Enhancement of the sensitivity of surface plasmon resonance (SPR) immunosensor for the detection of anti-GAD antibody by changing the pH for streptavidin immobilization

Surface plasmon resonance (SPR) immunobiosensor was developed for the detection of anti-glutamic acid decarboxylase (GAD) antibody. In this study, carboxylic terminated self-assembled monolayer, which was prepared by mixing of 3-mercaptopropionic acid (3-MPA) and 11-mercaptoundecanoic acid (11-MUA) (10:1 ratio), was used to evaluate the effect of external pH on the affinity between streptavidin and sensor surface. At pH values ranging from 4.0 to 5.5, it was found that streptavidin could more easily access onto the sensor surface at higher pH, and the enhanced binding of streptavidin at high pH allowed more extensive immobilization of biotin-GAD, which serves as the epitope for anti-GAD antibody. Consequently, the increase of RU caused by immuno-response between GAD and anti-GAD antibody was remarkably higher when streptavidin was bound on to the sensor surface at pH 5.5 than at pH 4.5. Therefore, we could conclude that the pH of coupling buffer greatly influences the sensitivity of immunosensor.     

Signal enhancement of surface plasmon resonance immunoassay using enzyme precipitation-functionalized gold nanoparticles: a femto molar level measurement of anti-glutamic acid decarboxylase antibody

Colloidal gold nanoparticles (AuNPs) and precipitation of an insoluble product formed by HRP-biocatalyzed oxidation of 3,3'-diaminobenzidine (DAB) in the presence of H2O2 were used to enhance the signal obtained from the surface plasmon resonance (SPR) biosensor. The AuNPs were synthesized and functionalized with HS-OEG3-COOH by self assembling technique. Thereafter, the HS-OEG3-COOH functionalized nanoparticles were covalently conjugated with horseradish peroxidase (HRP) and anti IgG antibody to form an enzyme-immunogold complex. Characterizations were performed by several methods: UV-vis absorption, DLS, HR-TEM and FT-IR. The Au-anti IgG-HRP complex has been applied in enhancement of SPR immunoassay using a sensor chip constructed by 1:9 molar ratio of HS-OEG6-COOH and HS-OEG3-OH for detection of anti-GAD antibody. As a result, AuNPs showed their enhancement as being consistent with other previous studies while the enzyme precipitation using DAB substrate was applied for the first time and greatly amplified the SPR detection. The limit of detection was found as low as 0.03 ng/ml of anti-GAD antibody (or 200 fM) which is much higher than that of previous reports. This study indicates another way to enhance SPR measurement, and it is generally applicable to other SPR-based immunoassays.     

Enhanced performance of a surface plasmon resonance immunosensor for detecting Ab-GAD antibody based on the modified self-assembled monolayers

To enhance the feasibility of surface plasmon resonance (SPR) immunosensor as a tool for diagnosing type I diabetes, we enhanced the sensitivity of immunoresponse for detecting the monoclonal anti-glutamic acid decarboxylase (GAD) antibody by modification of mixed self-assembled monolayers (SAMs). The effects of the different mixed SAMs were evaluated with respect to the degree of streptavidin immobilization, the degree of biotin-GAD immobilization, and the immunoresponse sensitivity. Consequently, the sensitivity of the immunoresponse for the detection of anti-GAD antibody was enhanced as a result of the reduction in steric hindrance brought about by using SAMs of heterogeneous lengths. The immunoresponse for detecting the monoclonal anti-GAD antibody was also enhanced with the reduction of the excess immobilization of biotin-GAD and the minimization of non-specific binding that resulted from the simple substitution of the spacer from a carboxylic-terminated SAM for the hydroxyl-terminated SAM.     

