Immobilization of antibody fragment for immunosensor application based on surface plasmon resonance

Biosurface fabrication using the Fab′ fragment of immunoglobulin (IgG) was carried out by self-assembly (SA) technique. The pepsin-digested monoclonal antibody (Mab) against bovine insulin containing the F(ab′)₂ fragment and residual proteins was separated using affinity chromatography and dialysis. To prevent the nonspecific binding of F(ab′)₂ onto gold (Au) substrate, the native disulfide bridge was reduced using dithiothreitol (DTT) to convert F(ab′)₂ into Fab′, which made the immobilization to be carried out via the native thiol (–SH) group. The fabricated biosurface using SA technique showed the formation of stable thin film through AFM topography. Through the concentration change of DTT and Fab′, the absorption characteristics against the Au surface were investigated using surface plasmon resonance (SPR) with the flow cell. The amount of immobilized antibody fragment and the antigen binding capacity were regulated with respect to the reduction state and concentration of F(ab′)₂. Based on the biosurface of the fabricated Fab′, the insulin-detection was carried out by the measurement of SPR. The proposed antibody surface could successfully detect the bovine insulin at the concentration from 100 ng/mL to 10 μg/mL.     

Comparative Assessment of Oriented Antibody Immobilization on Surface Plasmon Resonance Biosensing

Protein A and protein G are extremely useful molecules for the immobilization of antibodies. However, there are limited comparative reports available to evaluate their immobilization performance for use as biosensors. In this study, a comparative analysis was made of approaches that use protein A and protein G for avian leukosis virus detection. The antibody‐protein binding affinities were determined using surface plasmon resonance (SPR) analysis. The immobilization efficiency was obtained by calculating the number of the protein molecular binding sites. The positive influence of sensor response on antigen detection indicates that the amount of immobilized antibody plays a major role in the extent of immobilization. Moreover, the biosensors constructed using both proteins were found to be regenerative. The SPR results from this study suggest that the surfaces of protein G provide a better equilibrium constant and binding efficacy for immobilized antibodies, resulting in enhanced antigen detection.     

A self-assembled fusion protein-based surface plasmon resonance biosensor for rapid diagnosis of severe acute respiratory syndrome

A surface plasmon resonance (SPR)-based biosensor was developed for simple diagnosis of severe acute respiratory syndrome (SARS) using a protein created by genetically fusing gold binding polypeptides (GBPs) to a SARS coronaviral surface antigen (SCVme). The GBP domain of the fusion protein serves as an anchoring component onto the gold surface, exploiting the gold binding affinity of the domain, whereas the SCVme domain is a recognition element for anti-SCVme antibody, the target analyte in this study. SPR analysis indicated the fusion protein simply and strongly self-immobilized onto the gold surface, through GBP, without surface chemical modification, offering a stable and specific sensing platform for anti-SCVme detection. AFM and SPR imaging analyses demonstrated that anti-SCVme specifically bound to the fusion protein immobilized onto the gold-micropatterned chip, implying that appropriate orientation of bound fusion protein by GBP resulted in optimal exposure of the SCVme domain to the assay solution, resulting in efficient capture of anti-SCVme antibody. The best packing density of the fusion protein onto the SPR chip was achieved at the concentration of 10 μg mL⁻¹; this density showed the highest detection response (906 RU) for anti-SCVme. The fusion protein-coated SPR chip at the best packing density had a lower limit of detection of 200 ng mL⁻¹ anti-SCVme within 10 min and also allowed selective detection of anti-SCVme with significantly low responses for non-specific mouse IgG at all tested concentrations. The fusion protein provides a simple and effective method for construction of SPR sensing platforms permitting sensitive and selective detection of anti-SCVme antibody.     

Mass spectrometric proteome analyses of synovial fluids and plasmas from patients suffering from rheumatoid arthritis and comparison to reactive arthritis or osteoarthritis

