Analysis of agalacto-IgG in rheumatoid arthritis using surface plasmon resonance

It is well established that IgG from rheumatoid arthritis (RA) patients are less galactosylated than IgG from normal individuals. Determination of agalacto-IgG may therefore aid in diagnosis and treatment of RA. The decrease in galactosylation of IgG leads to an increase in terminal N-acetylglucosamine residues, which can be detected using a specific lectin from Psathyrella velutina. In the present study IgG from RA and control serum was purified using affinity chromatography. The samples were then, after reduction, analyzed on a BIOCORE® 2000 system with immobilized Psathyrella velutina lectin. Using this technique it was possible to discriminate between IgG from RA patients and IgG from control individuals with respect to its content of IgG with terminal N-acetylglucosamine. The affinity biosensor technique makes it possible to detect binding without labeling or using secondary antibodies.     

Circulating immune complexes contain citrullinated fibrinogen in rheumatoid arthritis

Introduction  

There is increasing evidence that autoantibodies and immune complexes (ICs) contribute to synovitis in rheumatoid arthritis (RA), yet the autoantigens incorporated in ICs in RA remain incompletely characterised.     

Mapping of linear B-cell epitopes of hepatitis B surface antigen

Eight monoclonal antibodies directed against the surface protein of hepatitis B virus (HBV) were tested using an epitope-mapping system (Pepscan) for characterizing antigenic domains. Four different amino acid sequences corresponding to linear epitopes were identified: one in pre-S1 corresponding to the sequence 29–36, two in pre-S2 corresponding to overlapping sequences 134–141 and 137–144, and one in the S region of the protein corresponding to the amino acid sequence 117–126.     

Quantitative serum proteomics from surface plasmon resonance imaging

The detection and quantification of specific proteins in complex mixtures is a major challenge for proteomics. For example, the development of disease-related biomarker panels will require fast and efficient methods for obtaining multiparameter protein profiles. We established a high throughput, label-free method for analyzing serum using surface plasmon resonance imaging of antibody microarrays. Microarrays were fabricated using standard pin spotting on bare gold substrates, and samples were applied for binding analysis using a camera-based surface plasmon resonance system. We validated the system by measuring the concentrations of four serum proteins using part of a 792-feature microarray. Transferrin concentrations were measured to be 2.1 mg/ml in human serum and 1.2 mg/ml in murine serum, which closely matched ELISA determinations of 2.6 and 1.2 mg/ml, respectively. In agreement with expected values, human and mouse albumin levels were measured to be 24.3 and 23.6 mg/ml, respectively. The lower limits of detection for the four measurements ranged from 14 to 58 ng/ml or 175 to 755 pm. Where purified target proteins are not available for calibration, the microarrays can be used for relative protein quantification. We used the antibody microarray to compare the serum protein profiles from three liver cancer patients and three non-liver cancer patients. Hierarchical clustering of the serum protein levels clearly distinguished two distinct profiles. Thirty-nine significant protein changes were detected (p < 0.05), 10 of which have been observed previously in serum. alpha-Fetoprotein, a known liver cancer marker, was observed to increase. These results demonstrate the feasibility of this high throughput approach for both absolute and relative protein expression profiling.     

Immobilization of carbohydrate epitopes for surface plasmon resonance using the Staudinger ligation

The detection of carbohydrate-protein interactions is often performed using techniques that require surface immobilization of the lectin or the glycan. A commonly used assay for lectin binding is surface plasmon resonance (SPR). We describe an implementation of the Staudinger ligation as a method to immobilize carbohydrate epitopes to a biosensor surface. This was accomplished by first introducing an azide functionality to a carboxymethyldextran surface, followed by reaction with a phosphane-modified carbohydrate ligand. The chemistry employed is extremely mild and was easily adapted to a commercial biosensor system. Using this approach, we investigated the binding of jacalin and wheat germ agglutinin (WGA) to galactose, lactose, and N-acetyl-lactosamine. We observed that WGA binding shows evidence of multivalent interaction with the surface. Additionally, we found that jacalin binding was influenced by the presence of a flexible and hydrophobic galactosyl aglycone.     