Reversible surface thiol immobilization of carboxyl group containing haptens to a BIAcore biosensor chip enabling repeated usage of a single sensor surface

We describe a reversible immobilization method for carboxyl group containing haptens that makes the repeated usage of a BIAcore biosensor chip possible. Haptens which are immobilized according to the surface thiol method can be removed completely from the sensor surface again by a reducing step. In the first part of our study, analogues of the herbicides 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid were immobilized in succession to a biosensor surface of a BIAcore surface plasmon resonance instrument according to the thiol coupling method. Direct kinetic analysis of these ligands to a polyclonal anti-2,4-dichlorophenoxyacetic acid antibody were performed using these biosensor surfaces. In the second part of the study, different amounts of 2,4-dichlorophenoxyacetic acid were sequentially immobilized onto the same biosensor surface in order to generate a calibration plot for 2,4-dichlorophenoxyacetic acid. Using this plot, the quantitative detection of the herbicide down to a concentration of 0.1 microg/mL, the maximum admissible concentration of pesticides in drinking water, is possible.     

Large-surface biosensor technology for enhanced recovery in protein characterization.

A large-surface biosensor technique using surface plasmon resonance (SPR) was tested for protein purification by recovery of a monoclonal antibody against human proinsulin C-peptide. Notably, both reversible attachment/desorption and actual purification of the antibody from a multi-component protein mixture was shown. For initial chip attachment of the peptide ligand, C-peptide was biotinylated and attached to neutravidin on plastic chips with a large gold surface (effective area 26 mm(2)). Antibody binding and desorption was monitored in real-time SPR, and for elution different conditions were employed. Five percent formic acid (in contact with the chip surface for 3 min) in a 60-mul segment between air bubbles was efficient for subsequent analysis. In this manner, protein amounts up to 35 pmoles were recovered in a single capture/elution cycle. Evaluation by SDS-PAGE showed essentially no carryover between fractions in this elution process, and also not with other proteins in the mixture after purification. Compared to existing commercial instruments, this technique gives higher recovery and makes it possible to monitor monitor protein binding/desorption. Recovery of affinity partners at the multi-pmole level is demonstrated for protein purification in SPR approaches.     

Detection of weakly interacting anti-carbohydrate scFv phages using surface plasmon resonance

Methods to characterise and confirm specificity of scFv displayed on phages are important during panning procedures, especially when selecting for antibody fragments with weak affinities in the millimole to micromole range. In this report the surface plasmon resonance (SPR) biosensor was used to study and verify specificity of phages displaying weak anti-carbohydrate scFvs. The variable immunoglobulin light (VL) and heavy (VH) chain genes of the weak monoclonal antibody 39.5 were amplified and cloned into a phagemid and displayed as a scFv-pIII fusion protein on filamentous phage. This monoclonal antibody recognises with weak affinity the structural sequence Glcalpha1-4Glc present in a variety of carbohydrate molecules. Injection of the 39.5 phages over a biosensor chip immobilised with a (Glc)4-BSA conjugate confirmed selective binding of the scFv to its antigen. Inhibition studies verified the specificity. These results clearly show that SPR technology can be used to evaluate in terms of binding and specificity weakly interacting scFv displayed on the phage surface.     

Analysis of protein interactions on protein arrays by a wavelength interrogation-based surface plasmon resonance biosensor

We have investigated whether surface plasmon resonance (SPR) sensors based on the wavelength interrogation are able to analyze protein interactions on protein arrays. The spectral SPR sensor was self-constructed and its detection limit, expressed as the minimal refractive index variation, was calculated to be 6.6x10(-5) with the signal fluctuation of 1.0x10(-5). The protein array surface was modified by a mixed thiol monolayer to immobilize proteins. Protein arrays were analyzed by the line-scanning mode of the SPR sensor, which scanned every 100 microm along the central line of array spots and the scanned results were presented by color spectra from blue to red. Glutathione S-transferase (GST)-rac1 caused a concentration-dependent increase of SPR wavelength shift on protein arrays. The surface structure of the protein arrays was analyzed by atomic force microscopy. Specific interactions of antigens with antibodies were analyzed on the protein arrays by using three antibodies and eight proteins. These results suggest that the wavelength interrogation-based SPR sensor can be used as the biosensor for the high-throughput analysis of protein interactions on protein arrays.     