Differential proteome analysis is used to study body fluids from patients suffering from rheumatoid arthritis (RA), reactive arthritis (reaA) or osteoarthritis (OA). Mass spectrometric structure characterization of gel-separated proteins provided a detailed view of the protein-processing events that lead to distinct protein species present in the respective body fluids. (i) Fibrin(ogen) beta-chain degradation products, presumably plasmin-derived, appeared solely in synovial fluids (SF) from both patient collectives, (ii) calgranulin B (MRP14) was exclusively identified in SF samples derived from 5 out of 6 patients suffering from RA. Calgranulin B was not observed in synovial fluids from OA patients, nor in plasmas from either patient group. In all cases where calgranulin B was detected, calgranulin C was identified as well. (iii) Serum amyloid A protein spots were determined in plasmas and synovial fluids from patients with RA, but not in patients with OA. In addition to disease-relevant differences, interindividual differences in haptoglobin patterns of the patients under investigation were observed. Hence, in-depth proteome analysis of body fluids has proven effective for identification of multiple molecular markers and determination of associated protein structure modifications, that are thought to play a role for specifically determining a defined pathological state of diseased joints.     

Specific detection of Campylobacter jejuni using the bacteriophage NCTC 12673 receptor binding protein as a probe

Campylobacter jejuni is found in the intestines of poultry, cattle, swine, wild birds and pet animals and is the major cause of foodborne gastroenteritis in developed countries. We report the use of the receptor binding protein (RBP) of Campylobacter bacteriophage NCTC 12673 for the specific capture of Campylobacter jejuni bacteria using RBP-derivatized capturing surfaces. The Gp48 RBP was expressed as a glutathione S-transferase-Gp48 (GST-Gp48) fusion protein and immobilized onto surface plasmon resonance (SPR) surfaces using glutathione self-assembled monolayers (GSH SAM). Bovine serum albumin (BSA) was used to block any non-specific binding. Glutathione SAM leads to an oriented attachment of the protein, resulting in a two- to three-fold improvement of bacterial capture when compared to dithiobis(succinimidyl propionate) (DTSP) SAM-based unoriented attachment. The specificity of recognition was confirmed using Salmonella enterica subsp. enterica serovar Typhimurium as a negative control, which indeed showed negligible binding. The detection limit of the RBP-derivatized SPR surfaces was found to be 10(2) cfu/ml. Finally, GST-Gp48 was also immobilized onto magnetic beads that were successfully used to capture and pre-concentrate the host pathogen from suspension.     

Solid-phase synthesis of BODIPY dyes and development of an immunoglobulin fluorescent sensor

The diversification of the BODIPY scaffold has been hindered by its controversial adaptability to solid-phase chemistry. Herein we report the first solid-phase synthesis of a BODIPY library in high purities. We screened the library against a set of proteins, identified an immunoglobulin fluorescent sensor (Ig Orange) and confirmed its binding by SPR experiments.     

Microchannel chips for the multiplexed analysis of human immunoglobulin G–antibody interactions by surface plasmon resonance imaging

We report the multiplexed, simultaneous analysis of antigen–antibody interactions that involve human immunoglobulin G (IgG) on a gold substrate by the surface plasmon resonance imaging method. A multichannel, microfluidic chip was fabricated from poly(dimethylsiloxane) (PDMS) to selectively functionalize the surface and deliver the analyte solutions. The sensing interface was constructed using avidin as a linker layer between the surface-bound biotinylated bovine serum albumin and biotinylated anti-human IgG antibodies. Four mouse anti-human IgG antibodies were selected for evaluation and the screening was achieved by simultaneously monitoring protein–protein interactions under identical conditions. Antibody–antigen binding affinities towards human immunoglobulin were quantitatively compared by employing Langmuir adsorption isotherms for the analysis of SPRi responses obtained under equilibrium conditions. We were able to identify two IgG samples with higher affinities towards the target, and the determined binding kinetics falls within the typical range of values reported in the literature. Direct measurement of proteins in serum samples by SPR imaging was achieved by developing methods to minimize nonspecific adsorption onto the avidin-functionalized surface, and a limit of detection (LOD) of 6.7 nM IgG was obtained for the treated serum samples. The combination of SPR imaging and multichannel PDMS chips offers convenience and flexibility for sensitive and label-free measurement of protein–protein interactions in complex conditions and enables high-throughput screening of pharmaceutically significant molecules. Figure Microchannel SPR imaging for protein–protein interactions     

SPR-based immunocapture approach to creating an interfacial sensing architecture: mapping of the MRS18-2 binding site on retinoblastoma protein

Biosensor technologies based on optical readout are widely used in protein–protein interaction studies. Here we describe a fast and simple approach to the creation of oriented interfacial architectures for surface plasmon resonance (SPR) transducers, based on conventional biochemical procedures and custom reagents. The proposed protocol permits the oriented affinity-capture of GST fusion proteins by a specific antibody which is bound to protein A, which in turn has been immobilized on the transducer surface (after the surface has been modified by guanidine thiocyanate). The applicability of the method was demonstrated by studying the interaction between retinoblastoma tumor suppressor protein (pRb) and MRS18-2 proteins. The formation of the pRb–MRS18-2 protein complex was examined and the pRb binding site (A-box–spacer–B-box) was mapped. We have also shown that MRS18-2, which was detected as the Epstein–Barr virus-encoded EBNA-6 binding partner using the yeast two-hybrid system, binds to pRb in GST pull-down assays.     

