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Search results for: Polyclonal

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#35044813   2022/01/19 To Up

From structure to sequence: Antibody discovery using cryoEM.

One of the rate-limiting steps in analyzing immune responses to vaccines or infections is the isolation and characterization of monoclonal antibodies. Here, we present a hybrid structural and bioinformatic approach to directly assign the heavy and light chains, identify complementarity-determining regions, and discover sequences from cryoEM density maps of serum-derived polyclonal antibodies bound to an antigen. When combined with next-generation sequencing of immune repertoires, we were able to specifically identify clonal family members, synthesize the monoclonal antibodies, and confirm that they interact with the antigen in a manner equivalent to the corresponding polyclonal antibodies. This structure-based approach for identification of monoclonal antibodies from polyclonal sera opens new avenues for analysis of immune responses and iterative vaccine design.
Aleksandar Antanasijevic, Charles A Bowman, Robert N Kirchdoerfer, Christopher A Cottrell, Gabriel Ozorowski, Amit A Upadhyay, Kimberly M Cirelli, Diane G Carnathan, Chiamaka A Enemuo, Leigh M Sewall, Bartek Nogal, Fangzhu Zhao, Bettina Groschel, William R Schief, Devin Sok, Guido Silvestri, Shane Crotty, Steven E Bosinger, Andrew B Ward

1013 related Products with: From structure to sequence: Antibody discovery using cryoEM.

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#35043970   2022/01/19 To Up

History and development of staining methods for skeletal muscle fiber types.

The contractile and metabolic properties of skeletal muscles depend on the composition of muscle fibers. There are two major fiber types: type 1 and type 2. Type 2 fibers are further subdivided into type 2A, 2X, and 2B fibers. Muscle fiber type composition is an important property that affects sports performance and metabolic ability in humans, and meat quality in domestic animals. In this review, we summarize the history of muscle fiber type classification based on various staining methods for skeletal muscle sections. The history illustrates the development of an experimental method to detect myosin heavy chain (MyHC) proteins, which are the most common marker molecules for muscle fiber type. Metabolic enzymes, such as nicotinamide adenine dinucleotide-tetrazolium reductase and succinate dehydrogenase are also described for histochemical staining combined with myosin ATPase staining. We found an improvement in the quality of antibodies used for immunostaining of MyHC, from polyclonal antibodies to monoclonal antibodies (mAbs) and then to mAbs produced by synthetic peptides as antigens. We believe that the information presented herein will assist researchers in selecting optimal staining methods, dependent on the experimental conditions and purposes.
Shoko Sawano, Wataru Mizunoya

2064 related Products with: History and development of staining methods for skeletal muscle fiber types.

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#35042674   2022/01/15 To Up

Development of IgM-ELISA for diagnosis of recent infection of Japanese encephalitis virus in equines.

Japanese encephalitis (JE) is a re-emerging mosquito borne disease, for which equines are most susceptible amongst all animals. Detection of specific immunoglobulin 'M' (IgM) is considered as an ideal way to diagnose recent JE virus infection in equines due to low virus load and short-term viremia. The present study was undertaken to develop a sensitive and specific recombinant NS1 protein based indirect IgM-ELISA and IgM capture (MAC) ELISA to diagnose recent infection of JEV in equines. Indirect IgM ELISA was standardized with relative diagnostic sensitivity and specificity of 100% and 88.5%, respectively. The validation of indirect IgM-ELISA in different laboratories revealed excellent reproducibility with Cohen's kappa value ranging between 0.84 and 1. The standardization of MAC ELISA was attempted using checker board titration method and non-specific binding of polyclonal anti-equine IgM capture antibody with anti-porcine IgG conjugate and with hyperimmune serum raised in swine against the antigen was observed. Hence, the MAC ELISA was standardized with monoclonal capture antibody; however, its diagnostic performance could not meet the satisfactory limit. Due to better sensitivity and less turnaround time, indirect IgM-ELISA was employed to screen 821 equine serum samples revealing 33.73% positivity of IgM antibodies against JEV in equine population of India. The high JEV sero-positivity warrants the need for vaccination in Indian equine population along with the demand for research focused towards anti-viral therapy. The indirect IgM-ELISA developed in the present study could be useful to diagnose acute or recent infection of JEV in equines as well as in sero-epidemiological studies.
Anamika Sahu, Himani Dhanze, Vijayta Singh, Deepa Mehta, Megha Gupta, Mithilesh Singh, V K Vinod, B R Gulati

2011 related Products with: Development of IgM-ELISA for diagnosis of recent infection of Japanese encephalitis virus in equines.

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#35039617   2022/01/17 To Up

Author Correction: Cell-autonomous megakaryopoiesis associated with polyclonal hematopoiesis in triple-negative essential thrombocythemia.


