofloxacin-screened HSA as an Immunogen

    Institute of Scientific and Technical Information of China (English)

    LIU Chune; LIN Hong; CAO Limin; JIANG Jie

    2005-01-01

    A two-step zero-length cross-linking procedure using active esters was successfully adopted for conjugating enrofloxacin (EF) to human serum albumin (HSA). The derived conjugate was characterized by UV spectrum and then used for immunization of BALB/C mice. In enzyme-linked immunosorbent assay (ELISA) and competitive inhibition ELISA experiments, the derived antiserum exhibited high antibody titer (greater than 1: 250 000) as well as varied cross-reactivity (from 97.8% to 161.7%) to three analogs of EF belonging to fluoroquinolones family. But over the concentration range studied, no significant cross-reactivity was observed to other group of antibiotics (chloramphenicol, oxytetracycline, sulphamethoxazole and nysfungin). It was confirmed that the synthesized immunogen was highly antigenic and elicited specific antibody responses in BALB/C mice against EF.

  19. Immune responses to HBsAg conjugated to protein D of non-typeable Haemophilus influenzae in mice.

    Directory of Open Access Journals (Sweden)

    Qiudong Su

    Full Text Available Hepatitis B vaccine that contains an aluminum hydroxide adjuvant induces apoptotic death of Hepa 1-6 cells. Difficult-to-degrade chemical additives in vaccines effectively enhance vaccine immunogenicity, but also affect the host tissue. Identification of bio-molecules that are readily degraded and compatible in vivo as an adjuvant is important for vaccine research. The hapten-carrier effect suggests that stimulation of helper T (Th cells by carrier adjuvants is feasible. Protein D (PD of non-typeable Haemophilus influenzae covalently conjugated to some polysaccharide vaccines has been confirmed to convert T-cell independent (TI antigens into T-cell dependent (TD antigens, and elicit strong T-cell responses ultimately. Herein, we would substitube PD for aluminum hydroxide adjuvant in Hepatitis B vaccine.Truncated PD (amino acids 20-364 was expressed in Escherichia coli and purified by (NH42SO4 precipitation and DEAE chromatography. After evaluation of antigenicity by western blotting, PD was covalently conjugated to yeast-derived recombinant HBsAg by cross-linking with glutaraldehyde. Intramuscular immunization with the conjugate induced higher level of HBsAg-specific antibody than did HBsAg alone (p < 0.05, and was comparable to commercial Hepatitis B vaccine. During the surveillance period (days 35-105, anti-HBs titers were hold high. Moreover, the conjugated vaccine enhanced Th1 immune responses, while Th2 responses were also activated and induced an antibody response, as determined by IFN-γ ELISPOT and IgG1/IgG2a ratio assays.Recombinant truncated PD covalently conjugated to HBsAg antigen enhanced the immunogenicity of the antigen in mice simultaneously by humoral and cellular immune response, which would facilitate therapeutic hepatitis B vaccines.

  20. Antibody mimetics: promising complementary agents to animal-sourced antibodies.

    Science.gov (United States)

    Baloch, Abdul Rasheed; Baloch, Abdul Wahid; Sutton, Brian J; Zhang, Xiaoying

    2016-01-01

    Despite their wide use as therapeutic, diagnostic and detection agents, the limitations of polyclonal and monoclonal antibodies have inspired scientists to design the next generation biomedical agents, so-called antibody mimetics that offer many advantages over conventional antibodies. Antibody mimetics can be constructed by protein-directed evolution or fusion of complementarity-determining regions through intervening framework regions. Substantial progress in exploiting human, butterfly (Pieris brassicae) and bacterial systems to design and select mimetics using display technologies has been made in the past 10 years, and one of these mimetics [Kalbitor® (Dyax)] has made its way to market. Many challenges lie ahead to develop mimetics for various biomedical applications, especially those for which conventional antibodies are ineffective, and this review describes the current characteristics, construction and applications of antibody mimetics compared to animal-sourced antibodies. The possible limitations of mimetics and future perspectives are also discussed. PMID:25264572

  1. Protective effects of anti-ricin A-chain antibodies delivered intracellularly against ricin-induced cytotoxicity

    Institute of Scientific and Technical Information of China (English)

    Frank; Martiniuk; Seth; Pincus; Sybille; Müller; Heinz; Kohler; Kam-Meng; Tchou-Wong

    2010-01-01

    AIM:To evaluate the ability of anti-ricin A-chain antibodies,delivered intracellularly,to protect against ricininduced cytotoxicity in RAW264.7 cells. METHODS:Anti-deglycosylated ricin A-chain antibody and RAC18 anti-ricin A-chain monoclonal antibody were delivered intracellularly by encapsulating in liposomes or via conjugation with the cell-penetrating MTS-transport peptide.RAW264.7 cells were incubatedwith these antibodies either before or after ricin exposure.The changes in cytotoxicity were estimated by MTT assay.Co-localization of internalized antibody and ricin was evaluated by fluorescence microscopy. RESULTS:Internalized antibodies significantly increased cell viability either before or after ricin exposure compared to the unconjugated antibodies.Fluorescence microscopy confirmed the co-localization of internalized antibodies and ricin inside the cells. CONCLUSION:Intracellular delivery of antibodies to neutralize the ricin toxin after cellular uptake supports the potential use of cell-permeable antibodies for postexposure treatment of ricin intoxication.

