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Microparticle immunocapture assay for quantitation of protein multimer amount and size.

ABSTRACT: Cellular stress and toxicity are often associated with the formation of protein multimers, or aggregates. Numerous degenerative disorders, including Alzheimer's, Parkinson's, and Huntington's disease, prion-propagated disease, amyotrophic lateral sclerosis, cardiac amyloidosis, and diabetes, are characterized by aggregated protein deposits. Current methods are limited in the ability to assess multimer size along with multimer quantitation and to incorporate one or more ancillary traits, including target specificity, operative simplicity, and process speed. Here, we report development of a microparticle immunocapture assay that combines the advantages inherent to a monoclonal antibody:protein interaction with highly quantitative flow cytometry analysis. Using established reagents to build our platform, and aggregation-prone amyloid beta 1-42 peptide (Aβ42) and alpha-synuclein to demonstrate proof of principle, our results indicate that this assay is a highly adaptable method to measure multimer size and quantity at the same time in a technically streamlined workflow applicable to laboratory and clinical samples.

SUBMITTER: Gutknecht MF

PROVIDER: S-EPMC9142677 | biostudies-literature | 2022 May

REPOSITORIES: biostudies-literature

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Microparticle immunocapture assay for quantitation of protein multimer amount and size.

Gutknecht Michael F MF Kaku Hiroaki H Rothstein Thomas L TL

Cell reports methods 20220523 5

Cellular stress and toxicity are often associated with the formation of protein multimers, or aggregates. Numerous degenerative disorders, including Alzheimer's, Parkinson's, and Huntington's disease, prion-propagated disease, amyotrophic lateral sclerosis, cardiac amyloidosis, and diabetes, are characterized by aggregated protein deposits. Current methods are limited in the ability to assess multimer size along with multimer quantitation and to incorporate one or more ancillary traits, includin ...[more]

PMID: 35637905

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Project description:Oral administration of protein-based therapeutics is highly desirable due to lower cost, enhanced patient compliance, and convenience. However, the harsh pH environment of the gastrointestinal tract poses significant challenges. Silica-based carriers have emerged as potential candidates for the delivery of protein molecules, owing to their tuneable surface area and pore volume. We explored the use of a commercial mesoporous silica carrier, SYLOID, for the delivery of octreotide and bovine serum albumin (BSA) using a solvent evaporation method in three different solvents. The loading of proteins into SYLOID was driven by diffusion, as described by the Stokes-Einstein equation. Various parameters were investigated, such as protein size, diffusion, and solubility. Additionally, 3D fluorescence confocal imaging was employed to identify fluorescence intensity and protein diffusion within the carrier. Our results indicated that the loading process was influenced by the molecular size of the protein as octreotide exhibited a higher recovery rate (71%) compared to BSA (32%). The methanol-based loading of octreotide showed uniform diffusion into the silica carrier, whereas water and ethanol loading resulted in the drug being concentrated on the surface, as shown by confocal imaging, and further confirmed by scanning electron microscopy (SEM). Pore volume assessment supported these findings, showing that octreotide loaded with methanol had a low pore volume (1.2 cc/g). On the other hand, BSA loading was affected by its solubility in the three solvents, its tendency to aggregate, and its low solubility in ethanol and methanol, which resulted in dispersed particle sizes of 223 and 231 μm, respectively. This reduced diffusion into the carrier, as confirmed by fluorescence intensity and diffusivity values. This study underscores the importance of protein size, solvent properties, and diffusion characteristics when using porous carriers for protein delivery. Understanding these factors allows for the development of more effective oral protein-based therapeutics by enhancing loading efficiency. This, in turn, will lead to advances in targeted drug delivery and improved patient outcomes.

Dataset Information

Microparticle immunocapture assay for quantitation of protein multimer amount and size.

Publications

Microparticle immunocapture assay for quantitation of protein multimer amount and size.

Similar Datasets

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