Targeting of anti-citrullinated protein/peptide antibodies in rheumatoid arthritis using peptides mimicking endogenously citrullinated fibrinogen antigens

IntroductionWe have previously identified endogenously citrullinated peptides derived from fibrinogen in rheumatoid arthritis (RA) synovial tissues. In this study, we have investigated the auto-antigenicity of four of those citrullinated peptides, and explored their feasibility to target anti-citrullinated protein/peptide antibodies (ACPA).MethodsThe autoantigenic potential of the fibrinogen peptides was investigated by screening 927 serum samples from the Epidemiological Investigation of RA (EIRA) cohort on a peptide microarray based on the ImmunoCAP ISAC® system. In order to assay for ACPA blocking, two independent pools of purified ACPA were incubated with the respective targeting peptide prior to binding to cyclic citrullinated peptide (CCP)2 using the CCPlus® ELISA kit.ResultsTwo peptides derived from the fibrinogen α chain, Arg573Cit (563-583) and Arg591Cit (580-600), referred to as Cit573 and Cit591, and two peptides from the fibrinogen β chain, Arg72Cit (62-81) and Arg74Cit (62-81) (Cit72 and Cit74), displayed 65 %, 15 %, 35 %, and 53 % of immune reactivity among CCP2-positive RA sera, respectively. In CCP2-negative RA sera, a positive reactivity was detected in 5 % (Cit573), 6 % (Cit591), 8 % (Cit72), and 4 % (Cit74). In the competition assay, Cit573 and Cit591 peptides reduced ACPA binding to CCP2 by a maximum of 84 % and 63 % respectively. An additive effect was observed when these peptides were combined. In contrast, Cit74 and Cit72 were less effective. Cyclization of the peptide structure containing Cit573 significantly increased the blocking efficiency.ConclusionsHere we demonstrate extensive autoantibody reactivity against in vivo citrullinated fibrinogen epitopes, and further show the potential use of these peptides for antagonizing ACPA.     

Compact, high performance surface plasmon resonance imaging system

We report the construction and characterization of a new compact surface plasmon resonance imaging instrument. Surface plasmon resonance imaging is a versatile technique for detection, quantification and visualization of biomolecular binding events which have spatial structure. The imager uses a folded light path, wide-field optics and a tilted detector to implement a high performance optical system in a volume 7 in. x 4 in. x 2 in. A bright diode light source and an image detector with fast frame rate and integrated digital signal processor enable real-time averaging of multiple images for improved signal-to-noise ratio. Operating angle of the imager is adjusted by linear translation of the light source. Imager performance is illustrated using resolution test targets, refractive index test solutions, and competition assays for the antiepileptic drug phenytoin. Microfluidic flowcells are used to enable simultaneous assay of three sample streams. Noise level of refractive index measurements was found to decrease proportional to the square root of the number of pixels averaged, reaching approximately 5 x 10(-7) refractive index units root-mean-square for 160 x 120 pixels image regions imaged for 1s. The simple, compact construction and high performance of the imager will allow the device to be readily applied to a wide range of applications.     

Nanostructured digital microfluidics for enhanced surface plasmon resonance imaging

The advances in genomics and proteomics have unveiled an exhaustive catalogue of biomarkers that can potentially be used as diagnostic and prognostic indicators of genetic and infectious diseases. Current thrust in biosensor development is towards rapid, real-time, label-free and highly sensitive detection of the indicative biomarkers. While surface plasmon resonance imaging (SPRi) biosensors could potentially be the best suited candidate for biomarker-based diagnosis, important milestones need to be reached. Commercially available SPRi instrumentation is currently limited by the flow-cell technology to serial-sample processing and has limited sensitivity for the detection of markers present at low concentration. In this paper, we have implemented an approach to enhance sample handling and increase the sensitivity of the SPRi detection technique. We have developed a digital microfluidic platform with an integrated nanostructured biosensor interface that allows for rapid, ultra-low volume, sensitive, and automated on-chip SPRi detection of DNA hybridization reactions. Through the exploitation of electromagnetic properties of nanofabricated periodic gold nanoposts, SPRi signal was increased by 200% with the estimated limit of detection of 500 pM (90 attomoles). Using the versatile fluidic manipulation provided by the digital microfluidics, rapid and parallel target identification was achieved on multiple array elements within 1 min using 180 nL sample volume. By delivering multiple target analytes in individually addressable low volume droplets, without external pumps and fluidic interconnects, the overall assay time, cost and complexity was reduced. The proposed platform allows extreme versatility in the manipulation of precious low volume samples which makes this technology very suitable for diagnostic applications.     