Tadaaki Inano, Marito Araki, Soji Morishita, Misa Imai, Yoshihiko Kihara, Maho Okuda, Yinjie Yang, Masafumi Ito, Satoshi Osaga, Hiroyuki Mano, Yoko Edahiro, Tomonori Ochiai, Kyohei Misawa, Yasutaka Fukuda, Jun Ando, Norio Komatsu

2083 related Products with: Author Correction: Cell-autonomous megakaryopoiesis associated with polyclonal hematopoiesis in triple-negative essential thrombocythemia.

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#35038922   2022/01/18 To Up

Cross-Reactive Antibody Responses against Nonpoliovirus Enteroviruses.

Enteroviruses are among the most common human viral pathogens. Infection with members of a subgroup of viruses within this genus, the nonpoliovirus enteroviruses (NPEVs), can result in a broad spectrum of serious illnesses, including acute flaccid myelitis (AFM), a polio-like childhood paralysis; neonatal sepsis; aseptic meningitis; myocarditis; and hand-foot-mouth disease. Despite the diverse primary sites of virus infection, including the respiratory and alimentary tracts, and an array of diseases associated with these infections, there is significant genetic and antigenic similarity among NPEVs. This conservation results in the induction of cross-reactive antibodies that are either able to bind and neutralize or bind but not neutralize multiple NPEVs. Using plaque reduction and enzyme-linked immunosorbent assay (ELISA)-based binding assays, we define the antigenic relationship among poliovirus and NPEVs, including multiple isolates of EV-D68, EV-A71, EV-D70, EV-94, EV-111, Coxsackievirus A24v, and rhinovirus. The results reveal extensive cross-reactivity among EVs that cannot be predicted from phylogenetic analysis. Determining the immunologic relationship among EVs is critical to understanding the humoral response elicited during homologous and heterologous virus infections. Enteroviruses (EVs) are common human pathogens. Although infection with EVs leads to cross-reactive antibodies, the clinical relevance of these antibodies is unclear given the estimated incidence of EV infections in the general population of one per year. The hypothesis that anti-EV cross-reactive antibodies can bind and neutralize heterologous EVs was investigated using polyclonal sera collected from animals immunized with individual EVs. Both binding and neutralization activities against heterologous EVs was observed in these sera, and we speculate that cross-reactive antibodies may modulate infection and disease severity. Defining the antigenic relationship among EVs may provide insights into the epidemiology and pathogenesis of enterovirus infections.
Amy B Rosenfeld, Edmund Qian Long Shen, Michaela Melendez, Nischay Mishra, W Ian Lipkin, Vincent R Racaniello

1895 related Products with: Cross-Reactive Antibody Responses against Nonpoliovirus Enteroviruses.

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#35036470   2021/12/06 To Up

Safe and efficient hematopoietic stem cell transduction in nonhuman primates using HDAd5/35++ vectors.

We tested a new hematopoietic stem cell (HSC) transduction/selection approach in rhesus macaques using HSC-tropic, integrating, helper-dependent adenovirus vectors (HDAd5/35++) designed for the expression of human γ-globin in red blood cells (RBCs) to treat hemoglobinopathies. We show that HDAd5/35++ vectors preferentially transduce HSCs after intravenous injection into granulocyte colony-stimulating factor (G-CSF)/AMD3100-mobilized animals and that transduced cells return to the bone marrow and spleen. The approach was well tolerated, and the activation of proinflammatory cytokines that are usually associated with intravenous adenovirus vector injection was successfully blunted by pre-treatment with dexamethasone in combination with interleukin (IL)-1 and IL-6 receptor blockers. Using our MGMT-based selection approach, γ-globin RBCs increased in all animals with levels up to 90%. After selection, the percentage of γ-globin RBCs declined, most likely due to an immune response against human transgene products. Our biodistribution data indicate that γ-globin RBCs in the periphery were mostly derived from mobilized HSCs that homed to the spleen. Integration site analysis revealed a polyclonal pattern and no genotoxicity related to transgene integrations. This is the first proof-of-concept study in nonhuman primates to show that HSC gene therapy could be feasible in humans without the need for high-dose chemotherapy conditioning and HSC transplantation.
Chang Li, Hongjie Wang, Sucheol Gil, Audrey Germond, Connie Fountain, Audrey Baldessari, Jiho Kim, Zhinan Liu, Aphrodite Georgakopoulou, Stefan Radtke, Tamás Raskó, Amit Pande, Christina Chiang, Eli Chin, Evangelia Yannaki, Zsuzsanna Izsvák, Thalia Papayannopoulou, Hans-Peter Kiem, André Lieber

2712 related Products with: Safe and efficient hematopoietic stem cell transduction in nonhuman primates using HDAd5/35++ vectors.

