scholarly journals Binding of a Monoclonal Antibody to Sporozoites of Sarcocystis singaporensis Enhances Escape from the Parasitophorous Vacuole, Which Is Necessary for Intracellular Development

2001 ◽  
Vol 69 (10) ◽  
pp. 6475-6482 ◽  
Author(s):  
T. Jäkel ◽  
E. Wallstein ◽  
F. Müncheberg ◽  
C. Archer-Baumann ◽  
B. Weingarten ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Specific Binding ◽  
Parasitophorous Vacuole ◽  
Intracellular Localization ◽  
Ultrastructural Analysis ◽  
Early Gene ◽  
Histone H4 ◽  
Acetylated Histone H4 ◽  
Development In Vitro

ABSTRACT Early intracellular development in vitro of the cyst-forming protozoon Sarcocystis singaporensis and the influence of a monoclonal antibody on invasion, intracellular localization, and development of sporozoites were studied. As revealed by immunofluorescence using parasite-specific antibodies which labeled the parasitophorous vacuole membrane (PVM) and by ultrastructural analysis, sporozoites invaded pneumonocytes of the rat via formation of a parasitophorous vacuole (PV). About half of the sporozoites left this compartment within the first 8 h postinfection to enter the host cell cytosol. By semiquantitative analysis of acetyl-histone H4 expression of sporozoites, a marker linked to early gene expression of eukaryotic cells, we show (supported by ultrastructural analysis) that escape from the PV appears to be necessary for early intracellular development. More than 90% of sporozoites located in the cytosol expressed high levels of acetylated histone H4 in the nucleus, whereas only a quarter of the intravacuolar sporozoites exhibited a similar signal. As revealed by ultrastructural analysis, young schizonts all resided in the cytosol. Specific binding of a monoclonal antibody (11D5/H3) to sporozoites before invasion significantly enhanced their escape from the PV, whereas cell invasion itself remained unaffected. The antibody actually increased proliferation of the parasites in vitro, providing a further link between residence in the cytosol and successful intracellular development. Monoclonal antibody 11D5/H3 precipitated a major 58-kDa antigen from oocyst-sporocyst extracts and reacted with the cytoplasm and the surface of sporozoites in immunofluorescence assays. Collectively, the observed antibody-parasite interaction suggests the existence of a signaling event that influences intracellular development of Sarcocystis.

scholarly journals Cerenkov luminescence imaging is an effective preclinical tool for assessing colorectal cancer PD-L1 levels in vivo

2020 ◽  
Author(s):  
Sheng Zhao ◽  
Wen-Bin Pan ◽  
Hui-Jie Jiang ◽  
Rong-Jun Zhang ◽  
Hao Jiang ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Monoclonal Antibody ◽  
Competitive Inhibition ◽  
Specific Binding ◽  
Luminescence Imaging ◽  
Tumor Bearing ◽  
Cerenkov Luminescence Imaging ◽  
The Status

Abstract Background : Preclinical and clinical studies have demonstrated that immunotherapy has effectively delayed tumor progression, and the clinical outcomes of anti-PD-1/PD-L1 therapy were related to PD-L1 expression level in the tumors. A 131 I-labeled anti-PD-L1 monoclonal antibody tracer, 131 I-PD-L1-Mab, was developed to study the target ability of non-invasive Cerenkov luminescence imaging in colorectal cancer xenograft mice.Method: Anti-PD-L1 monoclonal antibody labeled with 131 I( 131 I-PD-L1-Mab), and in vitro binding assays were used to evaluate the affinity of 131 I-PD-L1-Mab to PD-L1 and their binding level to different colorectal cancer cells, and compared with flow cytometry, western blot analysis, and immunofluorescence staining. The clinical application value of 131 I-PD-L1-Mab was evaluated through biodistribution and Cerenkov luminescence imaging, and different tumor-bearing models expressing PD-L1 were evaluated.Results: 131 I-PD-L1-Mab showed high affinity to PD-L1, and the equilibrium dissociation constant was 1.069×10 -9 M. The competitive inhibition assay further confirmed the specific binding ability of 131 I-PD-L1-Mab. In four different tumor-bearing models with different PD-L1 expression, the biodistribution and Cerenkov luminescence imaging showed that the RKO tumors demonstrated the highest uptake of the tracer 131 I-PD-L1-Mab, with a maximum uptake of 1.613 ± 0.738% ID/g at 120 h.Conclusions: There is a great potential for 131 I-PD-L1-Mab noninvasive Cerenkov luminescence imaging to assess the status of tumor PD-L1 expression and select patients for anti-PD-L1 targeted therapy.

