Electroimmobilization of proinsulin C-peptide to a quartz crystal microbalance sensor chip for protein affinity purification

Proinsulin C-peptide was electroimmobilized to a quartz crystal microbalance sensor chip, localizing this low-pI peptide for covalent attachment to activated surface carboxyl groups. The resulting chip was used in a continuous flow biosensor to capture anti-C-peptide antibodies, which could subsequently be eluted in 5% formic acid between air bubbles for efficient recovery and mass spectrometric identification. The method is reproducible through repeated cycles, providing affinity purification of proteins under real-time monitoring of the binding and elution processes.     

Use of a novel micro-fluidic device to create arrays for multiplex analysis of large and small molecular weight compounds by surface plasmon resonance

There is an increasing demand to develop biosensor monitoring devices capable of biomarker profiling for predicting animal adulteration and detecting multiple chemical contaminants or toxins in food produce. Surface plasmon resonance (SPR) biosensors are label free detection systems that monitor the binding of specific biomolecular recognition elements with binding partners. Essential to this technology are the production of biochips where a selected binding partner, antibody, biomarker protein or low molecular weight contaminant, is immobilised. A micro-fluidic immobilisation device allowing the covalent attachment of up to 16 binding partners in a linear array on a single surface has been developed for compatibility with a prototype multiplex SPR analyser. The immobilisation unit and multiplex SPR analyser were respectively evaluated in their ability to be fit-for-purpose for binding partner attachment and detection of high and low molecular weight molecules. The multiplexing capability of the dual technology was assessed using phycotoxin concentration analysis as a model system. The parent compounds of four toxin groups were immobilised within a single chip format and calibration curves were achieved. The chip design and SPR technology allowed the compartmentalisation of the binding interactions for each toxin group offering the added benefit of being able to distinguish between toxin families and perform concentration analysis. This model is particularly contemporary with the current drive to replace biological methods for phycotoxin screening.     

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.     

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.     

Surface plasmon resonance immunosensor for the detection of Salmonella typhimurium

An immunosensor based on surface plasmon resonance (SPR) using protein G was developed for the detection of Salmonella typhimurium. A protein G layer was fabricated by binding chemically to self-assembly monolayer (SAM) of 11-mercaptoundecanoic acid (MUA) on gold (Au) surface. The formation of protein G layer on Au surface modified with 11-MUA and the binding of antibody and antigen in series were confirmed by SPR spectroscopy. The effect of detergent such as Tween-20 on binding efficiency of antibody and antigen was investigated by SPR. The binding efficiency of antigen to the antibody immobilized on Au surface was improved up to about 85% and 100% by using protein G and Tween-20, respectively. The surface morphology analyses of 11-MUA monolayer on Au substrate, protein G layer on 11-MUA monolayer and antibody layer immobilized on protein G layer were performed by atomic force microscope (AFM). Consequently, an immunosensor based on SPR for the detection of S. typhimurium using protein G was developed with a detection range of 10(2) to 10(9)CFU/ml. The current fabrication technique of a SPR immunosensor for the detection of S. typhimurium could be applied to construct other immnosensors or protein chips.     

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.     

Rapid analysis of protein interactions: On-chip micropurification of recombinant protein expressed in Esherichia coli

We describe a rapid analysis of interactions between antibodies and a recombinant protein present in total cell lysates. Using a surface plasmon resonance biosensor, a low concentration of glutathione-S-transferase (GST) fused protein expressed in small scale Esherichia coli culture was purified on an anti-GST antibody immobilized sensor chip. The 'on-chip purification' was verified using matrix-assisted laser desorption/ionization-time of flight mass spectrometry by measuring the molecular masses of recombinant proteins purified on the sensor chip. The specific binding of monoclonal antibodies for the on-chip micropurified recombinant proteins can then be monitored, thus enabling kinetic analysis and epitope mapping of the bound antibodies. This approach reduced time, resources and sample consumption by avoiding conventional steps related to concentration and purification.     

