A novel ELISA using PVDF microplates.

Nader D Halim; Andrew W Joseph; Barbara K Lipska

doi:10.1016/j.jneumeth.2004.09.025

Home/Archive/PubMed

PubMed · 15814149

A novel ELISA using PVDF microplates.

Nader D Halim·Andrew W Joseph·Barbara K Lipska

View original source ↗Export metadata ↓

Abstract

Here we describe the development of a novel specific, rapid ELISA system, which is performed on modified microplates where polyvinylidine fluoride (PVDF) forms the base of each well. The use of microplates with PVDF membranes as the solid phase allows for a greater binding capacity of protein in comparison to the solid phases of traditional ELISAs. The increased binding capacity of the solid phase provides for the direct binding of antigens, which can subsequently be assayed using a single, specific and well-characterized antibody. This direct assay system eliminates the need for two distinct antibodies that are often necessary in conventional two site ELISA systems. The system is able to specifically detect purified proteins as well as antigens in crude preparations of tissue homogenates. The PVDF-based ELISA performs with similar sensitivity and reproducibility as conventional two site ELISAs in tissue homogenates. The intra- and inter-assay coefficients of variation for the measurement of actin in crude rat brain homogenate were 2.36 and 5.15%, respectively.

Explore related subjects

Actins Animals Brain Coated Materials, Biocompatible Enzyme-Linked Immunosorbent Assay Equipment Design Equipment Failure Analysis Humans Materials Testing Microchemistry Muscle, Skeletal Polyvinyls Rabbits Rats Tissue Distribution Fluorocarbon Polymers Evaluation Study Journal Article

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

Nader D Halim, Andrew W Joseph, Barbara K Lipska. 2004-11-30. A novel ELISA using PVDF microplates.. https://doi.org/10.1016/j.jneumeth.2004.09.025

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2009

Nap1-mediated actin remodeling is essential for mammalian myoblast fusion.

Myoblast fusion is crucial for the formation, growth, maintenance and regeneration of healthy skeletal muscle. Unfortunately, the molecular machinery, cell behaviors, and membrane and cytoskeletal remodeling events that govern fusion and myofiber formation remain poorly understood. Using time-lapse imaging approaches on mouse C2C12 myoblasts, we identify discrete and specific molecular events at myoblast membranes during fusion and myotube formation. These events include rearrangement of cell shape from fibroblast to spindle-like morphologies, changes in lamellipodial and filopodial extensions during different periods of differentiation, and changes in membrane alignment and organization during fusion. We find that actin-cytoskeleton remodeling is crucial for these events: pharmacological inhibition of F-actin polymerization leads to decreased lamellipodial and filopodial extensions and to reduced myoblast fusion. Additionally, shRNA-mediated inhibition of Nap1, a member of the WAVE actin-remodeling complex, results in accumulations of F-actin structures at the plasma membrane that are concomitant with a decrease in myoblast fusion. Our data highlight distinct and essential roles for actin cytoskeleton remodeling during mammalian myoblast fusion, provide a platform for cellular and molecular dissection of the fusion process, and suggest a functional conservation of Nap1-regulated actin-cytoskeleton remodeling during myoblast fusion between mammals and Drosophila.

Actins↗

PubMed2007

Shape-engineered vascular endothelial cells: nitric oxide production, cell elasticity, and actin cytoskeletal features.

Single cell shape determines cellular functions. Therefore, control of cell shape is of considerable importance for the tissue engineering field. This study was designed to assess the effect of surface-induced shaping of vascular endothelial cells (ECs) on the intracellular nitric oxide (NO) production level, the cell elasticity, and cytoskeletal (CSK) features on shape-engineered ECs (round, 90, 120 microm diameter; spindle-shaped, 20, 30, 40 microm width) prepared on a photolithographically microprocessed surface. Intracellular NO production was measured using a microscopic spectrometer with diaminofluorescein diacetate probe. Cell elasticity and actin CSK features were analyzed through microindentation measurement and fluorescence observations with fluorescence and atomic force microscopy. Results showed that spindle-shaped cells exhibited lower NO production, higher cell stiffness, and denser actin stress fibers than the round and nonrestrictedly cultured control cells. Relations between cell shape with NO production, cell elasticity, and actin CSK features were discussed.

Actins↗

PubMed2007

Benzyl butyl phthalate influences actin distribution and cell proliferation in rat Py1a osteoblasts.

We previously reported that transient administration of phthalates induced actin cytoskeleton disruption in Py1a osteoblasts. However, the mechanism of this transient effect was not elucidated. In this study we provided evidence that the actin cytoskeletal re-established conditions are dependent on new actin expression and synthesis. To assess the role of phthalates in modulating the distribution of actin, confocal and electron microscopy studies were carried out. Results indicated a modification of actin distribution after phthalate administration. In addition, a relation with the nucleoskeletal component lamin A supports the hypothesis that phthalates may participate in regulatory cell processes involving actin in Py1a osteoblasts. The present study also supports the mitogenic effects of phthalates, which involve microfilament disruption, nuclear actin and lamin A. In particular, the increased levels of cyclin D3, which in mammalian cells plays a critical role in G1 to S transition and is a putative proto-oncogene in benzyl butyl phthalate treated cells, suggested a possible effect of the endocrine disruptor in cancer processes.

Actins↗