Nano-scale probe fabrication using self-assembly technique and application to detection of<Emphasis Type="Italic">Escherichia coli</Emphasis> O 157∶H7

A self-assembled monolayer of protein G was fabricated to develop an immunosensor based on surface plasmon resonance (SPR), thereby improving the performance of the antibody-based biosensor through immobilizing the antibody molecules (IgG). As such, 11-mercaptoundecanoic acid (11-MUA) was adsorbed on a gold (Au) support, while the non-reactive hydrophilic surface was changed through substituting the carboxylic acid group (-COOH) in the 11-MUA molecule using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrocholide (EDAC). The formation of the self-assembled protein G layer on the Au substrate and binding of the antibody and antigen were investigated using SPR spectroscopy, while the surface topographies of the fabricated thin films were analyzed using atomic force microscopy (AFM). A fabricated monoclonal antibody (Mab) layer was applied for detectingE. coli O157∶H7. As a result, a linear relationship was achieved between the pathogen concentration and the SPR angle shift, plus the detection limit was enhanced up to 10² CFU/mL.     

Immunosensor for detection of Legionella pneumophila using surface plasmon resonance

Immunosensor using surface plasmon resonance (SPR) onto self-assembled protein G layer was developed for the detection of Legionella pneumophila. A self-assembled protein G layer on gold (Au) surface was fabricated by adsorbing a mixture of 11-mercaptoundecanoic acid (MUA) and hexanethiol (molar ratio of 1:2) and the activation process for chemical binding between free amine (-NH(2)) of protein G and 11-(MUA) using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC) in series. The formation of self-assembled protein G layer on Au substrate and the binding of antibody and antigen in series were confirmed by SPR spectroscopy. The surface morphology analyses of self-assembled protein G layer on Au substrate and monoclonal antibody against L. pneumophila immobilized on protein G were performed by atomic force microscope (AFM). The immunosensor for detection of L. pneumophila using SPR was developed and its detection limit could find up to 10(5) cells/ml.     

Screening of a specific monoclonal antibody against and detection ofListeria monocytogenes whole cells using a surface plasmon resonance biosensor

In this study, a specific monoclonal antibody againstListeria monocytogenes was screened using an SPR biosensor Monoclonal antibodies were bound to protein L, after which theL. monocytogenes cells were subjected to an affinity assay. Protein L was immobilized on a carboxymethyl dextran (CM-Dex) surface via an amine coupling method and utilized repeatedly by regeneration. The monoclonal antibody, ‘A18’, was selected and employed for the high-sensitivity detection ofL. monocytogenes. Under optimized conditions, 10³ cells/ml or 50 cells were detected by the SPR biosensor.     

Novel immunoassay for carcinoembryonic antigen based on protein A-conjugated immunosensor chip by surface plasmon resonance and cyclic voltammetry

In this study, an immunosensor chip utilizing surface plasmon resonance (SPR) and cyclic voltammetry (CV) was fabricated for detecting carcinoembryonic antigen (CEA). Specifically, we applied in parallel an SPR instrument and a CV device to monitor the assembly of carcinoembryonic antibody (anti-CEA) on a protein A-conjugated surface and the subsequent ligand reaction. The immunosensor chips were constructed by various concentrations of protein A. To determine the surface characteristics of different self-assembly monolayers (SAMs), several quantitative and kinetic measurements were carried out. The extent of immobilization of anti-CEA and the immune response of anti-CEA antibody against CEA were measured using the SPR instrument and CV device. The terminal functional groups of protein A have different effects on the adsorption and covalent binding of immunoprotein depending on the steric hindrance. Through the parallel measurements, we demonstrate that SPR and CV are sensitive to measure the antigen–antibody binding capacity.     

