Use of Fluorescence-Activated Cell Sorting to Select Hybrid Hybridomas Producing Bispecific Monoclonal Antibodies

1987 ◽  
pp. 367-368
Author(s):  
L. Karawajew ◽  
B. Micheel ◽  
O. Behrsing ◽  
M. Gaestel ◽  
G. Pasternak
Keyword(s):  
Monoclonal Antibodies ◽  
Cell Sorting ◽  
Fluorescence Activated Cell Sorting

Hapten-Specific Single-Cell Selection of Hybridoma Clones by Fluorescence-Activated Cell Sorting for the Generation of Monoclonal Antibodies

2017 ◽  
Vol 89 (7) ◽  
pp. 4007-4012 ◽  
Author(s):  
Martin Dippong ◽  
Peter Carl ◽  
Christine Lenz ◽  
Jörg A. Schenk ◽  
Katrin Hoffmann ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Single Cell ◽  
Cell Sorting ◽  
Cell Selection ◽  
Fluorescence Activated Cell Sorting ◽  
Selection Of

scholarly journals Myeloid-associated differentiation antigens on stem cells and their progeny identified by monoclonal antibodies

Blood ◽  
1983 ◽  
Vol 62 (1) ◽  
pp. 124-132 ◽  
Author(s):  
RG Andrews ◽  
B Torok-Storb ◽  
ID Bernstein
Keyword(s):  
Stem Cells ◽  
Monoclonal Antibodies ◽  
Cell Sorting ◽  
Bone Marrow Cells ◽  
Antigenic Determinants ◽  
Fluorescence Activated Cell Sorting ◽  
Monocytic Cells ◽  
Colony Forming Units ◽  
Potential Applications ◽  
A Minor

Abstract Within the hematopoietic system, monoclonal antibodies reactive with antigenic determinants, expressed in a lineage- and stage-restricted fashion, can be used to map myeloid differentiation. We have generated a series of monoclonal antibodies that reacts with myeloid-associated determinants on committed myeloid stem cells and their progeny. Their reactivity with peripheral blood cells was identified by immunofluorescence assays, with bone marrow cells by fluorescence- activated cell sorting, and with committed hematopoietic progenitor cells by both cytotoxic assays and fluorescence-activated cell sorting. Antibody 1G10, which has previously been reported to react with cells of the granulocytic lineage and with a minor subset of mature monocytes, was shown to react with granulocyte-macrophage colony- forming units (CFU-GM). Three antibodies not previously characterized (T5A7, L4F3, L1B2) were shown to react with both granulocytic and monocytic cells and in fluorescence-activated cell sorting studies to detectably stain granulocytic cells at different stages of maturation. These three antibodies also react with CFU-GM, two (L4F3 and L1B2) reacting with all CFU-GM, while T5A7 reacts with only a portion of the day 7 CFU-GM. Antibody L4F3 also reacts with a portion of erythroid burst-forming units (BFU-E). In contrast, the previously reported antibody 5F1, which reacts with monocytic cells, nucleated erythroid cells, and platelets, was shown to react with erythroid colony-forming units (CFU-E). Potential applications of these antibodies to studies of normal and malignant hematopoiesis are discussed.

scholarly journals Myeloid-associated differentiation antigens on stem cells and their progeny identified by monoclonal antibodies

Blood ◽  
1983 ◽  
Vol 62 (1) ◽  
pp. 124-132 ◽  
Author(s):  
RG Andrews ◽  
B Torok-Storb ◽  
ID Bernstein
Keyword(s):  
Stem Cells ◽  
Monoclonal Antibodies ◽  
Cell Sorting ◽  
Bone Marrow Cells ◽  
Antigenic Determinants ◽  
Fluorescence Activated Cell Sorting ◽  
Monocytic Cells ◽  
Colony Forming Units ◽  
Potential Applications ◽  
A Minor

