Dataset Information

Surface plasmon resonance (SPR) based binding studies of refolded single chain antibody fragments.

ABSTRACT: Recent advances in Recombinant antibody technology / Antibody Engineering has given impetus to the genetic manipulation of antibody fragments that has paved the way for better understanding of the structure and functions of immunoglobulins and also has escalated their use in immunotherapy. Bacterial expression system such as Escherichia coli has complemented this technique through the expression of recombinant antibodies. Present communication has attempted to optimize the expression and refolding protocol of single chain fragment variable (ScFv) and single chain antigen binding fragment (ScFab) using E.coli expression system. Efficiency of refolding protocol was validated by structural analysis by CD, native folding by fluorescence and functional analysis by its binding with full length HIV-1 gp120 via SPR. Results show the predominant ?-sheet (CD) as secondary structural content and native folding via red shift (tryptophan fluorescence). The single chain fragments have shown good binding with HIV-1 gp120 thus validating the expression and refolding strategy and also reinstating E.coli as model expression system for recombinant antibody engineering. SPR based binding analysis coupled with E.coli based expression and purification will have implication for HIV therapeutics and will set a benchmark for future studies of similar kind.

SUBMITTER: Singh P

PROVIDER: S-EPMC5986705 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Surface plasmon resonance (SPR) based binding studies of refolded single chain antibody fragments.

Singh Pranveer P

Biochemistry and biophysics reports 20180425

Recent advances in Recombinant antibody technology / Antibody Engineering has given impetus to the genetic manipulation of antibody fragments that has paved the way for better understanding of the structure and functions of immunoglobulins and also has escalated their use in immunotherapy. Bacterial expression system such as <i>Escherichia coli</i> has complemented this technique through the expression of recombinant antibodies. Present communication has attempted to optimize the expression and ...[more]

PMID: 29872739

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Project description:BackgroundSurface plasmon resonance imaging (SPRI) is a label-free technique that can image refractive index changes at an interface. We have previously used SPRI to study the dynamics of cell-substratum interactions. However, characterization of spatial resolution in 3 dimensions is necessary to quantitatively interpret SPR images. Spatial resolution is complicated by the asymmetric propagation length of surface plasmons in the x and y dimensions leading to image degradation in one direction. Inferring the distance of intracellular organelles and other subcellular features from the interface by SPRI is complicated by uncertainties regarding the detection of the evanescent wave decay into cells. This study provides an experimental basis for characterizing the resolution of an SPR imaging system in the lateral and distal dimensions and demonstrates a novel approach for resolving sub-micrometer cellular structures by SPRI. The SPRI resolution here is distinct in its ability to visualize subcellular structures that are in proximity to a surface, which is comparable with that of total internal reflection fluorescence (TIRF) microscopy but has the advantage of no fluorescent labels.ResultsAn SPR imaging system was designed that uses a high numerical aperture objective lens to image cells and a digital light projector to pattern the angle of the incident excitation on the sample. Cellular components such as focal adhesions, nucleus, and cellular secretions are visualized. The point spread function of polymeric nanoparticle beads indicates near-diffraction limited spatial resolution. To characterize the z-axis response, we used micrometer scale polymeric beads with a refractive index similar to cells as reference materials to determine the detection limit of the SPR field as a function of distance from the substrate. Multi-wavelength measurements of these microspheres show that it is possible to tailor the effective depth of penetration of the evanescent wave into the cellular environment.ConclusionWe describe how the use of patterned incident light provides SPRI at high spatial resolution, and we characterize a finite limit of detection for penetration depth. We demonstrate the application of a novel technique that allows unprecedented subcellular detail for SPRI, and enables a quantitative interpretation of SPRI for subcellular imaging.

Dataset Information

Surface plasmon resonance (SPR) based binding studies of refolded single chain antibody fragments.

Publications

Surface plasmon resonance (SPR) based binding studies of refolded single chain antibody fragments.

Similar Datasets

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