  2. Referencing cross-reactivity of detection antibodies for protein array experiments [version 1; referees: 1 approved, 2 approved with reservations

    Directory of Open Access Journals (Sweden)

    Darragh Lemass

    2016-01-01

    Full Text Available Protein arrays are frequently used to profile antibody repertoires in humans and animals. High-throughput protein array characterisation of complex antibody repertoires requires a platform-dependent, lot-to-lot validation of secondary detection antibodies. This article details the validation of an affinity-isolated anti-chicken IgY antibody produced in rabbit and a goat anti-rabbit IgG antibody conjugated with alkaline phosphatase using protein arrays consisting of 7,390 distinct human proteins. Probing protein arrays with secondary antibodies in absence of chicken serum revealed non-specific binding to 61 distinct human proteins. The cross-reactivity of the tested secondary detection antibodies points towards the necessity of platform-specific antibody characterisation studies for all secondary immunoreagents. Secondary antibody characterisation using protein arrays enables generation of reference lists of cross-reactive proteins, which can be then excluded from analysis in follow-up experiments. Furthermore, making such cross-reactivity lists accessible to the wider research community may help to interpret data generated by the same antibodies in applications not related to protein arrays such as immunoprecipitation, Western blots or other immunoassays.

  3. Conjugate Gradient Methods with Armijo-type Line Searches

    Institute of Scientific and Technical Information of China (English)

    Yu-Hong DAI

    2002-01-01

    Two Armijo-type line searches are proposed in this paper for nonlinear conjugate gradient methods.Under these line searches, global convergence results are established for several famous conjugate gradient method.

  4. Large Scale Generation and Characterization of Anti-Human IgA Monoclonal Antibody in Ascitic Fluid of Balb/c Mice

    Directory of Open Access Journals (Sweden)

    Fatemeh Ezzatifar

    2015-03-01

    Full Text Available Purpose: Monoclonal antibodies are potentially powerful tools used in biomedical research, diagnosis, and treatment of infectious diseases and cancers. The monoclonal antibody against Human IgA can be used as a diagnostic application to detect infectious diseases. The aim of this study was to improve an appropriate protocol for large-scale production of mAbs against IgA. Methods: For large-scale production of the monoclonal antibody, hybridoma cells that produce monoclonal antibodies against Human IgA were injected intraperitoneally into Balb/c mice that were previously primed with 0.5 ml Pristane. After ten days, ascitic fluid was harvested from the peritoneum of each mouse. The ELISA method was carried out for evaluation of the titration of produced mAbs. The ascitic fluid was investigated in terms of class and subclass by a mouse mAb isotyping kit. MAb was purified from the ascitic fluid by ion exchange chromatography. The purity of the monoclonal antibody was confirmed by SDS-PAGE, and the purified monoclonal antibody was conjugated with HRP. Results: Monoclonal antibodies with high specificity and sensitivity against Human IgA were prepared by hybridoma technology. The subclass of antibody was IgG1 and its light chain was the kappa type. Conclusion: This conjugated monoclonal antibody could have applications in designing ELISA kits in order to diagnose different infectious diseases such as toxoplasmosis and H. Pylori.

  5. Design of indirect solid-phase immunosorbent methods for detecting arenavirus antigens and antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Ivanov, A.P.; Rezapkin, G.V.; Dzagurova, T.K.; Tkachenko, E.A.

    1984-05-01

    Specifications have been elaborated for formulating indirect solid-phase enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (SPRIA) methods that employ anti-human and anti-mice G class immunoglobulin (IgG), conjugated with horseradish peroxidase and /sup 125/I for detecting the arenaviruses Junin, Machupo, Tacaribe, Amalpari, Tamiami, Lassa, and LCM (lymphocytic choriomeningitis). These methods make it possible to identify with a high degree of sensitivity arenavirus antigens and antibodies in various kinds of material.

  6. Interaction of antithrombin III with preadsorbed albumin-heparin conjugates

    OpenAIRE

    Hennink, W.E.; Ebert, C.D.; Kim, S. W.; Breemhaar, W.; Bantjes, A.; Feijen, J.

    1984-01-01

    The adsorption of antithrombin III (AT III) onto polystyrene surfaces preadsorbed with albumin or albuminheparin conjugates was studied using a two step enzyme immuno assay. When AT III-buffer solutions were used, the highest adsorption values were measured on high affinity albumin-heparin conjugate pretreated surfaces. Less AT III adsorption was found on nonfractionated albumin-heparin conjugate preadsorbed surfaces. AT III adsorption could also be detected on low affinity conjugate and albu...