Rapid purification of ribosomal particles assembled on histone H4 mRNA: a new method based on mRNA–DNA chimaeras

2013 ◽  
Vol 449 (3) ◽  
pp. 719-728 ◽  
Author(s):  
Lydia Prongidi-Fix ◽  
Laure Schaeffer ◽  
Angelita Simonetti ◽  
Sharief Barends ◽  
Jean-François Ménétret ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Specific Binding ◽  
Full Length ◽  
Molar Ratio ◽  
Detailed Knowledge ◽  
Histone H4 ◽  
Purification Method

Detailed knowledge of the structure of the ribosomal particles during their assembly on mRNA is a prerequisite for understanding the intricate translation initiation process. In vitro preparation of eukaryotic translation initiation complexes is limited by the rather tricky assembly from individually purified ribosomal subunits, initiation factors and initiator tRNA. In order to directly isolate functional complexes from living cells, methods based on affinity tags have been developed which, however, often suffer from non-specific binding of proteins and/or RNAs. In the present study we present a novel method designed for the purification of high-quality ribosome/mRNA particles assembled in RRL (rabbit reticulocyte lysate). Chimaerical mRNA–DNA molecules, consisting of the full-length mRNA ligated to a biotinylated desoxy-oligonucleotide, are immobilized on streptavidin-coated beads and incubated with RRL to form initiation complexes. After a washing step, the complexes are eluted by specific DNase I digestion of the DNA moiety of the chimaera, releasing initiation complexes in native conditions. Using this simple and robust purification setup, 80S particles properly programmed with full-length histone H4 mRNA were isolated with the expected ribosome/mRNA molar ratio of close to 1. We show that by using this novel approach purified ribosomal particles can be obtained that are suitable for biochemical and structural studies, in particular single-particle cryo-EM (cryo-electron microscopy). This purification method thus is a versatile tool for the isolation of fully functional RNA-binding proteins and macromolecular RNPs.

An ultrastructural analysis of chick embryonic skin development in vitro

1976 ◽  
Vol 197 (3) ◽  
pp. 389-401 ◽  
Author(s):  
P. Carinci ◽  
L. Simonelli ◽  
G. Bubola ◽  
P. Pettazzoni
Keyword(s):  
Ultrastructural Analysis ◽  
Skin Development ◽  
Embryonic Skin ◽  
Development In Vitro ◽  
Chick Embryonic Skin

scholarly journals Rapid production of SARS-CoV-2 receptor binding domain (RBD) and spike specific monoclonal antibody CR3022 in Nicotiana benthamiana

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Kaewta Rattanapisit ◽  
Balamurugan Shanmugaraj ◽  
Suwimon Manopwisedjaroen ◽  
Priyo Budi Purwono ◽  
Konlavat Siriwattananon ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Receptor Binding ◽  
Nicotiana Benthamiana ◽  
Specific Binding ◽  
Expression System ◽  
Disease Diagnosis ◽  
Binding Domain ◽  
Receptor Binding Domain ◽  
Global Public Health

Abstract Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is responsible for the ongoing global outbreak of coronavirus disease (COVID-19) which is a significant threat to global public health. The rapid spread of COVID-19 necessitates the development of cost-effective technology platforms for the production of vaccines, drugs, and protein reagents for appropriate disease diagnosis and treatment. In this study, we explored the possibility of producing the receptor binding domain (RBD) of SARS-CoV-2 and an anti-SARS-CoV monoclonal antibody (mAb) CR3022 in Nicotiana benthamiana. Both RBD and mAb CR3022 were transiently produced with the highest expression level of 8 μg/g and 130 μg/g leaf fresh weight respectively at 3 days post-infiltration. The plant-produced RBD exhibited specific binding to the SARS-CoV-2 receptor, angiotensin-converting enzyme 2 (ACE2). Furthermore, the plant-produced mAb CR3022 binds to SARS-CoV-2, but fails to neutralize the virus in vitro. This is the first report showing the production of anti-SARS-CoV-2 RBD and mAb CR3022 in plants. Overall these findings provide a proof-of-concept for using plants as an expression system for the production of SARS-CoV-2 antigens and antibodies or similar other diagnostic reagents against SARS-CoV-2 rapidly, especially during epidemic or pandemic situation.