Construction and Characterization of an Anti‐Prion scFv Fusion Protein Pair for Detection of Prion Protein on Antibody Chip

Abstract

A pair of single chain Fv fragment (scFv) fusion proteins were constructed and characterized. Antibody chips using the pair were designed for sensitive detection of prion protein. Phage displayed antibody library was synthesized by immunizing mice with thioredoxin‐mature bovine prion fusion protein (TrxA‐bPrP^c). After five rounds of panning against recombinant bovine prion protein (rb‐PrP^c) and ELISA test, two positive clones with high affinity to rb‐PrP^c, named Z163 and Z186, were obtained. They were conjugated with a linker‐streptavidin binding protein (SBP) or human IgG1 constant fragment (Fc) to form the scFv fusion protein pair Z186‐L‐SBP/Z163‐Fc. Western blot experiments showed that the scFv fusion pair specifically interacted with the line epitopes of the protease resistant core region bPrP27‐30. Surface plasmon resonance (SPR) sensorgrams revealed that the equilibrium dissociation constants of the interactions with rb‐PrP^c were 3.24×10^?8 M, 8.82×10^?8M, and 8.10×10^?9 M for Z186‐L‐SBP, Z163, and Z163‐Fc, respectively. All binding reactions followed rapid association and slow dissociation kinetics. As a detection pair, Z186‐L‐SBP functioned as a capture probe and was immobilized on the streptavidin coated slides to form reactive layer of the antibody chip, and Z163‐Fc labeled with fluorescence dye Cy3 functioned as a detection probe generating fluorescence signal. The antibody chip could detect existence of rb‐PrP^c with detection limit of 1 pg/ml.     

Mixed‐monolayer of N‐hydroxysuccinimide‐terminated cross‐linker and short alkanethiol to improve the efficiency of biomolecule binding for biosensing

The goal of this study was to use a novel surface chemistry for modifying gold surfaces to decrease the steric hindrance, minimize the nonspecific bindings while providing directed immobilization of proteins for advancing the transducer property and to provide a biosensing platform for surface plasmon resonance (SPR) applications. Mixed self‐assembled monolayers (mSAMs) were prepared using 3,3′‐Dithiodipropionic acid di (N‐hydroxysuccinimide ester) (DSP) and 6‐mercapto‐1‐hexanol (MCH) and the selected model proteins bovine serum albumin (BSA) and lysozyme were tested for binding efficiency. First, binding of these two proteins at constant concentration to different DSP:MCH mSAMs were compared to deduce the best molar ratio for forming mSAM using a continuous flow system coupled to SPR. Coincidently the maximum protein binding DSP:MCH mSAM were the same for both proteins. The change in Response Unit (∆RU) signal due to protein binding between DSP SAM and maximum protein binding DSP:MCH mSAM for lysozyme binding was more in comparison to BSA binding. Second, the effect of BSA and lysozyme concentration on binding efficiency to maximum protein binding DSP:MCH mSAM were compared and discussed. Lysozyme and BSA were shown to reach saturations on the same monolayer at concentrations of 5.7x10⁻⁵ and 8.96x10⁻⁶ [M] respectively, hence the molar ratio for limit concentrations is 6:1. The DSP SAM, MCH SAM, and DSP:MCH mSAMs where maximum and minimum protein binding occurs were also characterized with XPS and Attenuated total reflectance‐Fourier transform infrared (ATR‐FTIR) spectroscopy. Blank gold surface, maximum protein binding DSP:MCH mSAM and BSA immobilized DSP:MCH mSAM on gold surface were also investigated utilizing tapping mode AFM.     

improvement of the Specificity of Surface Plasmon Resonance with BSA-modified Chip