Development of a whole-cell biosensor by cell surface display of a gold-binding polypeptide on the gold surface

Cell surface display was used as a strategy to display the gold-binding polypeptide (GBP) fusion protein on the surface of Escherichia coli , and consequently to immobilize the cells on the gold surface. The DNA encoding the GBP was fused to the truncated fadL gene and was expressed by the tac promoter. For the display of the core streptavidin (cSA) of Streptomyces avidinii , the cSA gene was fused to the truncated oprF gene. After the dual display of FadL–GBP and OprF–cSA on the surface of E. coli , binding of cells on the gold surface and the interaction of OprF–cSA with the biotin–horseradish peroxidase (HRP) were studied by surface plasmon resonance (SPR) analysis. Cells displaying the FadL–GBP fusion protein could be immobilized on the SPR sensor chip as shown by the SPR angle shift of 0.5°, which was stably bound at least for 60 h with a washing solution. When the FadL–GBP and OprF–cSA fusion proteins were displayed on the same cell surface, the former was used to immobilize the cells on the gold surface and the latter was used for the interaction studies with the biotin–HRP, which demonstrates that the strategy should be useful for developing whole-cell biosensor chips.     

Continuous flow immunosensor for highly selective and real-time detection of sub-ppb levels of 2-hydroxybiphenyl by using surface plasmon resonance imaging

A biosensor based on surface plasmon resonance (SPR) is developed for the detection of 2-hydroxybiphenyl (HBP). A monoclonal antibody against HBP (abbreviated hereafter as HBP-mAb) is developed and used for the detection of HBP by competitive SPR-based immunoassay and enzyme linked immunosorbent assay (ELISA) methods. A novel HBP-hapten compound, HBP-bovine serum albumin conjugate (HBP-BSA), derived by binding several HBP units with BSA by an aliphatic chain spacer is used in the development of antibody and for the functionalization of immunoprobes. HBP-BSA linked to the Au surface of the SPR sensor chip undergoes inhibitive immunoreaction with HBP-mAb in the presence of free HBP. The SPR-based immunoassay provides a rapid determination (response time: approximately 20 min) of the concentration of HBP in the range of 0.1-1000 ppb (ng/ml). Regeneration of the sensor chip is gained by treating the antibody-anchored SPR sensor chip with a pepsin solution (100 ppm (microg/ml); pH 2.0) for few minutes. The SPR sensor chip is reusable for the detection of HBP for more than 20 cycles with average loss of 0.35% reactivity per regeneration step. HBP concentration is determined as low as 0.1 and 3 ppb using the SPR sensor and ELISA measurements, respectively. The developed SPR sensor for HBP is free from interference by coexisting benzo[a]pyrene (BaP), 2,4-dichlorophenoxyacetic acid (2,4-D) and benz[a]anthracene; SPR angle shift obtained to the flow of HBP is almost same irrespective to the presence or absence of a same concentration of these carcinogenic polycyclic aromatic hydrocarbons together. The SPR sensor for HBP is proved to be applicable in simultaneous detection of HBP and BaP in parallel with another SPR sensor for BaP.     

The fabrication of protein chip based on surface plasmon resonance for detection of pathogens

Protein chip based on surface plasmon resonance (SPR) was developed for detection of pathogens existing in contaminated environment, such as Escherichia coli O157:H7, Salmonella typhimurium, Legionella pneumophila, and Yersinia enterocolitica. Protein G was immobilized to endow the orientation of antibody molecules on the SPR surface. The pathogen binding of the protein chip was investigated by SPR spectroscopy. Consequently, it was found that the four kinds of pathogen could be selectively detected by using SPR-based protein chip.     

Physicochemical characterization of poly(ethylene glycol)-modified anti-GAD antibodies.