  7. Polarized immune responses modulated by layered double hydroxides nanoparticle conjugated with CpG.

    Science.gov (United States)

    Yan, Shiyu; Rolfe, Barbara E; Zhang, Bing; Mohammed, Yousuf H; Gu, Wenyi; Xu, Zhi P

    2014-11-01

    Modulation of the immune response is an important step in the induction of protective humoral and cellular immunity against pathogens. In this study, we investigated the possibility of using a nanomaterial conjugated with the toll-like receptor (TLR) ligand CpG to modulate the immune response towards the preferred polarity. MgAl-layered double hydroxide (LDH) nanomaterial has a very similar chemical composition to Alum, an FDA approved adjuvant for human vaccination. We used a model antigen, ovalbumin (OVA) to demonstrate that MgAl-LDH had comparable adjuvant activity to Alum, but much weaker inflammation. Conjugation of TLR9 ligand CpG to LDH nanoparticles significantly enhanced the antibody response and promoted a switch from Th2 toward Th1 response, demonstrated by a change in the IgG2a:IgG1 ratio. Moreover, immunization of mice with CpG-OVA-conjugated LDH before challenge with OVA-expressing B16/F10 tumor cells retarded tumor growth. Together, these data indicate that LDH nanomaterial can be used as an immune adjuvant to promote Th1 or Th2 dominant immune responses suitable for vaccination purposes. PMID:25145853

  8. Biocatalytic Synthesis of Fluorescent Conjugated Indole Oligomers

    Directory of Open Access Journals (Sweden)

    Ryan M. Bouldin

    2014-12-01

    Full Text Available Fluorescent conjugated materials exhibiting reasonable biocompatibility that are capable of interacting with biological molecules are of interest for bio-sensing and imaging applications. Traditional approaches do not allow for the synthesis of conjugated materials in the presence of biologically relevant substrates. Further conjugated polymers synthesized using conventional methods are doped and not fluorescent. Here we explore the possibility of synthesizing fluorescent oligomers of indole using enzymes as catalyst under mild conditions. The peroxidase catalyzed coupling reaction presented here creates a photoluminescent material that allows for direct utilization (without purification and separation of the dopant in biosensing applications. The polymerization reaction proceeds smoothly in just deionized water and ethanol. Monitoring of the absorption and fluorescence spectra over one hour shows that the concentration of both absorbing and emitting species grows steadily over time. The presence of anionic buffers and templates is shown to effectively retard the development of light emitting species and instead leads to the formation of an electrically doped conjugated polymer. Structural characterization through FTIR and 1H-NMR analysis suggests that the oligomer is coupled through the 2 and 3 positions on the indole ring.

  9. Women experiencing the intergenerationality of conjugal violence

    Directory of Open Access Journals (Sweden)

    Gilvânia Patrícia do Nascimento Paixão

    2015-10-01

    Full Text Available Objective: to analyze the family relationship, in childhood and adolescence, of women who experience conjugal violence.Method: qualitative study. Interviews were held with 19 women, who were experiencing conjugal violence, and who were resident in a community in Salvador, Bahia, Brazil. The project was approved by the Research Ethics Committee (N. 42/2011.Results: the data was organized using the Discourse of the Collective Subject, identifying the summary central ideas: they witnessed violence between their parents; they suffered repercussions from the violence between their parents: they were angry about the mother's submission to her partner; and they reproduced the conjugal violence. The discourse showed that the women witnessed, in childhood and adolescence, violence between their parents, and were injured both physically and psychologically. As a result of the mother's submission, feelings of anger arose in the children. However, in the adult phase of their own lives, they noticed that their conjugal life resembled that of their parents, reproducing the violence.Conclusion: investment is necessary in strategies designed to break inter-generational violence, and the health professionals are important in this process, as it is a phenomenon with repercussions in health. Because they work in the Family Health Strategy, which focuses on the prevention of harm and illness, health promotion and interdepartmentality, the nurses are essential in the process of preventing and confronting this phenomenon.

  10. Stochastic differential equations used to model conjugation

    DEFF Research Database (Denmark)

    Philipsen, Kirsten Riber; Christiansen, Lasse Engbo

    Stochastic differential equations (SDEs) are used to model horizontal transfer of antibiotic resis- tance by conjugation. The model describes the concentration of donor, recipient, transconjugants and substrate. The strength of the SDE model over the traditional ODE models is that the noise can...

  11. Charge Conjugation in the Galilean Limit

    OpenAIRE

    Socolovsky, Miguel

    2006-01-01

    Strictly working in the framework of the nonrelativistic quantum mechanics of a spin 1/2 particle coupled to an external electromagnetic field, we show, by explicit construction, the existence of a charge conjugation operator matrix which defines the corresponding antiparticle wave function and leads to the galilean and gauge invariant Schroedinger-Pauli equation satisfied by it.

  12. Conjugate Function Method for Numerical Conformal Mappings

    CERN Document Server

    Hakula, Harri; Rasila, Antti

    2011-01-01

    We present a method for numerical computation of conformal mappings from simply or doubly connected domains onto so-called canonical domains, which in our case are rectangles or annuli. The method is based on conjugate harmonic functions and properties of quadrilaterals. Several numerical examples are given.

