Antibody mix-and-read assays based on fluorescence intensity probes

A high-fat diet increases the risk of insulin resistance, type-2 diabetes, and non-alcoholic steato-hepatitis. Here we identified two heat-shock proteins, Heat-Shock-Protein70 and Glucose-Regulated Protein78, which are increased in the jejunum of rats on a high-fat diet. We demonstrated a causal link between these proteins and hepatic and whole-body insulin-resistance, as well as the metabolic response to bariatric/metabolic surgery. Long-term continuous infusion of Heat-Shock-Protein70 and Glucose-Regulated Protein78 caused insulin-resistance, hyperglycemia, and non-alcoholic steato-hepatitis in rats on a chow diet, while in rats on a high-fat diet continuous infusion of monoclonal antibodies reversed these phenotypes, mimicking metabolic surgery. Infusion of these proteins or their antibodies was also associated with shifts in fecal microbiota composition. Serum levels of Heat-Shock-Protein70 and Glucose-Regulated Protein78were elevated in patients with non-alcoholic steato-hepatitis, but decreased following metabolic surgery. Understanding the intestinal regulation of metabolism may provide options to reverse metabolic diseases.

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“…We studied mAb pharmacokinetics using a previously validated method based on fluorescence intensity to measure antibody concentrations 25 .…”

Section: Resultsmentioning

confidence: 99%

Upper gut heat shock proteins HSP70 and GRP78 promote insulin resistance, hyperglycemia, and non-alcoholic steatohepatitis

et al. 2022

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Fluorescent Reporter‐Based Fiber Optic Probe for Continuous Detection of Antibodies

Nandy,

Vu,

Maranholkar

et al. 2024

Biotech & Bioengineering

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Measurement of antibody and antibody fusion protein concentration is vital for process development and manufacturing. Continuous, in‐line monitoring of antibody concentration could be useful in a variety of applications, such as controlling the loading of protein A columns to prevent breakthrough, monitoring bioreactor titer, and detecting leaks past ultrafiltration/diafiltration membranes. Molecule‐specific monitoring techniques are advantageous for antibody detection in cell culture fluid in the presence of complex process impurities. Here we report a continuous in‐line, real‐time IgG monitoring platform using a fiber‐optic biosensor with a replaceable sensor tip covalently functionalized with a fluor‐labeled protein consisting of a pentamer of the Z domain (a more stable form of the B domain) of protein A. The sensor demonstrates concentration‐dependent fluorescence enhancement in the presence of human IgG (0.01–0.75 g/L), with consistent signals during five runs each with 1 and 0.1 g/L IgG, and maintains its specificity in the presence of Chinese hamster ovary (CHO) cell culture fluid. A 5% breakthrough of a typical 10 g/L load would be detected in less than 20 s in a flowing stream emerging from a protein A column without prior sample preparation. This sensor platform may be suitable for monitoring IgG and fragment, crystallizable (Fc) fusion proteins in diverse upstream and downstream bioprocess applications.

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Transforming waste into valuables: Preparation and evaluation of dual-ligand hydrophobic charge-induction chromatography using two poor performing ligands

Shi,

Zhang,

Fang

et al. 2024

Journal of Chromatography A

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Antibody mix-and-read assays based on fluorescence intensity probes

Cited by 3 publications

References 51 publications

Upper gut heat shock proteins HSP70 and GRP78 promote insulin resistance, hyperglycemia, and non-alcoholic steatohepatitis

Upper gut heat shock proteins HSP70 and GRP78 promote insulin resistance, hyperglycemia, and non-alcoholic steatohepatitis

Fluorescent Reporter‐Based Fiber Optic Probe for Continuous Detection of Antibodies

Transforming waste into valuables: Preparation and evaluation of dual-ligand hydrophobic charge-induction chromatography using two poor performing ligands

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