Recently, surface plasmon resonance (SPR) become more and more popular without the need of the label technology1-3. However, sometimes, a number of experimental artifacts complicate the final biosensor analysis4-7. The utilization of a reference surface c…     

Effects of solute-matrix interaction on monitoring the conformational changes of immobilized proteins by surface plasmon resonance sensor

A real-time and labeling-free surface plasmon resonance (SPR) sensor was used to monitor the conformational changes of immobilized globule proteins (RNase A and lysozyme) in chemical unfolding and refolding. The effects of chemical denaturants on the protein structures were investigated. The methodology in protein conformational study on the solid surface is refined through the theoretic calculations and the conformational information of native/denatured proteins in solution. Additionally, our observation illustrates that the ambient buffer solution is merit to influence the refractive index of immobilized protein films and directly be observed from the SPR resonance angle shifts.     

Immobilization of light-harvesting chlorophyll a/b complex (LHCIIb) studied by surface plasmon field-enhanced fluorescence spectroscopy

The major light-harvesting chlorophyll a/ b complex (LHCIIb) of the photosynthetic apparatus in green plants can be viewed as a protein scaffold binding and positioning a large number of pigment molecules that engage in rapid excitation energy transfer. This property makes LHCIIb potentially interesting as a light harvester (or a model thereof) in photoelectronic applications. Such applications would require the immobilization of LHCIIb (or similar dye-protein complexes) on a solid surface. In this work, the immobilization of recombinant LHCIIb is tested and optimized on functionalized gold surfaces via a histidine 6 tag (His tag) in the protein moiety. Immobilization efficiency and kinetics are analyzed by using surface plasmon resonance (SPR) and surface plasmon field-enhanced fluorescence spectroscopy (SPFS). The latter was also used to assess the integrity of immobilized LHCIIb by recording Chl b-sensitized Chl a emission spectra. Since His tags have been included in a substantial number of recombinant proteins, the immobilization technique developed here for LHCIIb presumably can be extended to a large range of other membrane and water-soluble proteins.     

Amplified Immunoassay of Human IgG Using Real—time Biomolecular Interaction ANalysis （BIA） Technology

An automated biomolecular interaction analysis instrument(BIAcore)based on surface plasmon resonance(SPR)has been used to determine human immunoglobulin G(IgG) in real time.Polyclonal anti-human IgG antibody was covalently immobilized to a carboxymethyldextran-modified gold film surface.The samples of human IgG prepared in HBS buffer were poured over the immpbilized surface.The signal amplification antibody was applied to amplify the response signal.After each measurement,the surface was regenerated with 0.1mol/L H3PO4.The assay was rapid,requiring only 30 min for antibody immobilization and 20 min for each subsequent process of immune binding,antibody amplification and regeneration.The antibody immobilized surface had good response to human IgG in the range of 0.12-60 nmol/L with a detection limit of 60 pmol/L.The same antibody immobilized surface could be used for more than 110 cycles of binding,amplification and regeneration.The results demonstrate that the sensitivity,specificity and reproducibility of amplified immunoassay using real-time BIA technology are satisfactory.     

Combination of cysteine- and oligomerization domain-mediated protein immobilization on a surface plasmon resonance (SPR) gold chip surface

Here we report an effective method for protein immobilization on a surface plasmon resonance (SPR) gold chip, describing the combination of cysteine- and oligomerization domain-mediated immobilization of enhanced green fluorescent protein (EGFP) as a model protein for the purpose of orientation-controlled surface density packing. In order to facilitate the oligomerization of EGFP, the dimeric and trimeric constructs derived from GCN4- leucine zipper domain were chosen for multimeric EGFP assembly. For orientation-controlled immobilization of the protein, EGFP modified with cysteine residues showing excellent orientation on a gold chip was used as a starting protein, as previously reported in our earlier study (Anal. Chem., 2007, 79, 2680-2687). Constructs of EGFP with oligomerization domains were genetically engineered, and corresponding fusion proteins were purified, applied to a gold chip, and then analyzed under SPR. The immobilized EGFP density on a gold chip increased according to the states of protein oligomerization, as dimeric and trimeric EGFPs displayed better adsorption capability than monomeric and dimeric forms, respectively. Fluorescence measurement corroborated the SPR results. Taken together, our findings indicated that the combination of cysteine- and oligomerization domain-mediated immobilization of protein could be used in SPR biosensor applications, allowing for an excellent orientation and high surface density simultaneously.     

