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Julie Rayes, Mathieu Ing, Sandrine Delignat, Ivan Peyron, Laurent Gilardin, Carl-Wilhelm Vogel, David C Fritzinger, Véronique Frémeaux-Bacchi, Srinivas V Kaveri, Lubka T Roumenina, Sébastien Lacroix-Desmazes
Development of neutralizing antibodies against therapeutic factor VIII (FVIII) is the most serious complication of the treatment of hemophilia A. Increasing evidence shows the multifactorial origin of the anti-FVIII immune response, combining both genetic and environmental factors. While a role for the complement system on innate as well as adaptive immunity has been documented, the implication of complement activation on the onset of the anti-FVIII immune response is unknown. Here, using in vitro assays for FVIII endocytosis by human monocyte-derived dendritic cells and presentation to T cells, as well as animal models of in vivo complement depletion, we show a novel role for complement C3 in enhancing the immune response against therapeutic FVIII...
November 16, 2017: Haematologica
Seda Onder, Alicia J Dafferner, Lawrence M Schopfer, Gaoping Xiao, Udaya Yerramalla, Ozden Tacal, Thomas A Blake, Rudolph C Johnson, Oksana Lockridge
Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are irreversibly inhibited by organophosphorus pesticides through formation of a covalent bond with the active site serine. Proteins that have no active site serine, for example albumin, are covalently modified on tyrosine and lysine. Chronic illness from pesticide exposure is not explained by inhibition of AChE and BChE. Our goal was to produce a monoclonal antibody that recognizes proteins diethoxyphosphorylated on tyrosine. Diethoxyphosphate-tyrosine adducts for 13 peptides were synthesized...
November 14, 2017: Chemical Research in Toxicology
Fernando Izquierdo, Hercules Moura, Fernando Jorge Bornay-Llinares, Rama Sriram, Carolina Hurtado, Ángela Magnet, Soledad Fenoy, Govinda Visvesvara, Carmen Del Aguila
BACKGROUND: Microsporidia are intracellular obligate parasites traditionally associated with immunosuppressed patients; their detection in immunocompetent patients has increased, highlighting their possible importance as emerging pathogens. Detection of spores in stools, urine, body fluids and tissues is difficult and immunological techniques such as immunofluorescence have proved to be a useful and reliable tool in the diagnosis of human microsporidiosis. For this reason, we have produced and characterized monoclonal antibodies (MAbs) specific for Encephalitozoon intestinalis (the second most frequent microsporidian infecting humans), and other Encephalitozoon species, that can be used in different diagnostic techniques...
November 9, 2017: Parasites & Vectors
Ki Soo Park, Hoyoung Kim, Soojin Kim, Kyungheon Lee, Sohyeon Park, Jun Song, Changwook Min, Farhana Khanam, Rasheduzzaman Rashu, Taufiqur Rahman Bhuiyan, Edward T Ryan, Firdausi Qadri, Ralph Weissleder, Jinwoo Cheon, Richelle C Charles, Hakho Lee
Pathogen-activated antibody-secreting cells (ASCs) produce and secrete antigen-specific antibodies. ASCs are detectable in the peripheral blood as early as 3 days after antigen exposure, which makes ASCs a potential biomarker for early disease detection. Here, we present a magnetic capture and detection (MCD) assay for sensitive, on-site detection of ASCs. In this approach, ASCs are enriched through magnetic capture, and secreted antibodies are magnetically detected by a miniaturized nuclear magnetic resonance (μNMR) system...
November 14, 2017: ACS Nano
Ricardo Jara-Acevedo, Paula Díez, María González-González, Rosa María Dégano, Nieves Ibarrola, Rafael Góngora, Alberto Orfao, Manuel Fuentes
Phage-display technology constitutes a powerful tool for the generation of specific antibodies against a predefined antigen. The main advantages of phage-display technology in comparison to conventional hybridoma-based techniques are: (1) rapid generation time and (2) antibody selection against an unlimited number of molecules (biological or not). However, the main bottleneck with phage-display technology is the validation strategies employed to confirm the greatest number of antibody fragments. The development of new high-throughput (HT) techniques has helped overcome this great limitation...
2018: Methods in Molecular Biology
Chai Fung Chin, Yee Siew Choong, Theam Soon Lim
Antibody phage display has been widely established as the method of choice to generate monoclonal antibodies with various efficacies post hybridoma technology. This technique is a popular method which takes precedence over ease of methodology, time- and cost-savings with comparable outcomes to conventional methods. Phage display technology manipulates the genome of M13 bacteriophage to display large diverse collection of antibodies that is capable of binding to various targets (nucleic acids, peptides, proteins, and carbohydrates)...
2018: Methods in Molecular Biology
Zhihao Wu, Brian H Santich, Hong Liu, Cheng Liu, Nai-Kong V Cheung
Antibodies that bind peptide-MHC (pMHC) complex in a manner akin to T-cell receptor (TCR) have not only helped in understanding the mechanism of TCR-pMHC interactions in the context of T-cell biology, but also spurred considerable interest in recent years as potential cancer therapeutics. Traditional methods to generate such antibodies using hybridoma and B-cell sorting technologies are sometimes inadequate, possibly due to the small contribution of peptide to the overall B-cell epitope space on the surface of the pMHC complex (typical peptide MW = 1 kDa versus MHC MW = 45 kDa), and to the multiple efficiency limiting steps inherent in these methods...