Automated SPR-LC-MS/MS system for protein interaction analysis

We have developed a novel automated system to analyze protein complexes by integrating a surface plasmon resonance (SPR) biosensor with highly sensitive nanoflow liquid chromatography-tandem mass spectrometry (LC-MS/MS). A His-tagged protein, which is also tagged with FLAG and biotinylated sequences, was expressed in mammalian cells. After purification by using the His tag from the cell lysate, the sample protein mixture was applied to an SPR biosensor and the protein complex was captured on the sensor chip. The automated SPR-LC-MS/MS was then performed: (1) two-step on-chip purification of the protein complex by using the FLAG and the biotinylated tags, (2) on-chip protease digestion of the complex, and (3) online nanoflow LC-MS/MS analysis of the resulting peptide fragments for protein identification. All of these processes could be monitored in real-time by the SPR biosensor. We validated the performance of the system using either FK506-binding protein 52 kDa (FKBP52) or ribosomal protein S19 (rpS19) as bait. Thus, the fully automated SPR-LC-MS/MS system appeared to be a powerful tool for functional proteomics studies, particularly for snapshot analysis of functional cellular complexes and machines.     

Optimization of a calcium-dependent Protein A-derived domain for mild antibody purification

ABSTRACT

As reported here, we developed and optimized a purification matrix based on a Protein A-derived domain, Z_Ca, displaying calcium-dependent antibody binding. It provides an alternative to the acidic elution conditions of conventional Protein A affinity chromatography for purification of sensitive antibodies and other Fc-based molecules. We describe the multimerization of Z_Ca to generate a chromatography resin with higher binding capacity. The highest order multimeric variant, Z_CaTetraCys, demonstrated a considerably high dynamic binding capacity (35 mg IgG/ml resin) while preserving the specificity for IgG. High recovery was obtained and host cell protein and DNA content in purified fractions proved to be comparable to commercial MabSelect SuRe and MabSelect PrismA. Various elution conditions for use of this domain in antibody purification were investigated. The purification data presented here revealed variations in the interaction of different subclasses of human IgG with Z_CaTetraCys. This resulted in diverse elution properties for the different IgGs, where complete elution of all captured antibody for IgG2 and IgG4 was possible at neutral pH. This optimized protein ligand and the proposed purification method offer a unique strategy for effective and mild purification of antibodies and Fc-fusion proteins that cannot be purified under conventional acidic elution conditions due to aggregation formation or loss of function.     

Highly sensitive and selective surface plasmon resonance sensor for detection of sub-ppb levels of benzo[a]pyrene by indirect competitive immunoreaction method

A surface plasmon resonance (SPR)-immunosensor for detection of benzo[a]pyrene (BaP) is developed by using a model BaP-hapten compound, BaP-bovine serum albumin conjugate (BaP-BSA), and an anti-BaP-BSA monoclonal antibody. BaP-BSA conjugate is immobilized on a gold thin-film sensor chip by means of simple physical adsorption. The number of BaP-hapten units in BaP-BSA conjugate is estimated to be 28 from the difference in molecular weight (MW) between BaP-BSA conjugate and BSA based on the results of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) measurement. Anti-BaP-BSA antibody on contact with the BaP-BSA conjugate immobilized sensor chip causes an increase in the incident angle of the sensor chip. Binding of anti-BaP-BSA antibody with surface-immobilized BaP-BSA conjugate is inhibited by the presence of BaP in analyte solution, because of the inhibition effect of BaP. The SPR immunosensor for BaP functioning with the indirect competitive immunoreaction of anti-BaP-BSA antibody between the analyte (BaP) in testing solution and the BaP-BSA conjugate immobilized on the sensor chip provides a rapid determination (response time: ca. 15 min) of BaP in the concentration range of 0.01-1000 ppb. The antibody anchored to the sensor chip by antigen-antibody binding is removed on treatment with a pepsin solution (pH 2.0) for few minutes. The SPR sensor chip is found to be reusable for more than 20 times with a little decrease (<7%) in the sensor response. Detection of BaP by direct competitive immunoreactions is also carried out by enzyme-linked immunosorbent assay (ELISA). The concentration of BaP could be determined as low as 0.01 ppb and 2 ppb using the SPR sensor and the ELISA method, respectively. The SPR sensor is found to detect BaP selectively in the presence of 2-hydroxybiphenyl (HBP); the incident angle shift of the SPR sensor for BaP is found to be same irrespective to the presence or the absence of a same concentration (as much as 30 ppb) of HBP together.     