  13. Predicting the optical gap of conjugated systems

    Science.gov (United States)

    Botelho, Andre Leitao

    The adapted Su-Schrieffer-Heeger model is developed in this work as a tool for in silico prediction of the optical gap of pi-conjugated systems for photovoltaic applications. Full transferability of the model ensures reliable predictive power - excellent agreement with 180 independent experimental data points covering virtually all existing conjugated system types with an accuracy exceeding the time-dependent density functional theory, one of the most accurate first-principles methods. Insights on the structure-property relation of conjugated systems obtained from the model lead to guiding rules for optical gap design: 1) fusing aromatic rings parallel to the conjugated path does not significantly lower the optical gap, 2) fusing rings perpendicularly lowers the optical gap of the monomer, but has a reduced benefit from polymerization, and 3) copolymers take advantage of the lower optical gap of perpendicular fused rings and benefit from further optical gap reduction through added parallel fused rings as electronic communicators. A copolymer of parallel and perpendicular benzodithiophenes, differing only in sulfur atom locations, is proposed as a candidate to achieve the optimal 1.2 eV donor optical gap for organic photovoltaics. For small-molecule organic photovoltaics, substituting the end pairs of carbon atoms on pentacene with sulfur atoms is predicted to lower the optical gap from 1.8 eV to 1.1 eV. Furthermore, the model offers an improvement of orders of magnitude in the computational efficiency over commonly used first-principles tools.

  14. Conjugate Problems in Convective Heat Transfer: Review

    Directory of Open Access Journals (Sweden)

    Abram Dorfman

    2009-01-01

    Full Text Available A review of conjugate convective heat transfer problems solved during the early and current time of development of this modern approach is presented. The discussion is based on analytical solutions of selected typical relatively simple conjugate problems including steady-state and transient processes, thermal material treatment, and heat and mass transfer in drying. This brief survey is accompanied by the list of almost two hundred publications considering application of different more and less complex analytical and numerical conjugate models for simulating technology processes and industrial devices from aerospace systems to food production. The references are combined in the groups of works studying similar problems so that each of the groups corresponds to one of selected analytical solutions considered in detail. Such structure of review gives the reader the understanding of early and current situation in conjugate convective heat transfer modeling and makes possible to use the information presented as an introduction to this area on the one hand, and to find more complicated publications of interest on the other hand.

  15. Conjugate problems in convective heat transfer

    CERN Document Server

    Dorfman, Abram S

    2009-01-01

    The conjugate heat transfer (CHT) problem takes into account the thermal interaction between a body and fluid flowing over or through it, a key consideration in both mechanical and aerospace engineering. Presenting more than 100 solutions of non-isothermal and CHT problems, this title considers the approximate solutions of CHT problems.

  16. Compositions for directed alignment of conjugated polymers

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jinsang; Kim, Bong-Gi; Jeong, Eun Jeong

    2016-04-19

    Conjugated polymers (CPs) achieve directed alignment along an applied flow field and a dichroic ratio of as high as 16.67 in emission from well-aligned thin films and fully realized anisotropic optoelectronic properties of CPs in field-effect transistor (FET).

  17. Bacillus thuringiensis Conjugation in Simulated Microgravity

    Science.gov (United States)

    Beuls, Elise; van Houdt, Rob; Leys, Natalie; Dijkstra, Camelia; Larkin, Oliver; Mahillon, Jacques

    2009-10-01

    Spaceflight experiments have suggested a possible effect of microgravity on the plasmid transfer among strains of the Gram-positive Bacillus thuringiensis, as opposed to no effect recorded for Gram-negative conjugation. To investigate these potential effects in a more affordable experimental setup, three ground-based microgravity simulators were tested: the Rotating Wall Vessel (RWV), the Random Positioning Machine (RPM), and a superconducting magnet. The bacterial conjugative system consisted in biparental matings between two B. thuringiensis strains, where the transfer frequencies of the conjugative plasmid pAW63 and its ability to mobilize the nonconjugative plasmid pUB110 were assessed. Specifically, potential plasmid transfers in a 0-g position (simulated microgravity) were compared to those obtained under 1-g (normal gravity) condition in each device. Statistical analyses revealed no significant difference in the conjugative and mobilizable transfer frequencies between the three different simulated microgravitational conditions and our standard laboratory condition. These important ground-based observations emphasize the fact that, though no stimulation of plasmid transfer was observed, no inhibition was observed either. In the case of Gram-positive bacteria, this ability to exchange plasmids in weightlessness, as occurs under Earth's conditions, should be seen as particularly relevant in the scope of spread of antibiotic resistances and bacterial virulence.

  18. Hyperbranched: a universal conjugated polymer platform.

    Science.gov (United States)

    Tolosa, Juan; Kub, Chris; Bunz, Uwe H F

    2009-01-01

    Out on a limb: Sonogashira coupling of a suitable AB(2) monomer containing two iodine and one alkyne group forms a hyperbranched conjugated polymer that is studded with iodine end groups (see picture: I purple). These iodine groups are a perfect handle for convenient, efficient, and high-yielding post-functionalization to access hyperbranched, fluorescent poly(phenyleneethynylene)s. PMID:19373821

  19. Conjugated Polymer Actuators: Prospects and Limitations

    DEFF Research Database (Denmark)

    Skaarup, Steen

    2007-01-01

    Actuators constructed with a conjugated polymer as the active part have been predicted to have a number of highly desirable properties: Large mechanical strength, high power density, i.e. high actuation speeds possible, sufficient maximum strain values, high reversibility and safe, low voltages (1...

  20. Continuous flow synthesis of conjugated polymers.

    Science.gov (United States)

    Seyler, Helga; Jones, David J; Holmes, Andrew B; Wong, Wallace W H

    2012-02-01

    A selection of conjugated polymers, widely studied in organic electronics, was synthesised using continuous flow methodology. As a result of superior heat transfer and reagent control, excellent polymer molecular mass distributions were achieved in significantly reduced reaction times compared to conventional batch reactions.