2018: Methods in Molecular Biology
Thi Thu Ha Nguyen, Jong Seo Lee, Hyunbo Shim
Rabbits have distinct advantages over mice as a source of target-specific antibodies. They produce higher affinity antibodies than mice, and may elicit strong immune response against antigens or epitopes that are poorly immunogenic or tolerated in mice. However, a great majority of currently available monoclonal antibodies are of murine origin because of the wider availability of murine fusion partner cell lines and well-established tools and protocols for fusion and cloning of mouse hybridoma. Phage-display selection of antibody libraries is an alternative method to hybridoma technology for the generation of target-specific monoclonal antibodies...
2018: Methods in Molecular Biology
Arnaud Avril, Sebastian Miethe, Michael Hust, Thibaut Pelat
Rapidly after the clinical success of the first murine therapeutic antibody licensed in 1985 (muromomab-CD3), the first limits of the therapeutic use of antibodies deriving from hybridoma technology appeared. Indeed, the nonhuman nature of these therapeutic antibodies makes them immunogenic when administrated to patients, which develop anti-drug antibodies (ADA). If repeated drug-administrations are needed, the immune response will accelerate the elimination of the drug, leading to a therapeutic failure, or in the worst case to an anaphylactic reaction against the murine protein...
2018: Methods in Molecular Biology
Meng Gao, Feng Zhang, Yun Zhu, Limei Gao, Yunshui Jiang, Yongneng Luo, Fangchang Zhuang, Zian Mao, Jiangsen Mao
The enterovirus 71 (EV71) SP70 epitope, derived from amino acids 208‑222 of VP1, is a neutralizing epitope. The present study aimed to assess the inter‑species differences of the antibodies induced by EV71‑based antigens in responses to SP70 mutant peptides. BALB/c mice and Lou/C rats were immunized with EV71 SP70. Monoclonal antibodies (Mabs) were produced by hybridoma clones. Serum polyclonal antibodies (Pabs) were produced from BALB/c mice and New Zealand white rabbits immunized with recombinant EV71 VP1 (rEV71‑VP1) protein or inactivated EV71...
November 6, 2017: Molecular Medicine Reports
J E Abud, C G Santamaría, E H Luque, H A Rodriguez
In the present study, we developed both a conventional enzyme-linked immunosorbent assay (ELISA) and a highly sensitive immuno-polymerase chain reaction (IPCR) assay specific for detection of human thyroid stimulating hormone (hTSH). Several anti-hTSH monoclonal antibodies (MAbs) were generated using hybridoma technology. Two pairs of MAbs (B-4 and B-9) were rationally selected and the optimal assay conditions of sandwich ELISAs were established. The ELISA prototypes were evaluated with standards calibrated with WHO 2nd International Reference Preparation for hTSH and in comparison with a commercial ELISA Kit...
October 27, 2017: Analytical Biochemistry
Satoshi Hiroi, Motoki Kuhara, Yoshiro Kishi, Ken-Ichiro Ono, Shun Matsuzawa, Naomasa Yamamoto, Jun Komano
Influenza virus causes acute respiratory infection in humans, and is a major public health concern globally. Antibodies play a central role in host protection against influenza virus. We isolated human monoclonal antibodies (hMAb) 206-2-4 and 201-6-8 by a human hybridoma protocol that neutralized various but distinct influenza virus (IFV) A/H1N1 strains, including 2009 pandemic strains. The half-inhibitory concentration of 206-2-4 and 201-6-8 against A/H1N1pdm09 strains was 2-100ng/mL and 5-20μg/mL, respectively...
October 18, 2017: Immunobiology
Edward A Greenfield
Immunologically active organs such as the spleen and lymph nodes are rich sources of antibodies; they are also major sites in the body where antibody-producing B cells accumulate. The lymph nodes, in particular, can be targeted when deciding where to inject an animal with antigen. Once an immunized animal has developed a sufficient serum antibody titer, these organs can be harvested for hybridoma fusion and monoclonal antibody production.
November 1, 2017: Cold Spring Harbor Protocols
Frances Weis-Garcia, Robert H Carnahan
For research groups needing to isotype a significant number of antibodies on a fairly regular basis, the protocol outlined here provides a cost-effective option. In this simple sandwich ELISA, the monoclonal antibody is first captured with antibodies that discriminate between heavy-chain isotypes and then is detected with antibodies specific for each light chain. This combination ensures that free light chain secreted by hybridomas does not yield a false-positive result, because in a true positive both the capture and detecting antibodies bind the monoclonal antibody...