Surface plasmon resonance immunosensor using self-assembled protein G for the detection of Salmonella paratyphi

A surface plasmon resonance (SPR) based immunosensor using self-assembled protein G was developed for the detection of Salmonella paratyphi. In order to endow a solid substrate binding affinity to protein G, the free amine (-NH2) of protein G was substituted into thiol (-SH) using 2-iminothiolane. Thus, self-assembled protein G was fabricated on gold (Au) substrate. The formation of protein G layer on Au surface, and the binding of antibody and antigen in series were confirmed by SPR spectroscopy. The surface morphology analysis of the protein G layer on Au surface was performed by atomic force microscope (AFM). Consequently, an immunosensor based on SPR for the detection of S. paratyphi using self-assembled protein G was developed with a detection range of 10(2)-10(7) CFU/ml. The current fabrication technique of a SPR immunosensor for the detection of S. paratyphi could be applied to construct other immnosensors or protein chips.     

Analysis of native proteins from biological fluids by biomolecular interaction analysis mass spectrometry (BIA/MS): exploring the limit of detection, identification of non-specific binding and detection of multi-protein complexes

Biomolecular interaction analysis mass spectrometry (BIA/MS) is a two-dimensional analytical technique that quantitatively and qualitatively detects analytes of interests. In the first dimension, surface plasmon resonance (SPR) is utilized for detection of biomolecules in their native environment. Because SPR detection is non-destructive, analyte(s) retained on the SPR-active sensor surface can be analyzed in a second dimension using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The qualitative nature of the MALDI-TOF MS analysis complements the quantitative character of SPR sensing and overcomes the shortcomings of the SPR detection stemming from the inability to differentiate and characterize multi-protein complexes and non-specific binding. In this work, the benefit of performing MS analysis following SPR sensing is established. Retrieval and detection of four markers present in biological fluids (cystatin C, beta-2-microglobulin, urinary protein 1 and retinol binding protein) was explored to demonstrate the effectiveness of BIA/MS in simultaneous detection of clinically related biomarkers and delineation of non-specific binding. Furthermore, the BIA/MS limit of detection at very low SPR responses was investigated. Finally, detection of in-vivo assembled protein complexes was achieved for the first time using BIA/MS.     

Characterization of a self-assembled monolayer of thiol on a gold surface and the fabrication of a biosensor chip based on surface plasmon resonance for detecting anti-GAD antibody

A biosensor chip utilizing surface plasmon resonance (SPR) was fabricated for detecting anti-glutamic acid decarboxylase (GAD) antibody, which is an indicator of the presence of type I diabetes mellitus. The sensor surfaces were constructed from various thiol mixtures of different molar ratios of 3-mercaptopropionic acid (3-MPA) to 11-mercaptoundecanoic acid (11-MUA). To determine the surface characteristics of the different alkanethiol monolayers, several quantitative and kinetic measurements were carried out. The extent of immobilization of streptavidin (SA) and biotin-GAD (the anti-GAD receptor) and the immune response of anti-GAD antibody against GAD were measured using the SPR biosensor. The terminal functional group of a thiol has different effects on the adsorption and covalent binding of protein depending on the steric hindrance. The protein chip described herein permits simple, rapid detection of anti-GAD antibody.     

Characterization of antibody-antigen interactions: comparison between surface plasmon resonance measurements and high-mass matrix-assisted laser desorption/ionization mass spectrometry

The interaction between the bovine prion protein (bPrP) and a monoclonal antibody, 1E5, was studied with high-mass matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) and surface plasmon resonance (SPR). In the case of MS a cross-linking stabilization was used prior to the analysis, whereas for SPR the antibody was immobilized and bPrP was injected. We compared the determination of parameters such as the epitope, the kinetics and binding strength, and the capacity of the antigen to bind two different antibodies. The two methods are highly complementary. SPR measurements require a lower amount of sample but are more time-consuming due to all of the necessary side steps (e.g., immobilization, regeneration). High-mass MALDI MS needs a higher overall amount of sample and cannot give direct access to the kinetic constants, but the analysis is faster and easier compared with SPR.     