  1. Conjugal Succession and the American Kinship System.

    Science.gov (United States)

    Furstenberg, Frank F., Jr.

    Although not the preferred type of family formation, conjugal succession is now an accepted, if not expected, alternative to continuous marriage in the United States. This new trend appears to be related to a shift in the meaning of matrimony. Previously, marriage was part of a cultural pattern of transitions and as such was closely timed to…

  2. Engineering antibodies by yeast display.

    Science.gov (United States)

    Boder, Eric T; Raeeszadeh-Sarmazdeh, Maryam; Price, J Vincent

    2012-10-15

    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.

  3. Antiphospholipid Antibody and Antiphospholipid Syndrome

    Institute of Scientific and Technical Information of China (English)

    吴竞生

    2008-01-01

    @@ 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.

  4. Synthesis of Indomethacin Conjugates with D-Glucosamine

    Institute of Scientific and Technical Information of China (English)

    Yi Chun ZHANG; Ying Xia LI; Hua Shi GUAN

    2005-01-01

    Two series of indomethacin conjugates with D-glucosamine were prepared with the objectives of reducing ulcerogenic potency, increasing the bioavailability of indomethacin and exerting the coordinative effects on osteoarthritis. The structures of the conjugates were identified by 1H NMR and 13C NMR. The ester conjugates inhibited edema as potent as indomethacin.

  5. Conjugated Educational System: Notion, Structure, Educational Potential 

    OpenAIRE

    Andrei A. Ostapenko; Dar'ya S. Tkach

    2012-01-01

    The article indicates the ways to decrease risk from teenagers and youth’s growing-up in today’s Russia by development of fundamental models of conjugated educational systems and their mass implementation in educational practice, introduces the notion of “conjugated educational system” for scientific use, describes types of conjugation and educational results of submitted models use.

  6. Resolution of Digitized Conjugate Tooth-Face Surface Based on the Theory of Digitized Conjugate Surfaces

    Institute of Scientific and Technical Information of China (English)

    XIAO Lai-yuan; LIAO Dao-xun; YI Chuan-yun

    2004-01-01

    According to the principle of meshing engagement and the theory of the digitized conjugate surface, this paper applies the software Conjugater-l. 0 that is developed by ourselves to compute, respectivcly, the digitized conjugate curved surfaces of the straight-tooth surface and drum-tooth surface,which will establish the theoretical and technical foundation for digitized engaging analysis, simulation, and digitized manufacturing technology of the diversified gears.

  7. lmmunofiuorescent Labeling of Human HepG2 Cells with CdTe Quantum Dot Probe Conjugated with Anti-pan CK MAb

    Institute of Scientific and Technical Information of China (English)

    SUI Yu-jie; ZHANG Gui-zhen; WANG Qian; WANG Ya-li; WU Mei; DU Zhen-wu; ZHANG Jie; JIANG Ri-hua

    2011-01-01

    A relatively sensitive, specific, and photostable method for the detection of cytokeratin of cancer cells via conjugation with cadmium telluride quantum dots(CdTe QDs) was described. Water soluble CdTe QDs were conjugated to anti-pan-cytokeratin(CK) monoclonal antibody(MAb) through coupling reagent [1-ethyi-3-(3-dimethylamino propyl)carbodiimide, EDC] and the conjugates were purified by dialysis. The expression of pan CK protein in HepG2 cells was observed by immunocytochemistry and direct immunofluorescence via QDs-Ab conjugates respectively. Fluorescence intensity and photostability of QDs were compared with those of FITC(fiuorescein isothiocyanate). The results show that the QDs-Ab conjugates recognized specifically pan CK protein in HepG2 cells. Compared with FITC, CdTe QDs had higher brightness and photostability without obvious photobleaching under continuous exciting light illumination for 30 min and after the placement at room temperature for 3 d. The results indicate that conjugates of CdTe quantum dot with anti-pan CK MAb can be used for labeling cancer cells derived from epithelial tissues, which provides the basis for the detection of circulating tumor cells(CTCs).

  8. Development of a Pfs25-EPA malaria transmission blocking vaccine as a chemically conjugated nanoparticle.

    Science.gov (United States)

    Shimp, Richard L; Rowe, Christopher; Reiter, Karine; Chen, Beth; Nguyen, Vu; Aebig, Joan; Rausch, Kelly M; Kumar, Krishan; Wu, Yimin; Jin, Albert J; Jones, David S; Narum, David L