Vesicle fusion studied by surface plasmon resonance and surface plasmon fluorescence spectroscopy

Substrate-supported planar lipid bilayers are generated most commonly by the adsorption and transformation of phospholipid vesicles (vesicle fusion). We have recently demonstrated that simultaneous measurements of surface plasmon resonance (SPR) and surface plasmon fluorescence spectroscopy (SPFS) are highly informative for monitoring lipid membranes on solid substrates. SPR and SPFS provide information on the amount and topography of adsorbed lipid membranes, respectively. In this study, the vesicle fusion process was studied in detail by measuring SPR-SPFS at a higher rate and plotting the obtained fluorescence intensity versus film thickness. We could track the initial adsorption of vesicles, the onset of vesicle rupture occurring at certain vesicle coverage of the surface, and the autocatalytic transformation into planar bilayers. We also monitored vesicle fusion of the same vesicle suspensions by quartz crystal microbalance with dissipation monitoring (QCM-D). We compared the results obtained from SPR-SPFS and QCM-D to highlight the unique information provided by SPR-SPFS.     

Quantification of antibody production of individual hybridoma cells by surface plasmon resonance imaging

Surface plasmon resonance imaging (SPRi) is most frequently used for the label-free measurement of biomolecular interactions. Here we explore the potential of SPRi to measure antibody production of individual hybridoma cells. As a model system, cells from a hybridoma, producing monoclonal antibodies recognizing epithelial cell adhesion molecule (EpCAM), were used. Recombinant human EpCAM protein was immobilized on an SPR sensor and hybridoma cells were introduced into an IBIS MX96 SPR imager and the SPRi response was followed for 10 h. SPRi responses were detected on the spots of the sensor only where ligands of the produced antibody were present. By measuring the SPRi signals on individual cells the antibody production of the individual cells was measured and production rates were calculated. For 53 single EpCAM hybridoma cells the production ranged from 0.16 to 11.95 pg (mean 2.96 pg per cell, SD 2.51) over a period of 10 h. Antibody excretion per cell per hour ranged from 0.02 to 1.19 pg (mean 0.30, SD 0.25). Here we demonstrate for the first time that antibody production of individual cells can be measured and quantified by SPRi, opening a new avenue for measuring excretion products of individual cells.     

A surface plasmon resonance array biosensor based on spectroscopic imaging

We have developed a multi-element transduction system which combines conventional SPR spectroscopy with one-dimensional SPR microscopy to create an effective platform for monitoring binding events on macro- or micro-patterned receptor arrays created on disposable sensor chips. This creates an effective platform for monitoring simultaneous binding events on each of the regions patterned with the receptors. This system has been specifically designed with commercially available components to allow relatively easy duplication. Furthermore, this system can use a proven, simple method to compensate for changes in the bulk index of refraction of the solution containing the analytes due to changes in temperature or solute concentration with simple modifications to the sensor chips alone. Preliminary results demonstrate how this system can be used to monitor several independent biospecific binding events simultaneously.     