Effects of an anti-exospore monoclonal antibody on microsporidial development in vitro

Parasitology ◽  
1998 ◽  
Vol 117 (6) ◽  
pp. 515-520 ◽  
Author(s):  
F. J. ENRIQUEZ ◽  
G. WAGNER ◽  
M. FRAGOSO ◽  
O. DITRICH
Keyword(s):  
Monoclonal Antibody ◽  
Host Cells ◽  
Infected Host ◽  
Encephalitozoon Cuniculi ◽  
Isotype Control ◽  
Encephalitozoon Intestinalis ◽  
Infected Cells ◽  
Pre Treatment ◽  
Development In Vitro

In this study we evaluated the effects of the anti-microsporidial exospore monoclonal antibody 3B6, recognizing 3 Encephalitozoon species, Encephalitozoon intestinalis (Syn. Septata intestinalis), Encephalitozoon cuniculi, and Encephalitozoon hellem on microsporidial growth in vitro. Pre-treatment of spores for 24 h with mAb 3B6 resulted in 21–29% fewer infected host cells 4 days after inoculation of the cultures compared to cultures pre-treated with medium or an irrelevant isotype control mAb (P<0·001). Fewer intracellular spores (1·2±0·2) in infected cells were found when mAb 3B6 was present in cultures compared to cultures with medium alone (4·3±0·8) or an irrelevant isotype control mAb (4·2±0·9; P<0·001). This decrease appeared not to be dependent on time of exposure, mAb concentration, or presence of complement. It is concluded that antibodies, particularly those directed to potential neutralizing-sensitive epitopes on spores, may have a role in the control of microsporidial growth in vitro.

A Therapeutic TCR Mimic Monoclonal Antibody for Intracellular PRAME Protein in Leukemias

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2527-2527 ◽  
Author(s):  
Aaron Chang ◽  
Tao Dao ◽  
Andrew Scott ◽  
Leonid Dubrovsky ◽  
Cheng Liu ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Retinoic Acid ◽  
Conflicts Of Interest ◽  
Specific Binding ◽  
Retinoic Acid Receptor ◽  
Pharmacokinetic Studies ◽  
Breast Cancers ◽  
Biodistribution Studies

Abstract Preferentially expressed antigen in melanoma (PRAME) is a well-validated target for T cell-based immunotherapy in leukemias and solid tumors. PRAME is a retinoic acid receptor binding protein that prevents retinoic acid-mediated differentiation, proliferation arrest, and apoptosis. As a cancer-testis antigen, PRAME has limited expression in healthy adult tissue that is restricted to the testes, ovaries, and endometrium. However, PRAME is over-expressed in multiple cancers including ALL, AML, melanomas, and breast cancers, making it a specific and highly attractive therapeutic target. PRAME is an intracellular protein making it impossible to target using traditional antibodies and it is not currently druggable. After proteasomal processing, the PRAME300-309 peptide is presented on the cell surface in the context of HLA*A02:01 molecules, for recognition by CD8 T cells. We therefore hypothesized that a TCR-mimic (TCRm) monoclonal antibody that recognizes surface PRAME300-309 presented by HLA*A02:01 could have therapeutic activity. Here, we describe Pr20, a therapeutic TCRm antibody, specific for the PRAME300-309 peptide in complex with HLA*A02:01, identified through a human scFv phage display library screen. Pr20 was engineered into full length human IgG1. Pr20 exhibited specific binding to PRAME300-309 -pulsed TAP-deficient T2 cells and bound PRAME+/ HLA*A02:01+ Ph+ ALL and AML, demonstrating that endogenously presented PRAME300-309 could be recognized by Pr20. Pr20 was determined to have 4 nM binding affinity by scatchard plot analysis. The specific epitope was mapped using alanine substitutions of non-anchor residues in the PRAME300-309 peptide and determined to primarily require the C-terminal residues. Pr20M, an afucosylated form of the antibody with enhanced Fc binding, mediated antibody-dependent cellular cytotoxicity (ADCC) in-vitro in a PRAME+/ HLA*A02:01+ restricted manner. Pharmacokinetic studies in C57BL/6 mice indicated that Pr20M was stable in-vivo and biodistribution studies in HLA*A02:01 transgenic mice suggested that there was no significant antibody sink. Pr20M was therapeutically active in established xenograft leukemia models in NSG mice (T, B, and NK-deficient). Interestingly, Pr20 binding to PRAME+/HLA*A02:01+ melanomas was minimally detectable, but was dramatically increased upon treatment with IFNγ, which also led to an increased sensitivity to ADCC. The data provide rationale for developing TCRm antibodies against intracellular oncoproteins as therapeutics. Disclosures No relevant conflicts of interest to declare.