Within the hematopoietic system, monoclonal antibodies reactive with antigenic determinants, expressed in a lineage- and stage-restricted fashion, can be used to map myeloid differentiation. We have generated a series of monoclonal antibodies that reacts with myeloid-associated determinants on committed myeloid stem cells and their progeny. Their reactivity with peripheral blood cells was identified by immunofluorescence assays, with bone marrow cells by fluorescence- activated cell sorting, and with committed hematopoietic progenitor cells by both cytotoxic assays and fluorescence-activated cell sorting. Antibody 1G10, which has previously been reported to react with cells of the granulocytic lineage and with a minor subset of mature monocytes, was shown to react with granulocyte-macrophage colony- forming units (CFU-GM). Three antibodies not previously characterized (T5A7, L4F3, L1B2) were shown to react with both granulocytic and monocytic cells and in fluorescence-activated cell sorting studies to detectably stain granulocytic cells at different stages of maturation. These three antibodies also react with CFU-GM, two (L4F3 and L1B2) reacting with all CFU-GM, while T5A7 reacts with only a portion of the day 7 CFU-GM. Antibody L4F3 also reacts with a portion of erythroid burst-forming units (BFU-E). In contrast, the previously reported antibody 5F1, which reacts with monocytic cells, nucleated erythroid cells, and platelets, was shown to react with erythroid colony-forming units (CFU-E). Potential applications of these antibodies to studies of normal and malignant hematopoiesis are discussed.

scholarly journals New Monoclonal Antibodies to Mesothelin Useful for Immunohistochemistry, Fluorescence-Activated Cell Sorting, Western Blotting, and ELISA

2005 ◽  
Vol 11 (16) ◽  
pp. 5840-5846 ◽  
Author(s):  
Masanori Onda ◽  
Mark Willingham ◽  
Satoshi Nagata ◽  
Tapan K. Bera ◽  
Richard Beers ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Western Blotting ◽  
Cell Sorting ◽  
Fluorescence Activated Cell Sorting

scholarly journals A High-Throughput Screening System Based on Fluorescence-Activated Cell Sorting for the Directed Evolution of Chitinase A

2021 ◽  
Vol 22 (6) ◽  
pp. 3041
Author(s):  
Gheorghita Menghiu ◽  
Vasile Ostafe ◽  
Radivoje Prodanović ◽  
Rainer Fischer ◽  
Raluca Ostafe
Keyword(s):  
High Throughput ◽  
Cell Sorting ◽  
High Throughput Screening ◽  
Screening Method ◽  
Enzymatic Reaction ◽  
Hydrolytic Enzymes ◽  
Screening System ◽  
Fluorescence Activated Cell Sorting ◽  
Fungal Cell ◽  
Chitinase A

Chitinases catalyze the degradation of chitin, a polymer of N-acetylglucosamine found in crustacean shells, insect cuticles, and fungal cell walls. There is great interest in the development of improved chitinases to address the environmental burden of chitin waste from the food processing industry as well as the potential medical, agricultural, and industrial uses of partially deacetylated chitin (chitosan) and its products (chito-oligosaccharides). The depolymerization of chitin can be achieved using chemical and physical treatments, but an enzymatic process would be more environmentally friendly and more sustainable. However, chitinases are slow-acting enzymes, limiting their biotechnological exploitation, although this can be overcome by molecular evolution approaches to enhance the features required for specific applications. The two main goals of this study were the development of a high-throughput screening system for chitinase activity (which could be extrapolated to other hydrolytic enzymes), and the deployment of this new method to select improved chitinase variants. We therefore cloned and expressed the Bacillus licheniformis DSM8785 chitinase A (chiA) gene in Escherichia coli BL21 (DE3) cells and generated a mutant library by error-prone PCR. We then developed a screening method based on fluorescence-activated cell sorting (FACS) using the model substrate 4-methylumbelliferyl β-d-N,N′,N″-triacetyl chitotrioside to identify improved enzymes. We prevented cross-talk between emulsion compartments caused by the hydrophobicity of 4-methylumbelliferone, the fluorescent product of the enzymatic reaction, by incorporating cyclodextrins into the aqueous phases. We also addressed the toxicity of long-term chiA expression in E. coli by limiting the reaction time. We identified 12 mutants containing 2–8 mutations per gene resulting in up to twofold higher activity than wild-type ChiA.

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