A fusion protein expression analysis using surface plasmon resonance imaging

A surface plasmon resonance (SPR) imaging system was constructed and used to detect the affinity-tagged recombinant proteins expressed in Escherichia coli. With regards to model proteins, the hexahistidine-ubiquitin-tagged human growth hormone (His(6)-Ub-hGH), glutathione S-transferase-tagged human interleukin-6 (GST-hIL6), and maltose-binding protein-tagged human interleukin-6 (MBP-hIL6) expressed in E. coli were analyzed. The cell lysates were spotted on gold thin films coated with 11-mercaptoundecanol (MUOH)/dextran derivatized with Ni(II)-iminodiacetic acid (IDA-Ni(II)), glutathione, or cyclodextrin. After a brief washing of the gold chip, SPR imaging measurements were carried out in order to detect the bound affinity-tagged fusion proteins. Using this new approach, rapid high-throughput expression analysis of the affinity-tagged proteins were obtained. The SPR imaging protein chip system used to measure the expression of affinity-tagged proteins in a high-throughput manner is expected to be an attractive alternative to traditional laborious and time-consuming methods, such as SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blots.     

Carbohydrate-lectin interactions assessed by surface plasmon resonance

The specificity, the strength, the kinetics and some thermodynamic parameters of sugar-protein interactions are easily assessed by surface plasmon resonance (SPR). This paper intends to present both theoretical and practical considerations. This includes: the principle of SPR, the analysis according to Langmuir and Scatchard, the problems linked either to mass transport limitation, to the heterogeneity of the immobilized ligand density or to the non-linearity due to cluster effects. The non-linearity may be taken into account by either one of two ways: the fractal or the Sips approaches that have been developed with the aim of linearizing the data. In addition, selected data obtained by using either immobilized carbohydrates or immobilized lectins are summarized. The SPR has also been found useful to collect information concerning oligosaccharide structure as well as lectin-sugar specificity and to develop new tools with medical applications. Finally, a series of practical considerations are gathered in the hope of avoiding some of the common pitfalls arising in sugar-lectin interaction studies based on the use of SPR.     

Substrate-supported phospholipid membranes studied by surface plasmon resonance and surface plasmon fluorescence spectroscopy

Substrate-supported planar lipid bilayer membranes are attractive model cellular membranes for biotechnological applications such as biochips and sensors. However, reliable fabrication of the lipid membranes on solid surfaces still poses significant technological challenges. In this study, simultaneous surface plasmon resonance (SPR) and surface plasmon fluorescence spectroscopy (SPFS) measurements were applied to the monitoring of adsorption and subsequent reorganization of phospholipid vesicles on solid substrates. The fluorescence intensity of SPFS depends very sensitively on the distance between the gold substrate and the fluorophore because of the excitation energy transfer to gold. By utilizing this distance dependency, we could obtain information about the topography of the adsorbed membranes: Adsorbed vesicles could be clearly distinguished from planar bilayers due to the high fluorescence intensity. SPSF can also incorporate various analytical techniques to evaluate the physicochemical properties of the adsorbed membranes. As an example, we demonstrated that the lateral mobility of lipid molecules could be estimated by observing the recovery of fluorescence after photobleaching. Combined with the film thickness information obtained by SPR, SPR-SPFS proved to be a highly informative technique to monitor the lipid membrane assembly processes on solid substrates.     

Diagnostic utility of anti-cyclic citrullinated peptide and anti-modified citrullinated vimentin antibodies in rheumatoid arthritis