Surface plasmon resonance analysis of interactions between diacylglycerol acyltransferase and its interacting molecules

To measure the interactions of diacylglycerol acyltransferase (DGAT) by surface plasmon resonance (SPR), we immobilized Saccharomyces cerevisiae DGAT2 encoded by DGA1 on a BIACORE sensor chip surface. We used N-terminally truncated Dga1p with a FLAG tag at the C-terminus, which was purified to apparent homogeneity, maintaining significant DGAT activity (Kamisaka et al., Appl. Microbiol. Biotechnol., 88, 105-115 (2010)). Truncated Dga1p with a FLAG tag was immobilized with an anti-FLAG antibody that had been coupled with an L1 chip surface consisting of a carboxymethyl dextran matrix with additional hydrophobic alkane groups. The Dga1p-immobilized chip surface was analyzed for interactions of Dga1p with oleoyl-CoA, its substrate, and anti-Dga1p IgG, its interacting protein, by SPR. The binding of these analytes with the Dga1p-immobilized chip surface was specific, because butyryl-CoA, which cannot be used as a substrate for DGAT, and anti-glyceraldehyde-3-phosphate dehydrogenase IgG, did not induce any signals on SPR. Furthermore, injection of organic compounds such as xanthohumol, a DGAT inhibitor, into the Dga1p-immobilized chip surface induced significant SPR signals, probably due to interaction with DGAT. Another DGAT inhibitor, piperine, did not induce SPR signals on application, but induced them due to piperine on application together with oleoyl-CoA, in which piperine can be incorporated into the micelles of oleoyl-CoA. The results indicate that the Dga1p-immobilized L1 chip surface recognized DGAT inhibitors. Taking all this together, SPR measurement using the Dga1p-immobilized L1 chip surface provided a useful system to elucidate the structure-function relationships of DGAT and screen DGAT inhibitors.     

In-process monitoring of protein purification with thin film silicon sensor technology

We have developed a rapid and sensitive thin film assay for in-process monitoring of target protein purification. This novel biosensor method provides rapid (5-min) visual evaluation of column purification fractions. The method can be used to monitor the efficiency of purification and potential loss of protein if the column binding capacity is exceeded. The eluted fractions containing the highest yield of target protein can be quickly identified, pooled, and processed. This convenient platform, known as the SILAS product, is a thin-film detection technology in which specific molecular interactions are transduced into visible color changes based on changes in the optical thickness of layers on a silicon surface. The results are interpreted without instrumentation. Proteins eluted from a purification column are adsorbed to the assay surface, and the ligand of interest (target) can be identified with specific binding reagents. Here we demonstrate two protein purification applications for the SILAS technology product: monitoring antibody elution from a Protein G column and evaluating the efficiency of purification of a glutathione-S-transferase (GST)-tagged recombinant protein through each step of the purification process.     

Immunosensor for the detection of Vibrio cholerae O1 using surface plasmon resonance

An immunosensor for the detection of Vibrio cholerae O1 was developed on the basis of surface plasmon resonance (SPR). A protein G layer was fabricated by means of the chemical coupling between the free amine (-NH₂) groups of protein G and the activated carboxyl groups present on a self-assembled monolayer (SAM) consisting of a mixture of 11-mercaptoundecanoic acid (MUA) and hexanethiol (molar ratio of 1:2). A monoclonal antibody, which was confirmed to be specific to V. cholera O1 by the Western blotting technique, was immobilized on the protein G layer. The formation of the SAM, the protein G layer and the sequential binding of the antibody against V. cholera O1 were investigated with SPR spectroscopy. As the number of fabricated layers increased, the minimum angle of plasmon resonance was increased accordingly. The target bacteria, V. cholera O1, was measured with the fabricated immunosensor, whose detection range was between 10⁵ and 10⁹ cells/mL.     