Immobilization of histidine-tagged recombinant proteins onto micropatterned surfaces for cell-based functional assays

This letter describes a method for preparing protein microarrays that allow the functional analysis of proteins at a cellular level. This method involves the utilization of recombinant proteins genetically engineered to carry a fusion tag that has an affinity for metal ions. A micropatterned alkanethiol monolayer was used to prepare a microarray having multiple spots with immobilized metal ions. The fusion protein was chelated to the spots under physiological conditions. The feasibility of the method was demonstrated by culturing neural stem cells on the microarray that displayed oligohistidine-tagged epidermal growth factor.     

Surface plasmon resonance-enabled mass spectrometry arrays

Biosensors that utilize surface plasmon resonance (SPR) as a method of detection of protein interactions can be used for selective separation of proteins prior to MS analysis. The combination of SPR and MS results in a unique multiplexed detection technology capable of both quantitative and qualitative protein analysis. To further the development of a high-throughput SPR-MS approach, the possibility of arraying binding ligands on SPR chips for affinity capture of proteins and their MS analysis was explored. Antibodies to beta-2-microglobulin, cystatin C, transferrin, and insulin-like growth factors I and II were arrayed on a large number of SPR chips. Human plasma samples were injected over the antibody array chips in an SPR Biosensor, after which on-chip MS analysis was performed to detect the bound proteins. Signals from the targeted proteins were observed for each antibody-derivatized chip, indicating successful antibody immobilization and protein capture. The SPR-MS arrays are robust, highly reproducible, and are capable of high-throughput analysis.     

Adsorptive detagging of poly-histidine tagged protein using hexa-histidine tagged exopeptidase

The ubiquitous use of poly-histidine fusion tags has made the purification of the recombinant target proteins much simpler, although the presence of residual fusion tags can generate immunogenic products or products with changed biological activities. This work presents a generic method of removing poly-histidine fusion tags from recombinant proteins through the use of a hexa-histidine tagged exopeptidase (DAPase) when both tagged species are adsorbed to the immobilized metal affinity chromatography (IMAC) adsorbent. Adsorptive detagging was performed in the presence of 50mM imidazole in order to allow the cleavage reaction by the hexa-histidine tagged DAPase to occur. The progress of batch and adsorptive detagging by DAPase of maltose binding protein (MBP) tagged with two variants of hexa-histidine fusion tag was successfully monitored using cationic exchange chromatography. A single-step, column-based detagging strategy was then optimized to maximize the recovery of native MBP. The kinetics of batch and on-column digestion for both HT6 and HT15 fusion tags were investigated. The process involved the sequential removal of dipeptides during the digestion of full-length fusion protein down to its fully detagged native form. During the course of tag digestion, 4 and 7 different intermediates were detected for HT6 and HT15 tagged MBP respectively. The characteristics of on-column cleavage of poly-histidine fusion tags by DAPase as a function of incubation temperature and amount of protease activity used were examined. It was found that the influence of fusion tag design on the batch and column-based detagging yield and efficiency was substantial. In addition, the structural difference of fusion tags affects the binding strength of the fusion protein, which can influence the resulting product purity. Despite being a longer tag, HT15 fusion tag was the preferred sequence for shortening the time needed for on-column detagging. These results can be applied to the wider use of the proposed platform protocol for the on-column cleavage of poly-histidine tagged proteins using exopeptidases.     

Weak interaction between inhibition peptides and a soluble receptor of fusion protein in the liquid phase.

Fluorescence polarization analysis (FPA) of a liquid-phase method was carried out with a glycosylphosphatidylinositol (GPI) anchored membrane receptor bone marrow stromal cell antigen 1 (BST-1, CD157) as a model receptor for medical screening. A soluble receptor, BST1-Fc, was prepared by fusing the extracellular domain of BST-1 and the Fc region of human immunoglobulin G (IgG). The binding curves of BST1-Fc with a fluorescently labeled ligand peptide, or its three derivatives, were developed using ordinary FPA in the liquid phase. The obtained dissociation constants (Kd) were comparable with those reported as measured with SPR of a solid-phase method, except for one derivative peptide with Kd larger than 7000 nM. Competitive FPA was carried out, and it was demonstrated that a very weak interaction, which would be difficult to detect with SPR or other solid-phase methods, could be analyzed with both ordinary and competitive FPA.