Determination of the binding affinity of an anti-CD34 single-chain antibody using a novel, flow cytometry based assay

1997 ◽  
Vol 201 (2) ◽  
pp. 223-231 ◽  
Author(s):  
Chris A Benedict ◽  
Albert J MacKrell ◽  
W.French Anderson
Keyword(s):  
Flow Cytometry ◽  
Binding Affinity ◽  
Single Chain ◽  
Single Chain Antibody

scholarly journals Structure Determination of Inactive-State GPCRs with a Universal Nanobody

2021 ◽  
Author(s):  
Michael J. Robertson ◽  
Feng He ◽  
Justin G. Meyerowitz ◽  
Alpay B. Seven ◽  
Ouliana Panova ◽  
...  
Keyword(s):  
Somatostatin Receptor ◽  
Protein Structures ◽  
Single Chain ◽  
Intracellular Loop ◽  
Single Chain Antibody ◽  
Inactive State ◽  
X Ray Crystallography ◽  
High Resolution Map ◽  
Μ Opioid Receptor

Cryogenic electron microscopy (cryo-EM) has widened the field of structure-based drug discovery by allowing for routine determination of membrane protein structures previously intractable. However, despite representing one of the largest classes of therapeutic targets, most inactive-state G protein-coupled receptors (GPCRs) have remained inaccessible for cryo-EM because their small size and membrane-embedded nature impedes projection alignment for high-resolution map reconstructions. Here we demonstrate that a camelid single-chain antibody (nanobody) recognizing a grafted intracellular loop can be used to obtain cryo-EM structures of different inactive-state GPCRs at resolutions comparable or better than those obtained by X-ray crystallography. Using this approach, we obtained the structure of human neurotensin 1 receptor (NTSR1) bound to antagonist SR48692, of μ-opioid receptor (MOR) bound to the clinical antagonist alvimopan, as well as the structure of the previously uncharacterized somatostatin receptor 2 (SSTR2) in the apo state; each of these structures yields novel insights into ligand binding and specificity. We expect this rapid, straightforward approach to facilitate the broad structural exploration of GPCR inactive states without the need for extensive engineering and crystallization.

Determination of active single chain antibody concentrations in crude periplasmic fractions

1996 ◽  
Vol 194 (2) ◽  
pp. 201-209 ◽  
Author(s):  
Bert Kazemier ◽  
de Haard Hans ◽  
Piet Boender ◽  
Bob van Gemen ◽  
Hennie Hoogenboom
Keyword(s):  
Single Chain ◽  
Single Chain Antibody

scholarly journals Development of Anti-CD99scFvs for the Treatment of Acute Myeloid Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4345-4345
Author(s):  
Yang Zhao ◽  
Jiawei Li ◽  
Vijaya Pooja Vaikari ◽  
Albert Lam ◽  
Atham Ali ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Acute Myeloid Leukemia ◽  
Mouse Model ◽  
Binding Affinity ◽  
Myeloid Leukemia ◽  
Single Chain ◽  
Xenograft Mouse Model ◽  
Acute Myeloid