Trypsin purification by affinity binding to small unilamellar liposomes

A novel protein purification process using affinity-ligand-modified liposomes and membrane ultrafiltration is described. The feasibility of the process was tested using trypsin as the model protein and p-aminobenzamidine (PAB) as the affinity ligand for trypsin. The affinity liposomes were prepared by covalently attaching PAB to the surface of small unilamellar liposomes via the hydrophilic spacer arm diglycolic acid. The liposomes were comprised of dimyristoyl phosphatidyl choline, cholesterol, and dimyristoyl phosphatidyl ethanolamine to which the diglycolic acid was attached. The equilibrium binding constant between trypsin and immobilized PAB was shown to be dependent on the PAB density of the liposome surface. Bound trypsin was eluted from the liposomes by the trypsin inhibitor benzamidine. Trypsin was purified from a trypsin/chymotrypsin mixture and from one of its naturally occurring sources, porcine pancreatic extract. A recovery yield from the crude mixture of 68% was obtained with a trypsin purity of 98%. The affinity-modified liposomes were stable in the complex mixture and retained their trypsin binding capacity after multiple adsorption/elution cycles over a 30-day period.     

Surface plasmon resonance imaging analysis of hexahistidine-tagged protein on the gold thin film coated with a calix crown derivative

A surface plasmon resonance (SPR) imaging system was constructed and used to detect the hexahistidine-ubiquitin-tagged human parathyroid hormone fragment (His₆-Ub-hPTHF(1–34)) expressed inEscherichia coli. The hexahistidine-specific antibody was immobilized on a thin gold film coated with ProLinker^TM B, a novel calixcrown derivative with a bifunctional coupling property that permits efficient immobilization of capture proteins on solid matrices. The soluble and insoluble fractions of anE. coli cell lysate were spotted onto the antibody-coated gold chip, which was then washed with buffer (pH 7.4) solution and dried. SPR imaging measurements were carried out to detect the expressed His₆-Ub-hPTHF (1–34). There was no discernible protein image in the uninduced cell lysate, indicating that non-specific binding of contaminant proteins did not occur on the gold chip surface. It is expected that the approach used here to detect affinity-tagged recombinant proteins using an SPR imaging technique could be used as a powerful tool for the analyses of a number of proteins in a high-throughput mode.     

Optimizing recombinant antibody function in SPR immunosensing. The influence of antibody structural format and chip surface chemistry on assay sensitivity

BACKGROUND: Recombinant antibody fragments are valuable tools for SPR-based detection of small molecules such as illicit drugs. However, the multiple structural formats of recombinant antibody fragments are largely uncharacterised with respect to their respective performance in SPR sensing. We have expressed a model anti-M3G antibody in both scFv and chimeric Fab formats to examine its sensitivity and binding profiles in a microplate immunoassay format and Biacore. We have further examined the influence of scFv multimerisation, Fab constant region stability and SPR chip surface coating chemistry, on anti-hapten SPR assay development. RESULTS: Under optimised competition ELISA conditions, the anti-M3G scFv was found to have an IC(50) value of 30 ng/ml, while the most stable Fab construct exhibited an IC(50) value of 2.4 ng/ml. In SPR competition assay on an M3G-OVA-coated SPR chip surface, the two constructs again differed in sensitivity, with IC(50) values of 117 and 19 ng/ml for the scFv and Fab, respectively (the scFv also exhibiting poor linearity of response). However, when the SPR chip surface was directly coated with M3G, both antibody constructs exhibited good linearity of response, similar high sensitivity IC(50) values (scFv 30 ng/ml, Fab 14 ng/ml) and high reproducibility (50 effective regenerations for M3G-OVA, 200 for M3G direct). During SPR assay development it was noticed that scFv and Fab constructs gave differing off-rate profiles. Subsequent HPLC, ELISA and electrophoretic analyses then confirmed that a portion of the scFv population multimerises. Bivalent scFv was found to profoundly affect the dissociation curve for scFv in stringent SPR kinetic analyses, leading to a 40-fold difference in calculated off-rate values (Fab off rate 4.7 x 10(-3)S(-1), scFv off rate 1.03 x 10(-2)S(-1)). CONCLUSION: The structural format of recombinant antibody fragments and chip functionalisation methodology can both profoundly affect the function of anti-M3G SPR assay, with direct coating and Fab format proving to be optimal. The confirmation of scFv multimerisation and resulting changes in SPR kinetics profile, in comparison with a Fab, further suggest that caution must be taken in the interpretation of SPR sensorgrams, which are commonly used in the 'affinity ranking' of scFv panels in which the extent of dimerisation in each sample is unknown.