Generation and Validation of Monoclonal Antibodies Against the Maltose Binding Protein

Park, Yang-Nim; Glover, Rebecca A.; Daniels, Karla J.; Soll, David R.

doi:10.1089/mab.2015.0072

Cited by 6 publications

(3 citation statements)

References 18 publications

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“…Cell fusion was performed as previously described [ 47 , 48 ], with minor modifications. Following euthanizing protocols approved by the Office of the Institutional Animal Use Committee, the spleen was removed, and splenocytes isolated and resuspended in Iscove’s modified Dulbecco’s medium (IMDM; Cat.# 12440053; ThermoFisher Scientific) supplemented with 1% glutamax, 1% sodium pyruvate, 0.1% penicillin streptomycin, and 0.1% gentamicin and 15% fetal bovine serum.…”

Section: Methodsmentioning

confidence: 99%

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New monoclonal antibodies that recognize an unglycosylated, conserved, extracellular region of CD44 in vitro and in vivo, and can block tumorigenesis

et al. 2021

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CD44 is a transmembrane glycoprotein that binds to hyaluronic acid, plays roles in a number of cellular processes and is expressed in a variety of cell types. It is up-regulated in stem cells and cancer. Anti-CD44 monoclonal antibodies affect cell motility and aggregation, and repress tumorigenesis and metastasis. Here we describe four new anti-CD44 monoclonal antibodies originating from B cells of a mouse injected with a plasmid expressing CD44 isoform 12. The four monoclonal antibodies bind to the terminal, extracellular, conserved domain of CD44 isoforms. Based on differences in western blot patterns of cancer cell lysates, the four anti-CD44 mAbs separated into three distinct categories that include P4G9, P3D2, and P3A7, and P3G4. Spot assay analysis with peptides generated inEscherichia colisupport the conclusion that the monoclonal antibodies recognize unglycosylated sequences in the N-terminal conserved region between amino acid 21–220, and analyses with a peptide generated in human embryonic kidney 293 cells, demonstrate that these monoclonal antibodies bind to these peptides only after deglycosylation. Western blots with lysates from three cancer cell lines demonstrate that several CD44 isoforms are unglycosylated in the anti-CD44 target regions. The potential utility of the monoclonal antibodies in blocking tumorigenesis was tested by co-injection of cells of the breast cancer-derived tumorigenic cell line MDA-MB-231 with the anti-CD44 monoclonal antibody P3D2 into the mammary fat pads of mice. All five control mice injected with MDA-MB-231 cells plus anti-IgG formed palpable tumors, while only one of the six test mice injected with MDA-MB-231 cells plus P3D2 formed a tiny tumor, while the remaining five were tumor-free, indicating that the four anti-CD44 mAbs may be useful therapeutically.

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Section: Methodsmentioning

confidence: 99%

“…After the beads were resuspended, the isotyping strip was placed in the preparation and the isotype identified. Enzyme-linked immunosorbent assay (ELISA) was performed as previously described with minor modifications [ 48 ]. One hundred ng of a recombinant CD44 (CD44 std.)…”

Section: Methodsmentioning

confidence: 99%

New monoclonal antibodies that recognize an unglycosylated, conserved, extracellular region of CD44 in vitro and in vivo, and can block tumorigenesis

et al. 2021

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“…Hybridomas were cultured in Hyclone ADCF mAb medium (GE Healthcare, Cat.# SH30349.02), containing 1% glutamax, 1% sodium pyruvate, 1% penicillin streptomycin (all Thermo Fisher Scientific) supplemented with 0.1% gentamycin (IBI Scientific, Cat.# IB02030), at 37°C in 7.5% CO 2 . [82][83][84] In all cases, supernatants were harvested from hybridoma cultures that had reached concentrations of 1.5 to 2.0 × 10 6 cells per ml. The mAbs were affinity purified using a Protein G HP spin trap column (GE Healthcare, Cat.#28903134) and concentrated using an ultra-centrifugal filter (EMD Millipore, Cat.# UFC905024).…”

Section: Monoclonal Antibodiesmentioning

confidence: 99%

Integrin α-3 ß-1’s central role in breast cancer, melanoma and glioblastoma cell aggregation revealed by antibodies with blocking activity

Lusche

Klemme

Soll

et al. 2019

mAbs

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Breast cancer, melanoma and glioblastoma cells undergo cell-mediated aggregation and aggregate coalescence in a transparent 3D Matrigel environment. Cells from normal tissue and non-tumorigenic cell lines do not exhibit these behaviors. Here, 266 monoclonal antibodies (mAbs) demonstrated to interact with a wide variety of membrane, secreted and matrix proteins, have been screened for their capacity to block these tumorigenic cell-specific behaviors in a 3D environment. Remarkably, only six of the 266 tested mAbs exhibited blocking activity, four targeting integrin ß-1, one targeting integrin α-3 and one targeting CD44. Colocalization of integrins ß-1 and α-3 in fixed cells and in live aggregates suggests that the integrin α-3 ß-1 dimer plays a central role in cancer cell aggregation in the 3D environment provided by Matrigel. Our results suggest that blocking by anti-integrin and anti-CD44 mAbs involves interference in cell-cell interactions.

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Opportunities and challenges of the tag-assisted protein purification techniques: Applications in the pharmaceutical industry

Gomari

Saraygord‐Afshari

Farsimadan

et al. 2020

Biotechnology Advances

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Generation and Validation of Monoclonal Antibodies Against the Maltose Binding Protein

Cited by 6 publications

References 18 publications

New monoclonal antibodies that recognize an unglycosylated, conserved, extracellular region of CD44 in vitro and in vivo, and can block tumorigenesis

New monoclonal antibodies that recognize an unglycosylated, conserved, extracellular region of CD44 in vitro and in vivo, and can block tumorigenesis

Integrin α-3 ß-1’s central role in breast cancer, melanoma and glioblastoma cell aggregation revealed by antibodies with blocking activity

Opportunities and challenges of the tag-assisted protein purification techniques: Applications in the pharmaceutical industry

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