    2013-06-19

    Successful efforts to control infectious diseases have often required the use of effective vaccines. The current global strategy for control of malaria, including elimination and eradication will also benefit from the development of an effective vaccine that interrupts malaria transmission. To this end, a vaccine that disrupts malaria transmission within the mosquito host has been investigated for several decades targeting a 25 kDa ookinete specific surface protein, identified as Pfs25. Phase 1 human trial results using a recombinant Pfs25H/Montanide ISA51 formulation demonstrated that human Pfs25 specific antibodies block parasite infectivity to mosquitoes; however, the extent of blocking was likely insufficient for an effective transmission blocking vaccine. To overcome the poor immunogenicity, processes to produce and characterize recombinant Pfs25H conjugated to a detoxified form of Pseudomonas aeruginosa exoprotein A (EPA) have been developed and used to manufacture a cGMP pilot lot for use in human clinical trials. The Pfs25-EPA conjugate appears as a nanoparticle with an average molar mass in solution of approximately 600 kDa by static light scattering with an average diameter 20 nm (range 10-40 nm) by dynamic light scattering. The molar ratio of Pfs25H to EPA is about 3 to 1 by amino acid analysis, respectively. Outbred mice immunized with the Pfs25-EPA conjugated nanoparticle formulated on Alhydrogel(®) had a 75-110 fold increase in Pfs25H specific antibodies when compared to an unconjugated Pfs25H/Alhydrogel(®) formulation. A phase 1 human trial using the Pfs25-EPA/Alhydrogel(®) formulation is ongoing in the United States.

  9. Do pneumococcal conjugate vaccines provide any cross-protection against serotype 19A?

    Directory of Open Access Journals (Sweden)

    Hoet Bernard

    2010-02-01

    Full Text Available Abstract Background Introduction of the 7-valent pneumococcal conjugate vaccine (7vCRM in several countries has led to a rapid, significant drop in vaccine-type invasive pneumococcal disease (IPD in immunized children. In the United States and some other countries with high antibiotic use, a subsequent rise in serotype 19A IPD has been taken to indicate that the 19F conjugate in the vaccine provides no cross-protection against the immunologically related 19A. Discussion We systematically assessed the clinical efficacy and effectiveness of 19F-containing vaccines against 19A disease or nasopharyngeal carriage by searching English-language articles in the electronic databases PubMed, Current contents, Scopus, and Embase from 1985 to 2008. The vaccine efficacy and effectiveness point estimates were consistently positive for modest protection against 19A IPD and acute otitis media (AOM. However, statistical significance was not reached in any individual study. No consistent impact of 7vCRM on 19A nasopharyngeal colonization could be detected. These findings are discussed in context of immunogenicity analyses indicating that 7vCRM induces functionally active anti-19A antibodies after the booster dose, and that other 19F-containing vaccine formulations may elicit higher levels of such antibodies after both primary and booster doses. Summary Taken together, these results suggest that 19F-conjugates can provide some protection against 19A disease. The magnitude of this protection in a given setting will likely depend on several factors. These include the anti-19A immunogenicity of the specific vaccine formulation, the number of doses of that formulation needed to elicit the response, and the burden of 19A disease that occurs after those doses. It is possible that a modest protective effect may be obscured by the presence of countervailing selection pressures (such as high antibiotic use that favor an increase in colonization with antibiotic

  10. 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.

    2004-03-17

    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

  11. New Insights into the Functional Behavior of Antibodies as Revealed by Binding Studies on an Anti-Uranium Monoclonal Antibody

    International Nuclear Information System (INIS)

    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

  12. The tumor-inhibitory effectiveness of a novel anti-Trop2 Fab conjugate in pancreatic cancer.

    Science.gov (United States)

    Mao, Yuan; Wang, Xiaoying; Zheng, Feng; Wang, Changjun; Tang, Qi; Tang, Xiaojun; Xu, Ning; Zhang, Huiling; Zhang, Dawei; Xiong, Lin; Liang, Jie; Zhu, Jin

    2016-04-26

    Human trophoblastic cell surface antigen 2 (Trop2) has been reported to act oncogenically. In this study, one-step quantitative real-time polymerase chain reaction (qPCR) test and immunohistochemistry (IHC) analysis with were employed to evaluate the relationship between Trop2 expression and the clinicopathological features of patients with PC. Then a novel anti-Trop2 Fab antibody was conjugated with Doxorubicin (DOX) to form Trop2Fab-DOX, an antibody-drug conjugate. This Trop2Fab-DOX conjugate was characterized by cell ELISA and immunofluorescence assay. MTT and wound healing analyses were used to evaluate the inhibitory effect of Trop2Fab-DOX on PC cell growth in vitro, while xenograft nude mice model was established to examine the tumor-inhibitory effects of PC in vivo. High Trop2 expression was observed in PC tissues and Trop2 expression was associated with several malignant attributes of PC patients, including overall survival. Trop2Fab-DOX can bind to the Trop2-expressing PC cells and provide an improved releasing type of DOX. In addition, Trop2Fab-DOX inhibited the proliferation and suppressed the migration of PC cells in a dose-dependent manner in vitro, while inhibited the growth of PC xenografts in vivo. Trop2 is a specific marker for PC, and a novel Trop2Fab-DOX ADC has a potent antitumor activity.

  13. Synthesis and evaluation of a conjugate vaccine composed of Staphylococcus aureus poly-N-acetyl-glucosamine and clumping factor A.