Abstract CD99 has gained much attention in recent years as a novel therapeutic target in hematological malignancies, due to its upregulation in acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL). We have recently shown that targeting CD99 with a knockdown approach or with commercial antibodies results in antileukemia activity in AML cells. We have also developed anti-CD99 nanoparticles and demonstrated excellent in vitro and in vivo antileukemia activity. Here we report the development of human single chain variable fragment targeting CD99 (anti-CD99 scFv) and the preclinical activity in AML cells and in AML xenograft mouse model. The anti-CD99 scFv was developed by inserting CD99scFv DNA sequence into the pFUSE vector, encoding pATL103-CD99 scFvs. To produce anti-CD99 scFv, the plasmids were transfected in Expi293 (HEK293T) cells. Then the medium was harvested and underwent two cycles of dialysis. To determine product purity, proteins were analyzed on SDS-PAGE gel stained with Coomassie blue. For each 240ml cell culture media with recombinant CD99 plasmids, we obtained about 1-2mg anti-CD99 scFv. The binding affinity of CD99 scFv to CD99 surface protein was assessed in 293T cells (CD99 null cells) which exhibited no binding and in MOLM-13 cells and MV4-11 (CD99 positive AML cells) which demonstrated strong binding. Treatment with 5uM of anti-CD99 scFv significantly reduced cell viability in both leukemic cell line MOLM-13 and MV4-11 (MOLM-13: 35.14%, P= 0.008; MV4-11: 30.17%, P=0.002) and primary AML patient blasts (29.37%, P=0.048) compared with control cells. Colony forming assay showed that anti-CD99 scFv treated AML blasts exhibited less number of colonies compared with control cells (plating efficiency (PE): 0.035% vs 0.12%). We also established the in vivo antileukemia activity of anti-CD99 scFv using MOLM-13 cells (FLT3-ITD positive AML cells) NOD scid gamma (NSG) xenograft mouse model. MOLM-13 (2.5x10^6) cells were engrafted into NSG mice via IV tail injection (N=4 mice per group). Mice were treated with PBS (group 1) or 4mg/kg of anti-CD99 scFV on days 10, 14, 18 and 22 post cell engraftment. Mice were euthanized on day 24 and levels of leukemia engraftment were assessed by flow cytometry measurements of huCD45 staining of cells collected from the bone marrow and the peripheral blood and compared between the two groups. Mice treated with four doses of 4mg/kg CD99scFv demonstrated significant reduction in leukemia engraftment in the bone marrow assessed by flow cytometry measurements of huCD45 staining compared with the PBS mice group (huCD45%: 39.7 vs 56, P = 0.0017). In conclusion, we report the development of anti-CD99 single chain variable fragments for the treatment of AML. Our study demonstrates good binding affinity and specificity and a promising preclinical antileukemia activity both in AML cells and in xenograft mouse model. Disclosures Yaghmour: Jazz: Consultancy, Honoraria; Astellas: Consultancy; Takeda: Consultancy; Incyte: Consultancy.

Co-expression of recombinant single chain variable fragment recognizing blood antigen fused with sumo and chaperones in Escherichia coli

2018 ◽  
Vol 40 (4) ◽  
Author(s):  
Dang Thi Ngoc Ha ◽  
Le Thi Thu Hong ◽  
Truong Nam Hai
Keyword(s):  
Recombinant Antibody ◽  
Blood Type ◽  
Soluble Form ◽  
Supernatant Fraction ◽  
Blood Group Antigens ◽  
Single Chain ◽  
E Coli ◽  
Recombinant Scfv ◽  
Single Chain Variable Fragments

Single chain variable fragments (scFv) have widely been used in research, diagnosis and treatment, but the scFv is considered as difficult protein for expression in E. coli. In previous studies, we expressed a construction of recombinant single chain variable fragments again antigen specific for blood type A (antiA-scFv) individually or fused with Trx or SUMO. However, soluble fraction was low abandant and only approximately 40% when fused with Trx, the other cases were expressed in form of inclusion body. Therefore, it was difficult for purification, refolding and activity assesment. In thispaper, we demonstrated a suitable construction for soluble production of antiA-scFv fused with SUMO (SM/antiA-scFv) in presence of chaparones. Under fermentation with 0.1 mM IPTG at 20oC, the SM/antiA-scFv was entirely expressed in soluble form. Importantly, after cleavage from SUMO with SUMOprotease, antiA-scFv was still maintained in the supernatant fraction. Therefore, it can help ensure bioactivity and is useful for purification process. To the best of our knowledge, this is the first report showing soluble recombinant scFv fused with SUMO in presence of chaperone for determination of blood group antigens. Thus, this result facilitates the optimal study of soluble expression, purification and bioactivity determination of the antiA-scFv recombinant antibody. 