Cellular localization and photodynamic effect of chlorin e6-monoclonal antibody conjugate

2005 ◽  
Vol 09 (02) ◽  
pp. 138-141 ◽  
Author(s):  
Shun-ichiro Ogura ◽  
Toshiaki Kamachi ◽  
Ichiro Okura
Keyword(s):  
Monoclonal Antibody ◽  
Tumor Cells ◽  
Binding Affinity ◽  
Cellular Localization ◽  
Specific Binding ◽  
Intracellular Localization ◽  
Chlorin E6 ◽  
Binding Affinities ◽  
High Accumulation ◽  
Peptide Bonds

Chlorin e6(Ce6) was conjugated with anti-tumor monoclonal antibody ( IgG ) to increase its binding affinity for tumors. Ce 6 was activated by N -hydroxysulfosuccinimide and conjugated with IgG via peptide bonds, and Ce 6 molecules were conjugated with IgG ( IgG - Ce 6) and their binding affinities to tumor cells were investigated. Intracellular localization of IgG - Ce 6 was observed, and IgG - Ce 6 was accumulated in tumor cells much higher than Ce 6, indicating that the IgG - Ce 6 has specific binding affinity to tumor cells. The effective photocytotoxicity of the cells with IgG - Ce 6 is caused by the high accumulation of IgG - Ce 6 in tumor cells.

scholarly journals Evaluation the Reactivity of a Peptide-Based Monoclonal Antibody with Drug Resistant Pulsotypes of Acinetobacter Baumannii as Potential Therapeutic Approach

2021 ◽  
Author(s):  
Omid Yeganeh ◽  
Mahdi Shabani ◽  
Parviz Pakzad ◽  
Nariman Mosaffa ◽  
Ali Hashemi
Keyword(s):  
Monoclonal Antibody ◽  
Acinetobacter Baumannii ◽  
Therapeutic Approach ◽  
Specific Binding ◽  
Protein A ◽  
Treatment Strategies ◽  
Passive Immunization ◽  
Hybridoma Cells ◽  
Affinity Constant

Abstract Background: Acinetobacter baumannii is an opportunistic and antibiotic-resistant pathogen that predominantly causes nosocomial infections. There is urgent need for development nonantibiotic-based treatment strategies. We developed novel monoclonal antibody (mAb) against a peptide of conserved outer membrane protein A (OmpA) and evaluated its reactivity with different pulsotypes of A.baumannii. Materials and Methods: Peptide derived from A.baumannii OmpA was conjugated to keyhole limpet hemocyanin and injected into Balb/c mice. Splenocytes of immunized mice were fused with SP2/0 myeloma cells followed by selection of antibody-producing hybridoma cells. After screening of different hybridoma colonies by ELISA, one monoclone was selected as 3F10-C9 and the antibody was tested for reaction with five different Acinetobacter pulsotypes that were resistant to carbapenem antibiotics. The affinity constant was measured by ELISA. The ELISA, Western blotting, indirect immunofluorescence (IFA), and in vitro Opsonophagocytosis assays were used to evaluate the reactivity of generated mAb. Results: The anti-OmpA antibody reacted with the immunizing peptide and had a high affinity (around 1.94 × 10 − 9 M) for its antigen in the ELISA. Specific binding of mAb to OmpA was confirmed in Western blot. IFA assays revealed that mAb recognized specific OmpA on the pulsotypes. Opsonophagocytosis assays showed that the mAb increased bactericidal activity of macrophage cells. The antibody function was higher in the presence of serum complement. Conclusion: The peptide-based mAb demonstrated optimal performance in laboratory experiments which may be appropriate in investigation on OmpA in Acinetobacter pathogenesis and development of passive immunization as novel therapeutic approach.

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