    Directory of Open Access Journals (Sweden)

    Tomás Maira-Litrán

    Full Text Available The increasing frequency, severity and antimicrobial resistance of Staphylococcus aureus infections has made the development of immunotherapies against this pathogen more urgent than ever. Previous immunization attempts using monovalent antigens resulted in at best partial levels of protection against S. aureus infection. We therefore reasoned that synthesizing a bivalent conjugate vaccine composed of two widely expressed antigens of S. aureus would result in additive/synergetic activities by antibodies to each vaccine component and/or in increased strain coverage. For this we used reductive amination, to covalently link the S. aureus antigens clumping factor A (ClfA and deacetylated poly-N-β-(1-6-acetyl-glucosamine (dPNAG. Mice immunized with 1, 5 or 10 µg of the dPNAG-ClfA conjugate responded in a dose-dependent manner with IgG to dPNAG and ClfA, whereas mice immunized with a mixture of ClfA and dPNAG developed significantly lower antibody titers to ClfA and no antibodies to PNAG. The dPNAG-ClfA vaccine was also highly immunogenic in rabbits, rhesus monkeys and a goat. Moreover, affinity-purified, antibodies to ClfA from dPNAG-ClfA immune serum blocked the binding of three S. aureus strains to immobilized fibrinogen. In an opsonophagocytic assay (OPKA goat antibodies to dPNAG-ClfA vaccine, in the presence of complement and polymorphonuclear cells, killed S. aureus Newman and, to a lower extent, S. aureus Newman ΔclfA. A PNAG-negative isogenic mutant was not killed. Moreover, PNAG antigen fully inhibited the killing of S. aureus Newman by antisera to dPNAG-ClfA vaccine. Finally, mice passively vaccinated with goat antisera to dPNAG-ClfA or dPNAG-diphtheria toxoid conjugate had comparable levels of reductions of bacteria in the blood 2 h after infection with three different S. aureus strains as compared to mice given normal goat serum. In conclusion, ClfA is an immunogenic carrier protein that elicited anti-adhesive antibodies that fail to

  14. Impaired Antigen-Specific Immune Response to Vaccines in Children with Antibody Production Defects.

    Science.gov (United States)

    Szczawinska-Poplonyk, Aleksandra; Breborowicz, Anna; Samara, Husam; Ossowska, Lidia; Dworacki, Grzegorz

    2015-08-01

    The impaired synthesis of antigen-specific antibodies, which is indispensable for an adaptive immune response to infections, is a fundamental pathomechanism that leads to clinical manifestations in children with antibody production defects. The aim of this study was to evaluate the synthesis of antigen-specific antibodies following immunization in relation to peripheral blood B cell subsets in young children with hypogammaglobulinemia. Twenty-two children, aged from 8 to 61 months, with a deficiency in one or more major immunoglobulin classes participated in the study. Postvaccination antibodies against tetanus and diphtheria toxoids, the surface antigen of the hepatitis B virus, and the capsular Haemophilus influenzae type b polysaccharide antigen were assessed along with an immunophenotypic evaluation of peripheral blood B lymph cell maturation. A deficiency of antibodies against the tetanus toxoid was assessed in 73% of cases and that against the diphtheria toxoid was assessed in 68% of cases, whereas a deficiency of antibodies against the surface antigen of the hepatitis B virus was revealed in 59% of the children included in the study. A defective response to immunization with a conjugate vaccine with the Haemophilus influenzae type b polysaccharide antigen was demonstrated in 55% of hypogammaglobulinemic patients. Increased proportions of transitional B lymph cells and an accumulation of plasmablasts accompanied antibody deficiencies. The defective response to vaccine protein and polysaccharide antigens is a predominating disorder of humoral immunity in children with hypogammaglobulinemia and may result from a dysfunctional state of the cellular elements of the immune system. PMID:26018535

  15. Efficient, chemoselective synthesis of immunomicelles using single-domain antibodies with a C-terminal thioester

    Directory of Open Access Journals (Sweden)

    Raats Jos MH

    2009-07-01

    Full Text Available Abstract Background Classical bioconjugation strategies for generating antibody-functionalized nanoparticles are non-specific and typically result in heterogeneous compounds that can be compromised in activity. Expression systems based on self-cleavable intein domains allow the generation of recombinant proteins with a C-terminal thioester, providing a unique handle for site-specific conjugation using native chemical ligation (NCL. However, current methods to generate antibody fragments with C-terminal thioesters require cumbersome refolding procedures, effectively preventing application of NCL for antibody-mediated targeting and molecular imaging. Results Targeting to the periplasm of E. coli allowed efficient production of correctly-folded single-domain antibody (sdAb-intein fusions proteins. On column purification and 2-mercapthoethanesulfonic acid (MESNA-induced cleavage yielded single-domain antibodies with a reactive C-terminal MESNA thioester in good yields. These thioester-functionalized single-domain antibodies allowed synthesis of immunomicelles via native chemical ligation in a single step. Conclusion A novel procedure was developed to obtain soluble, well-folded single-domain antibodies with reactive C-terminal thioesters in good yields. These proteins are promising building blocks for the chemoselective functionalization via NCL of a broad range of nanoparticle scaffolds, including micelles, liposomes and dendrimers.