Single-chain Antibody Against Reg4 Suppresses Gastric Cancer Cell Growth and Enhances 5-FU-induced Cell Death in vitro

2019 ◽  
Vol 19 (5) ◽  
pp. 610-619 ◽  
Author(s):  
Xue-Qing Zhang ◽  
Lu-Ting Yu ◽  
Pei Du ◽  
Tian-Qi Yin ◽  
Zhi-Yuan Zhang ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cell Proliferation ◽  
Cell Death ◽  
Cancer Cell ◽  
Gastric Cancer Cell ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Ags Cells ◽  
Thiazolyl Blue Tetrazolium Bromide

Background:Regenerating islet-derived gene family member 4 (Reg4), a well-investigated growth factor in the regenerative pancreas, has recently been reported to be highly associated with a majority of gastrointestinal cancers. Pathological hyper-expression or artificial over-expression of Reg4 causes acceleration of tumor growth, migration, and resistance to chemotherapeutic 5-Fluorouracil (5-FU). Until now, no method has been successfully established for eliminating the effects of Reg4 protein.Methods:This study reports the production of an engineered immunoglobin, a single-chain variable fragment (scFv-Reg4), to specifically bind Reg4 and block the bioactivity. The complementary-determining regions (CDRs) against Reg4 were assigned using MOE and ZDOCK servers. The binding affinity (KD) was determined by bio-layer interferometry (BLI). MKN45 and AGS cell proliferation was determined by Thiazolyl blue tetrazolium bromide (MTT) method and the cell apoptosis was detected by flow cytometry assay.Results:The KD of scFv-Reg4 to Reg4 was determined to be 1.91×10-8. In MKN45 and AGS cell lines, scFv- Reg4 depressed Reg4-stimulated cell proliferation and the inhibitory rates were 27.7±1.5% and 17.3±2.6%, respectively. Furthermore, scFv significantly enhanced 5-FU-induced cell death, from 23.0±1.0% to 28.4±1.2% in MKN45 and 28.2±0.7% to 36.6±0.6% in AGS cells. Treatment with scFv alone could lyse cancer cells to a certain extent, but no significance has been observed.Conclusion:The single-chain antibody (scFv-Reg4) significantly inhibited gastric cancer cell proliferation and synergistically enhanced the lethal effect of 5-FU. Thus, traditional chemo-/radio- therapeutics supplemented with scFv-Reg4 may provide advances in the strategy for gastrointestinal cancer treatment.

Single-chain antibody-decorated Au nanocages@liposomal layer nanoprobes for targeted SERS imaging and remote-controlled photothermal therapy of melanoma cancer cells

2021 ◽  
Vol 124 ◽  
pp. 112086
Author(s):  
Ghazal Farahavar ◽  
Samira Sadat Abolmaali ◽  
Foroogh Nejatollahi ◽  
Amin Safaie ◽  
Sanaz Javanmardi ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Photothermal Therapy ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Melanoma Cancer ◽  
Sers Imaging

scholarly journals The Past, Present and Future of Flow Cytometry in Central Nervous System Malignancies

2021 ◽  
Vol 4 (1) ◽  
pp. 11
Author(s):  
Evrysthenis Vartholomatos ◽  
George Vartholomatos ◽  
George A. Alexiou ◽  
Georgios S. Markopoulos
Keyword(s):  
Central Nervous System ◽  
Flow Cytometry ◽  
Nervous System ◽  
Therapeutic Agents ◽  
Phenotypic Characterization ◽  
Cell Phenotype ◽  
Analytical Technique ◽  
The Past ◽  
Current Review

Central nervous system malignancies (CNSMs) are categorized among the most aggressive and deadly types of cancer. The low median survival in patients with CNSMs is partly explained by the objective difficulties of brain surgeries as well as by the acquired chemoresistance of CNSM cells. Flow Cytometry is an analytical technique with the ability to quantify cell phenotype and to categorize cell populations on the basis of their characteristics. In the current review, we summarize the Flow Cytometry methodologies that have been used to study different phenotypic aspects of CNSMs. These include DNA content analysis for the determination of malignancy status and phenotypic characterization, as well as the methodologies used during the development of novel therapeutic agents. We conclude with the historical and current utility of Flow Cytometry in the field, and we propose how we can exploit current and possible future methodologies in the battle against this dreadful type of malignancy.

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