Monoclonal antibodies against glutamic acid decarboxylase (anti-GAD) were modified with poly(ethylene glycol) (PEG), and the resulting conjugates were characterized. Monoclonal anti-GAD antibodies were purified from ATCC HB184 hybridoma cells by either cell culture supernatant or ascites fluid from BALB/c mice. Polyclonal rabbit IgG antibodies were also used as a model protein. Polyclonal rabbit IgG or purified anti-GAD was modified by PEG (MW = 5000 or 20000 Da) through either the lysine residues or through the carbohydrate moiety. Lysine modification was performed in PBS (pH 7.4) or 0.1 M borate (pH 9.2) by adding a molar excess (5-80) of a succinimidyl activated propionic acid terminated mPEG (SPA-PEG) while stirring at room temperature. Carbohydrate modifications were performed in PBS (pH 6.2) by first oxidizing the antibody with sodium periodate followed by incubation with hydrazide-terminated PEG followed by reduction with sodium cyanoborohydride. The degree of modification was assessed by 1H NMR or TNBS (trinitrobenzenesulfonic acid). Circular dichroism (CD) spectra were obtained for lysine-modified rabbit IgG at various degrees of modification ranging from 5 to 60 PEG per antibody. Binding was assessed using an ELISA method with GAD or rabbit anti-mouse-IgG (H+L) coated plates. The TNBS and 1H NMR analysis of the modified antibody showed reasonably similar results from 5 to 60 PEG per antibody. The 1H NMR method showed greater sensitivity at low modifications (below 20:1) and was fairly linear up to about 60 PEG per antibody. The CD spectra of the polyclonal rabbit IgG showed only small differences at variously modified antibody. The binding affinity of anti-GAD is lower for all PEG modifications with respect to unmodified anti-GAD. Modifications at pH 7.4 show lower binding to GAD than modifications at pH 9.2. Binding to GAD or anti-mouse-IgG is decreased as the degree of modification is increased. Lysine modifications showed lower binding to GAD or anti-mouse-IgG than carbohydrate modifications. Binding to GAD or anti-mouse-IgG is lower for PEG20000-modified anti-GAD with respect to PEG5000-modified anti-GAD.     

High-throughput analysis of GST-fusion protein expression and activity-dependent protein interactions on GST-fusion protein arrays with a spectral surface plasmon resonance biosensor

We modified gold arrays with a glutathione (GSH) surface, and investigated high-throughput protein interactions with a spectral surface plasmon resonance (SPR) biosensor. We fabricated the GSH exterior on gold surfaces by successive modification with aminoethanethiol, 4-maleimidobutyric acid N-hydroxysuccinimide ester and GSH. We immobilized GST-Rac1, GST-RhoA, the GST-Rho-binding domain of rhotekin and the GST-p21-binding domain of PAK1 onto the GSH surface, and observed specific antigen-antibody interactions on the GST-fusion protein arrays. We determined the expression of GST-fusion proteins in Escherichia coli on the GSH surface with the SPR biosensor. We then analyzed the interactions of tissue transglutaminase (tTGase), a Ca2+-dependent enzyme, with RhoA and Rac1 on the GST-fusion protein arrays with the SPR biosensor. We found that tTGase interacted with RhoA and Rac1 in a Ca2+-dependent manner, indicating that the interactions were dependent on tTGase activity. In addition, transamidation of Rac1 by tTGase was dependent on Ca2+ concentration. We obtained similar results with GST pull-down assays. Thus, protein arrays prepared on the GSH surface provide a useful system for the high-throughput analysis of GST-fusion protein expression and activity-dependent protein interactions with the spectral SPR biosensors.     

Catalytic enzyme activity on a biosensor chip: combination of surface plasmon resonance and mass spectrometry

Surface plasmon resonance (SPR) as a label-free biosensor technique has become an important tool in drug discovery campaigns during the last couple of years. For good assay performance, it is of high interest to verify the functional activity on the immobilization of the target protein on the chip. This study illustrates the verification of the catalytic activity of the drug target protein PqsD by monitoring substrate conversion as a decrease in SPR signal and product detection by ultra high-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS(2)). This assay would be applicable to control surface activity of immobilized ligands.     