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#35033254   2021/11/20 To Up

Molecularly imprinted polymer as a synthetic antibody for the biorecognition of hazelnut Cor a 14-allergen.

Artificial receptors that mimic their natural biological counterparts have several advantages, such as lower production costs and increased shelf-life stability/versatility, while overcoming the ethical issues related to raising antibodies in animals. In this work, the proposed tailor-made molecularly imprinted polymer (MIP)-allergen receptors aimed at substituting or even transcending the performance of biological antibodies. For this purpose, a MIP was proposed as an artificial antibody for the recognition of hazelnut Cor a 14-allergen. The target protein was grafted onto the conducting polypyrrole receptor film using gold screen-printed electrodes (Au-SPE). The electrochemical assessment presented a linear response for the dynamic range of 100 fg mL-1 μg mL and a LOD of 24.5 fg mL, as determined by square wave voltammetry from the calibration curves prepared with standards diluted in phosphate buffer. Surface plasmon resonance (SPR) was used as a secondary transducer to evaluate the performance of the Cor a 14-MIP sensor, enabling a linear dynamic range of 100 fg mL- 0.1 μg mL and a LOD of 18.1 fg mL. The selectivity of the tailored-made Cor a 14-MIP was tested against potentially cross-reactive plant/animal species based on the rebinding affinity (Freundlich isotherm-K) of homologues/similar proteins, being further compared with custom-made polyclonal anti-Cor a 14 IgG immunosensor. Results evidenced that the MIP mimics the biorecognition of biological antibodies, presenting higher selectivity (only minor cross-reactivity towards walnut and Brazil nut 2S albumins) than the Cor a 14/anti-Cor a 14 IgG immunosensor. The application of electrochemical Cor a 14-MIP sensor to model mixtures of hazelnut in pasta enabled quantifying hazelnut down to 1 mg kg (corresponding to 0.16 mg kg of hazelnut protein in the matrix). To the best of our knowledge, Cor a 14-MIP is the first sensor based on an artificial/synthetic biorecognition platform for the specific detection of hazelnut allergens, while presenting high-performance parameters with demonstrated application in food safety management.
Renata Costa, Joana Costa, Patrícia Moreira, Ana T S C Brandão, Isabel Mafra, A Fernando Silva, Carlos M Pereira

1296 related Products with: Molecularly imprinted polymer as a synthetic antibody for the biorecognition of hazelnut Cor a 14-allergen.

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#35027426   // To Up

Large-scale manufacturing and characterization of CMV-CD19CAR T cells.

Adoptive transfer of CD19-specific chimeric antigen receptor (CD19CAR) T cells can induce dramatic disease regression in patients with B cell malignancies. CD19CAR T cell therapy may be limited by insufficient engraftment and persistence, resulting in tumor relapse. We previously demonstrated a proof of principle that cytomegalovirus (CMV)-specific T cells can be isolated and enriched prior to CD19CAR transduction to produce CMV-CD19CAR T cells, and that these CMV-CD19CAR T cells can be expanded in vivo through CMV vaccination, resulting in better tumor control in a murine model. Here we developed a clinical platform for generating CMV-CD19CAR T cells.
Xiuli Wang, Ryan Urak, Miriam Walter, Min Guan, Tianxu Han, Vibhuti Vyas, Sheng-Hsuan Chien, Brenna Gittins, Mary C Clark, Sally Mokhtari, Angelo Cardoso, Don J Diamond, John Zaia, Stephen J Forman, Ryotaro Nakamura

2664 related Products with: Large-scale manufacturing and characterization of CMV-CD19CAR T cells.

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#35017838   2021/11/12 To Up

Expression of the recombinant C-terminal of the S1 domain and N-terminal of the S2 domain of the spike protein of porcine epidemic diarrhea virus.

Porcine epidemic diarrhea virus (PEDV) causes severe diarrhea in suckling piglets, leading to severe economic losses in the swine industry. Commercial vaccines have limited effectiveness against different genogroups of PEDV and the shedding of virus. The C-terminal of the S1 domain and the N-terminal of the S2 domain (S1-2) protein of the spike (S) protein have four neutralizing epitopes. However, research on the expression of the S1-2 segment of the S gene has been limited. In this study, we expressed a recombinant S1-2 protein of the S protein of the PEDV Thai isolate and characterized the immunological properties of the recombinant S1-2 protein.
Jiraporn Sritun, Natnaree Inthong, Siriluk Jala, Sakuna Phatthanakunanan, Khomson Satchasataporn, Kaitkanoke Sirinarumitr, Preeda Lertwatcharasarakul, Theerapol Sirinarumitr

1641 related Products with: Expression of the recombinant C-terminal of the S1 domain and N-terminal of the S2 domain of the spike protein of porcine epidemic diarrhea virus.

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