Development of a dot-blot assay for screening monoclonal antibodies to low-molecular-mass drugs

Rupprecht, Kevin; Nair, Rad K.; Harwick, L. C.; Grote, Jonathan; Beligere, Gangamani S.; Rege, Sushil; Chen, Yon-Yih; Lin, Zhen; Fishpaugh, Jeffrey R.

doi:10.1016/j.ab.2010.08.003

Cited by 10 publications

(8 citation statements)

References 27 publications

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“…Because plasma proinsulin is a risk factor for diabetes 42 , we also determined if proinsulin is secreted from MIN6 cells treated with CPA 43 (Supplementary Fig. 2a, b ).…”

Section: Resultsmentioning

confidence: 99%

Adaptation to chronic ER stress enforces pancreatic β-cell plasticity

et al. 2022

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Pancreatic β-cells are prone to endoplasmic reticulum (ER) stress due to their role in insulin secretion. They require sustainable and efficient adaptive stress responses to cope with this stress. Whether episodes of chronic stress directly compromise β-cell identity is unknown. We show here under reversible, chronic stress conditions β-cells undergo transcriptional and translational reprogramming associated with impaired expression of regulators of β-cell function and identity. Upon recovery from stress, β-cells regain their identity and function, indicating a high degree of adaptive plasticity. Remarkably, while β-cells show resilience to episodic ER stress, when episodes exceed a threshold, β-cell identity is gradually lost. Single cell RNA-sequencing analysis of islets from type 1 diabetes patients indicates severe deregulation of the chronic stress-adaptation program and reveals novel biomarkers of diabetes progression. Our results suggest β-cell adaptive exhaustion contributes to diabetes pathogenesis.

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“…Because plasma proinsulin is a risk factor for diabetes 42 , we also determined if proinsulin is secreted from MIN6 cells treated with CPA 43 (Supplementary Fig. 2a, b ).…”

Section: Resultsmentioning

confidence: 99%

Adaptation to chronic ER stress enforces pancreatic β-cell plasticity

et al. 2022

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“…A dot blot assay ( Rupprecht et al, 2010 ) was modified to screen transformed colonies in order to select clones with higher TGM1 secretion yields. Nitrocellulose membranes, 7 × 7 cm, were marked with a grid of 100 small squares and placed on a 10 cm diameter Fisherbrand™ Petri Dishes (Fisher Scientific, Cat #FB0875713) of TAP agar.…”

Section: Methodsmentioning

confidence: 99%

Oral delivery of a functional algal-expressed TGF-β mimic halts colitis in a murine DSS model

Smyth

Ren

White

et al. 2021

Journal of Biotechnology

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Inflammatory bowel disease (IBD) is a set of immunological disorders which can generate chronic pain and fatigue associated with the inflammatory symptoms. The treatment of IBD remains a significant hurdle with current therapies being only partially effective or having significant side effects, suggesting that new therapies that elicit different modes of action and delivery strategies are required. TGM1 is a TGF-β mimic that was discovered from the intestinal helminth parasite Heligmosomoides polygyrus and is thought to be produced by the parasite to suppress the intestinal inflammation response to help evade host immunity, making it an ideal candidate to be developed as a novel anti-inflammatory bio-therapeutic. Here we utilized the expression system of the edible green algae Chlamydomonas reinhardtii in order to recombinantly produce active TGM1 in a form that could be ingested. C. reinhardtii robustly expressed TGM1, and the resultant recombinant protein is biologically active as measured by regulatory T cell induction. When delivered orally to mice, the algal expressed TGM1 is able to ameliorate weight loss, lymphadenopathy, and disease symptoms in a mouse model of DSS-induced colitis, demonstrating the potential of this biologic as a novel treatment of IBD.

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“…Modification of the Dot immunoblotting assay (23, 24) was used to validate the affinity and specificity of PL5DLLD4 antibody with human PlGF. rhPlGF protein (200 ng; 264‐PGB‐010/CF; R&D Systems), VEGF‐165 (200 ng, 293‐VE‐010/CF; R&D Systems), and 500 ng bovine serum albumin (BSA) (MilliporeSigma) were deposited in the volume of 2 μl on dry nitrocellulose membrane (Bio‐Rad) and immobilized by 15 min incubation at room temperature The membranes were blocked with 5% nonfat milk (Bio‐Rad) in PBS at room temperature for 1 h and then incubated overnight at 4°C with mouse PL5DLLD4 antibody 0.5 μg/ml.…”

Section: Methodsmentioning

confidence: 99%

Placental growth factor negatively regulates retinal endothelial cell barrier function through suppression of glucose‐6‐phosphate dehydrogenase and antioxidant defense systems

Lennikov

Saddala

et al. 2019

The FASEB Journal

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We report that placental growth factor (PlGF) negatively affects the endothelial cell (EC) barrier function through a novel regulatory mechanism. The PlGF mAb promotes (but recombinant protein disrupts) EC barrier function, thus affecting the barrier‐forming protein levels, membrane distribution, and EC monolayer impedance by the electrical cell‐impedance sensing system, Western blot, and immunofluorescence staining. RNA sequencing‐based transcriptome analysis identified the up‐regulation of the pentose phosphate pathway (PPP) and the antioxidant defense protein by PlGF blockade. The PlGF and PlGF/VEGF dimers (but not VEGF‐A) down‐regulated the protein expression of glucose‐6‐phosphate dehydrogenase (G6PD) and peroxiredoxin (PRDX). G6PD inhibition and gene silencing (small interfering RNA) abolished the beneficial effects of PlGF inhibition on EC barrier function and PRDX3/6 protein expression. VEGF receptor (VEGFR)1 or VEGFR2 blockade prevented the inhibitory effect of PlGF on G6PD protein expression and EC barrier function. The PRDX6 played dual roles in EC barrier function through glutathione peroxidase and phospholipase A2 activity. In sum, PlGF negatively regulates EC barrier function through the activation of VEGFR1 and VEGFR2 and the suppression of the G6PD/PPP and the antioxidant pathways.—Huang, H., Lennikov, A., Saddala, M. S., Gozal, D., Grab, D. J., Khalyfa, A., Fan, L. Placental growth factor negatively regulates endothelial cell barrier function through suppression of glucose‐6‐phosphate dehydrogenase and antioxidant defense systems. FASEB J. 33, 13695‐13709 (2019). https://www.fasebj.org

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Development of a dot-blot assay for screening monoclonal antibodies to low-molecular-mass drugs

Cited by 10 publications

References 27 publications

Adaptation to chronic ER stress enforces pancreatic β-cell plasticity

Adaptation to chronic ER stress enforces pancreatic β-cell plasticity

Oral delivery of a functional algal-expressed TGF-β mimic halts colitis in a murine DSS model

Placental growth factor negatively regulates retinal endothelial cell barrier function through suppression of glucose‐6‐phosphate dehydrogenase and antioxidant defense systems

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