Development of a surface plasmon resonance biosensor for the identification of Campylobacter jejuni

The purpose of this study was to develop a biosensor based on surface plasmon resonance (SPR) for the rapid identification of C. jejuni in broiler samples. We examined the specificity and sensitivity of commercial antibodies against C. jejuni with six Campylobacter strains and six non-Campylobacter bacterial strains. Antigen-antibody interactions were studied using enzyme-linked immunosorbent assay (ELISA) and a commercially available SPR biosensor platform (Spreeta). Campylobacter cells killed with 0.5% formalin had significant lower antibody reactivity when compared to live cells, or cells inactivated with 0.5% thimerosal or heat (70 degrees C for 3 min) using ELISA. The SPR biosensor showed a good sensitivity with commercial antibodies against C. jejuni at 10(3) CFU/ml and a low cross reactivity with Salmonella serotype typhimurium. The sensitivity of the SPR was similar when testing spiked broiler meat samples. However, research is still needed to reduce the high background observed when sampling meat products.     

Epitope mapping analysis of apolipoprotein B-100 using a surface plasmon resonance-based biosensor

Using a surface plasmon resonance (SPR)-based biosensor (BIA-technology), we have studied the interaction of ten different murine monoclonal antibodies (mAbs, all IgG₁), raised against the main protein constituent of human low density lipoprotein (LDL), i.e. the apolipoprotein B-100 (apoB-100). These mAbs identify distinct domains on apoB-100, relevant to LDL-receptor interaction: epitopes in the amino-terminal region (mAbs L7, L9, L10 and L11: aa 1–1297) and in the middle region (mAb 6B: aa 1480–1693; mAbs 2A, 3B: aa 2152–2377; mAbs 9A, L2 and L4: aa 2657–3248) of native apoB-100. A multisite binding analysis was performed to further characterize the epitopes recognized by all these mAbs. A rabbit anti-mouse IgG₁-Fc antibody (RAM.Fc) was first coupled to the gold surface in order to capture one anti-human apoB-100 mAb. ApoB-100 protein was subsequently injected and allowed to react with this immobilized, oriented antibody. Multisite binding assays were then performed, by sequentially flowing other mAbs, in different orders, over the sensing surface. The capacity of each mAb to interact with the entrapped apoB-100 in a multimolecular complex was monitored in real time by SPR. The results achieved were comparable to those obtained by western immunoblotting using the same reagents. However, SPR ensures a more detailed epitope identification, demonstrating that BIA-technology can be successfully used for mapping distinct epitopes on apoB-100 protein in solution dispensing with labels and secondary tracers; moreover, compared with conventional immunoassays, it is significantly time saving (CNR-P.F. MADESS 2).     

High-throughput analysis of mumps virus and the virus-specific monoclonal antibody on the arrays of a cationic polyelectrolyte with a spectral SPR biosensor

We investigated the potential use of a spectral surface plasmon resonance (SPR) biosensor in a high-throughput analysis of mumps virus and a mumps virus-specific mAb on the arrays of a cationic polyelectrolyte, poly(diallyldimethylammonium chloride) (PDDA). The PDDA surface was constructed by electrostatic adsorption of the polyelectrolyte onto a monolayer of 11-mercaptoundecanoic acid (MUA). Poly-L-lysine was also adsorbed onto the MUA monolayer and compared with the PDDA surface in the capacity of mumps virus immobilization. The PDDA surface showed a higher adsorption of mumps virus than the poly-L-lysine surface. The SPR signal caused by the virus binding onto the PDDA surface was proportional to the concentration of mumps virus from 0.5 x 10(5) to 14 x 10(5) pfu/mL. The surface structure of the virus arrays was visualized by atomic force microscopy. Then, a dose-dependent increase in the SPR signal was observed when various concentrations of the antimumps virus antibody in buffer or human serum were applied to the virus arrays, and their interaction was specific. Thus, it is likely that the spectral SPR biosensor based on the cationic polyelectrolyte surface may provide an efficient system for a high-throughput analysis of intact virus and serodiagnosis of infectious diseases.