November 1, 2017: Cold Spring Harbor Protocols
Hua Dai, Haixia Zhang, Meiling Wang, Fan Ge, Ying Shen
Objective To prepare recombinant protein of human neuropilin 1 b1b2 domain (HuNRP1(b)) and monoclonal antibodies (mAbs) against the recombinant HuNRP1(b) (rHuNRP1(b)). Methods The coding sequence of HuNRP1(b) was amplified and cloned into vector pET22b to construct recombinant plasmid pET-HuNRP1(b). After analyzed by restriction enzyme digestion and DNA sequencing, pET-HuNRP1(b) was transformed into Escherichia coli and induced to express rHuNRP1(b) with histidine tag (His-HuNRP1(b)), which was identified by SDS-PAGE analysis...
September 2017: Xi Bao Yu Fen Zi Mian Yi Xue za Zhi, Chinese Journal of Cellular and Molecular Immunology
Jian Xu, Jing Wu, Bo Jiang, Houjun He, Xixi Zhang, Xiaoyang Li, Dawei Yang, Xiufen Huang, Joshua E Sealy, Munir Iqbal, Yongqing Li
Glycoprotein D (gD) of bovine herpesvirus-1 (BoHV-1) is essential for attachment and penetration of cells during infection and is a major target for neutralizing antibodies during an adaptive immune response. Currently there are no recombinant antibodies capable of binding gD epitopes for use in treating BoHV-1 infection. In this study, a bovine scFv gene derived from a hybridoma secreting monoclonal antibodies (McAbs) against the amino acid motif MEESKGYEPP of gD was expressed in E. coli. Molecular modeling, western blot and ELISA analysis showed that this scFv had a high affinity for BoHV-1 gD, with a Kd of 161...
December 2017: Applied Microbiology and Biotechnology
Pan Kyeom Kim, Sun Ju Keum, Modupe O V Osinubi, Richard Franka, Ji Young Shin, Sang Tae Park, Man Su Kim, Mi Jung Park, Soo Young Lee, William Carson, Lauren Greenberg, Pengcheng Yu, Xiaoyan Tao, Wang Lihua, Qing Tang, Guodong Liang, Madhusdana Shampur, Charles E Rupprecht, Shin Jae Chang
Current post-exposure prophylaxis for rabies virus infection has several limitations in terms of supply, cost, safety, and efficacy. Attempts to replace human or equine rabies immune globulins (HRIG or ERIG) have been made by several companies and institutes. We developed potent monoclonal antibodies to neutralize a broad spectrum of rabies viruses by screening hybridomas received from the U.S. Centers for Disease Control and Prevention (CDC). Two kinds of chimeric human antibodies (chimeric #7 and #17) were constructed by cloning the variable regions from selected hybridomas and the constant region of a human antibody...
2017: PloS One
Tomohiro Makino, Ryuichi Nakamura, Maki Terakawa, Satoshi Muneoka, Kazuhiro Nagahira, Yuriko Nagane, Jyoji Yamate, Masakatsu Motomura, Kimiaki Utsugisawa
The majority of patients with myasthenia gravis (MG), an organ-specific autoimmune disease, harbor autoantibodies that attack the nicotinic acetylcholine receptor (nAChR-Abs) at the neuromuscular junction of skeletal muscles, resulting in muscle weakness. Single cell manipulation technologies coupled with genetic engineering are very powerful tools to examine T cell and B cell repertoires and the dynamics of adaptive immunity. These tools have been utilized to develop mAbs in parallel with hybridomas, phage display technologies and B-cell immortalization...
2017: PloS One
Sarina Nurgul, Abeldenov Sailau, Turgimbayeva Aigerim, Zhylkibayev Assylbek, Ramankulov Erlan, Khassenov Bekbolat, Eskendirova Saule
Human epidermal growth factor receptor 2 (HER2) is an important biomarker for detection and treatment of different types of cancers such as breast, ovarian, stomach cancer. In this study, we developed a monoclonal antibody against the extracellular domain (ECD) of HER2 biomarker of breast cancer. For this purpose, the ECD-HER2 gene was amplified and cloned into an expression vector. Gene was generated in Escherichia coli BL21 (DE3) strain for expression of recombinant protein. The expressed protein was separated by SDS-PAGE and detected by anti-his monoclonal antibody in immunoblotting...
September 8, 2017: Human Antibodies
Yuehong Hu, Zimin Chen, Yuxiang Chen, Yinghui Yang, Shuying Wei, Liuwei Song, Guoliang Zhou, Shengxiang Ge
The aim of this study is to prepare and characterize cardiac troponin T (cTnT) monoclonal antibodies (mAb), and further develop a chemiluminescence quantitative detection assay for cTnT. BALB/c mice were immunized with recombinant cTnT antigen, and specific mAbs were prepared using conventional hybridoma technique and screened by indirect ELISA method. To identify the epitopes, several cTnT peptide fragments were synthesized or expressed by genetic engineering. A double antibody sandwich ELISA method was used to screen the mAb pairs for cTnT detection, and the automatic chemiluminescence detection assay for cTnT was developed...
December 25, 2016: Sheng Wu Gong Cheng Xue Bao, Chinese Journal of Biotechnology
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