  16. Production of antibodies against secretin and their use for radioimmunoassay of secretin

    International Nuclear Information System (INIS)

    Synthetic thyroglobulin was bonded to bovine albunia and thyroglobulin according to the principle of the carbodiimide condensation reaction. 16 rabbits were immunized with these conjugates and with unconjugated secretin. Secretin labelling with 125I was carried out by the chloramin-T method. The tracer has a specific activity of 15.45 mCi/mg. A secretin RIA was developed using the double antibody method. The sensitivity of the system could be raised by variation of the specific activity of the tracer and optimisation of the incubation parameters. Antisera were compared. The titers of secretin/bovine albumine conjugate antisera were similar to the antisera against secretin thyroglobulin conjugate. The sensitivity of the standard curves was higher for secretin/bovine albumin conjugate antisera than for thyroglobulin conjugate antisera. Two antisera were tested for specificity. The detection threshold of antiserum S 5 IX was 12,43 pmol/l while the 50% intercept was at 55.45 pmol/l. This antiserum is particularly suitable for a secretin RIA. (orig./MS)

  17. Glutaraldehyde mediated conjugation of amino-coated magnetic nanoparticles with albumin protein for nanothermotherapy.

    Science.gov (United States)

    Zhao, Lingyun; Yang, Bing; Dai, Xiaochen; Wang, Xiaowen; Gao, Fuping; Zhang, Xiaodong; Tang, Jintian

    2010-11-01

    A novel bioconjugation of amino saline capped Fe3O4 magnetic nanoparticles (MNPs) with bovine serum albumin (BSA) was developed by applying glutaraldehyde as activator. Briefly, Fe3O4 MNs were synthesized by the chemical co-precipitation method. Surface modification of the prepared MNPs was performed by employing amino saline as the coating agent. Glutaraldehyde was further applied as an activation agent through which BSA was conjugated to the amino-coated MNPs. The structure of the BSA-MNs was confirmed by FTIR analysis. Physico-chemical characterizations of the BSA-MNPs, such as surface morphology, surface charge and magnetic properties were investigated by Transmission Electron Microscopy (TEM), zeta-Potential and Vibrating Sample Magnetometer (VSM), etc. Magnetic inductive heating characteristics of the BSA-MNPs were analyzed by exposing the MNPs suspension (magnetic fluid) under alternative magnetic field (AMF). The results demonstrate that BSA was successfully conjugated with amino-coated MNs mediated through glutaraldehyde activation. The nanoparticles were spherical shaped with approximately 10 nm diameter. Possessing ideal magnetic inductive heating characteristics, which can generate very rapid and efficient heating while upon AMF exposure, BSA-MNPs can be applied as a novel candidature for magnetic nanothermotherapy for cancer treatment. In vitro cytotoxicity study on the human hepatocellular liver carcinoma cells (HepG-2) indicates that BSA-MNP is an efficient agent for cancer nanothermotherapy with satisfied biocompatibility, as rare cytotoxicity was observed in the absence of AMF. Moreover, our investigation provides a methodology for fabrication protein conjugated MNPs, for instance monoclonal antibody conjugated MNPs for targeting cancer nanothermotherapy. PMID:21137877

  18. 77 FR 9678 - Prospective Grant of Exclusive License: Photosensitizing Antibody-Fluorophore Conjugates for...

    Science.gov (United States)

    2012-02-17

    ... anti-HER1 (Panitumumab, for colon cancer), anti-HER2 (Trastuzumab, for breast cancer) and anti-PSMA...-immunotherapy of cancer'' and may be further limited to certain types of cancer and/or specific platforms. Upon... Trademark Office or the World Intellectual Property Organization. SUPPLEMENTARY INFORMATION: The...

  19. Development and evaluation of drug-antibody conjugates for the treatment of human myelogenous leukemia

    International Nuclear Information System (INIS)

    An immune serum to the Ph1+ human myelogenous leukemia cell line, K-562, was developed in goats. Following exhaustive absorptions, the antiserum and its immunoglobulin (Ig) fraction were highly cytotoxic for the homologenous cells in vitro in the presence of complement. In a nude mouse-human myelogenous leukemia model system, the Ig inhibits the growth and proliferation of myelosarcomas made up of K-562 cells. At the concentration of 6 mg or more, and beginning at 7 days after transplantation of myelosarcomas in nude mice, the administration of immunoglobulins resulted in the total suppression and subsequent elimination of the tumors. The dose-response relationship between the amount of Ig injected and the growth of myelosarcomas was demonstrated to be linear i.e., the extent of inhibition of tumor growth was directly dependent upon the dose of Ig given. Also, the uptake of 125I-labelled immunoglobulins by the K-562 myelosarcomas was at least 6-fold higher than that of the corresponding preimmune globulins

  20. Application of optical phase conjugation to plasma diagnostics (invited)

    Energy Technology Data Exchange (ETDEWEB)

    Jahoda, F.C.; Anderson, B.T.; Forman, P.R.; Weber, P.G.

    1985-05-01

    Several possibilities for plasma diagnostics provided by optical phase conjugation and, in particular, self-pumped phase conjugation in barium titanate (BaTiO/sub 3/) are discussed. These include placing a plasma within a dye laser cavity equipped with a phase conjugate mirror for intracavity absorption measurements, time differential refractometry with high spatial resolution, and simplified real-time holographic interferometry. The principles of phase conjugation with particular reference to photorefractive media and the special advantages of self-pumped phase conjugation are reviewed prior to the discussion of the applications. Distinctions are made in the applications between those for which photorefractive conjugators are essential and those for which they only offer experimental simplification relative to other types of phase conjugators.