Several autoantibodies found in RA are directed to epitopes in citrullinated proteins. One of them is anti modified citrullinated vimentin (Anti-MCV). We tested the value a newly developed ELISA for the detection of antibodies against a genetically modified citrullinated vimentin (anti-MCV) in comparison with an anti-CCP based ELISA system for the diagnosis of RA. Thirty-five patients with RA (mean age; 42.6 +/- 10.87 years, mean disease duration; 9.37 +/- 3.98 years) were enrolled in this study. Twenty -five ankylosing spondylitis (mean age; 35.88 +/- 6.64 years, mean disease duration; 10.25 +/- 4.61 years), and 19 healthy subjects (mean age; 40.26 +/- 5.11 years) served as controls. Anti-CCP antibodies and Anti-MCV antibodies were measured using ELISA. In all RA patients, mean anti- CCP level was 69.07 +/- 90.43 U/ml and anti-MCV level was 665.77 +/- 1040.19 U/ml. In patients with AS, the mean anti-CCP level was 10.7 +/- 5.22 U/ml and anti-MCV level was 40.54 +/- 20.15 U/ml. In healthy controls, the mean anti-CCP level was 11.11 +/- 7.65 U/ml, anti-MCV level was 23.12 +/- 12.04 U/ml. In patients with active RA, the mean serum anti-CCP level was 100.54 +/- 98.07 U/ml and anti-MCV level was 998.74 +/- 1154.93 U/ml. In patients with inactive RA, the mean serum anti-CCP level was 8.77 +/- 1.55 U/ml and anti-MCV level was 27.59 +/- 23.10 U/ml. According to these results; In patients with RA, the mean serum anti-MCV and anti-CCP levels were significantly high compared to patients with AS and healthy controls (p=0.002, p=0.001, p=0.002, p=0.001 respectively). The mean serum anti-MCV and anti- CCP levels were significantly higher in active patients with RA than in inactive patients with RA patients (p=0.001 and p=0.001 respectively). In inactive patients with RA, the mean serum anti-MCV and anti-CCP levels were similar in patients with AS and patients (p=0.484, p=0.308, p=0.09 and p=0.222 respectively). The mean serum anti-MCV levels were correlated with DAS 28 (r=0.531, p=0.001), VAS score (r=0.332, p=0.01), ESR (r=0.458, p=0.001), serum CRP levels (r=0.568, p=0.01), serum RF levels (r=0.529, p=0.001), swollen joints number (r=0.525, p=0.001) and tender joints number (r=0.638, p=0.001). As a result; measurement of serum anti-MCV levels is useful for diagnosis of RA and combined use of anti-MCV and RF may be more useful prognostic factor than either method alone, RF and anti-CCP.     

Study of cell-matrix adhesion dynamics using surface plasmon resonance imaging ellipsometry

The interaction of cells with extracellular matrix, termed cell-matrix adhesions, importantly governs multiple cellular phenomena. Knowledge of the functional dynamics of cell-matrix adhesion could provide critical clues for understanding biological phenomena. We developed surface plasmon resonance imaging ellipsometry (SPRIE) to provide high contrast images of the cell-matrix interface in unlabeled living cells. To improve the contrast and sensitivity, the null-type imaging ellipsometry technique was integrated with an attenuated total reflection coupler. We verified that the imaged area of SPRIE was indeed a cell-matrix adhesion area by confocal microscopy imaging. Using SPRIE, we demonstrated that three different cell types exhibit distinct features of adhesion. SPRIE was applied to diverse biological systems, including during cell division, cell migration, and cell-cell communication. We imaged the cell-matrix anchorage of mitotic cells, providing the first label-free imaging of this interaction to our knowledge. We found that cell-cell communication can alter cell-matrix adhesion, possibly providing direct experimental evidence for cell-cell communication-mediated changes in cell adhesion. We also investigated shear-stress-induced adhesion dynamics in real time. Based on these data, we expect that SPRIE will be a useful methodology for studying the role of cell-matrix adhesion in important biological phenomena.     

Detection of autoantibodies to citrullinated BiP in rheumatoid arthritis patients and pro-inflammatory role of citrullinated BiP in collagen-induced arthritis

Introduction

Anti-citrullinated protein/peptide antibodies (ACPAs) are highly specific to rheumatoid arthritis (RA) patients and are thought to have a close relationship with the pathogenesis of arthritis. Several proteins, including fibrinogen, vimentin, and alpha-enolase, were reported as ACPA-target antigens, and their importance in RA pathogenesis was widely proposed. We identified citrullinated immunoglobulin binding protein (citBiP) as another ACPA target in RA patients and examined its pro-inflammatory role in arthritis.

Methods

We measured the levels of anti-citBiP, anti-BiP, and anti-cyclic citrullinated peptide (CCP) antibodies in the serum of RA patients (n = 100), systemic lupus erythematosus (SLE) patients (n = 60), and healthy controls (n = 30) using ELISA and immunoblotting. Epitope mapping was performed using 27 citBiP-derived peptides. In the mouse study, after DBA/1J mice were immunized with BiP or citBiP, serum titers of ACPAs were measured by ELISA and immunohistochemistry. The development of collagen-induced arthritis (CIA) was observed in BiP- or citBiP-pre-immunized mice.

Results

The serum levels of anti-BiP and anti-citBiP antibodies were significantly increased in RA patients, although only anti-BiP antibodies were slightly increased in SLE patients. Interestingly, anti-citBiP antibody levels were higher than anti-BiP antibody levels in 72% of RA patients, whereas no significant increase in anti-citBiP antibody levels was detected in SLE patients and healthy controls. The serum levels of anti-CCP antibodies were correlated with those of anti-citBiP antibodies in RA patients (R² = 0.41). Several citrulline residues of citBiP were determined to be major epitopes of anti-citBiP antibodies, one of which showed cross-reactivity with CCP. Immunization of DBA/1J mice with citBiP induced several kinds of ACPAs, including anti-CCP and anti-citrullinated fibrinogen antibodies. Pre-immunization with citBiP exacerbated CIA, and anti-CCP antibody levels were increased in citBiP-pre-immunized CIA mice.

Conclusions

CitBiP is a newly described ACPA target that may play a pro-inflammatory role in arthritis.     

Epitope mapping by surface plasmon resonance in the BIAcore

An epitope may be defined as a specific site on an antigen module characterized by the binding of one monoclonal antibody (MAb). Epitope mapping by surface plasmon resonance in the BIAcore biosensor may be performed to characterize an antigen or a group of specific MAbs or both. This article describes the BIAcore instrument and methods for such mapping. Examples include molecular interaction studies with simple and complex proteins, such as myoglobin and calprotectin, respectively.     

Integrated-optic biosensor by electro-optically modulated surface plasmon resonance

We present a new integrated-optic surface plasmon resonance (SPR) biosensor based on electro-optical modulation. The SPR characteristics for the analyte concentration detection can be electro-optically modulated by applying the voltage on the electrodes of the biosensor fabricated on lithium niobate, which is an excellent electro-optic material. Two measurement methods, electro-optically modulated SPR spectral measurement and electro-optically modulated SPR intensity measurement, are demonstrated and their measurands are the SPR wavelength and the output optical intensity, respectively. Human serum albumin is coated on the gold film surface of the proposed biosensor to detect the concentration of beta-blocker, which is a remedy for heart disease. As the applied voltage increases such that the effective index of guided mode rises, the SPR wavelength shifts toward the long wavelength side and the output optical intensity at the wavelength of 632.8 nm diminishes. The linear regression slope of the relation between the measurand and the applied voltage is dependent on the analyte concentration and can be used to determine the concentration variation. Experimental results measured by the electro-optically modulated SPR methods are compared with those measured by the conventional spectral and intensity methods, and the effects of waveguide width on the biosensor performance are discussed.     

Analysis of protein interactions on protein arrays by a novel spectral surface plasmon resonance imaging

We presented a novel surface plasmon resonance (SPR) imaging method for analysis of protein arrays based on a wavelength interrogation-based SPR biosensor. The spectral imaging was performed by the combination of position control and resonance wavelengths calculated from SPR reflectivity spectra. The imaging method was evaluated by analyzing interactions of glutathione S-transferase-fusion proteins with their antibodies. Antigen-antibody interactions were successfully analyzed on glutathione S-transferase-fusion protein arrays by using the spectral imaging method, and the results were confirmed by a parallel analysis using a previously used spectral SPR biosensor based on wavelength interrogation. Specific binding of anti-Rac1 and anti-RhoA to Rac1 and RhoA on the protein arrays was qualitatively and quantitatively analyzed by the spectral SPR imaging. Thus, it was suggested that the novel spectral SPR imaging was a useful tool for the high-throughput analysis of protein-protein interactions on protein arrays.