Sensitivity enhancement of surface plasmon resonance biosensor with colloidal gold

We enhanced the sensitivity of surface plasmon resonance biosensor by the conversion of the real-time direct binding immunoassay into the sandwich immunoassay, in which colloidal gold particles coated with anti-mouse IgG was used. By the immobilization of anti-mouse IgG onto the carboxymethyl dextran surface of thin gold film, the direct binding of analyte (mouse IgG) onto the sensor chip, and the injection of colloidal gold particles coated with antimouse IgG, about 100 times of sensitivity enhancement was obtained. This result suggests that nanoparticles, which has a high refractive index, homogeneous ultrafine structure and capability of size control, would be applicable for the detection of very small quantity of biomaterial.     

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.     

Characterization of the binding epitope of the monoclonal antibody DMAb-1 to ganglioside GM2.

Several derivatives of ganglioside GM2 were synthesized for mapping of the binding epitope of a monoclonal antibody raised against this ganglioside. The GM2 ganglioside was modified in both the hydrophobic and the hydrophobilic part of the molecule. The synthesized derivatives were characterized with fast atom bombardment mass spectrometry (FAB-MS). Affinity of the monoclonal antibody for the GM2 derivatives was determined by enzyme-linked immunosorbent assay (ELISA) on microtitre plates or by TLC immunostaining. Modifying the GM2 sialic acid by deacetylation or blocking of the carboxyl moiety abolished the binding to the monoclonal antibody while the cleaving of the glycol group on the sialic acid tail led to a 70% reduced binding affinity. Removal of the fatty acid (lyso-GM2) eliminated the binding to the antibody. GM2 derivatives with fatty acid moieties of 8 carbon atoms or less showed almost no reactivity. GM2 with saturated fatty acids 16:0, 18:0 and 20:0 had binding affinity similar to natural GM2, while the 24:0 fatty acid had only half the binding affinity. The results demonstrate the importance of ganglioside fatty acid composition with regard to ligand binding between the monoclonal antibody and its specific ganglioside antigen. Thus, caution must be shown in the application of immunaffinity methods with monoclonal antibodies for the quantitative determination of glycosphingolipids from different tissues.     

A metal-chelating piezoelectric sensor chip for direct detection and oriented immobilization of polyHis-tagged proteins

A metal-chelating piezoelectric (PZ) chip for direct detection and controlled immobilization of polyHis-tagged proteins has been demonstrated. The chip was prepared by covalently binding a hydrogel matrix complex of oxidized dextran and nitrilotriacetic acid (NTA) ligand onto an activated alkanethiol-modified PZ crystal. The resulting chip effectively captured Ni2+ ions onto its NTA surface, as disclosed by the resonant frequency shift of the crystal and an X-ray photoelectron spectroscopy analysis. The real-time frequency analysis revealed that the bare NTA chip was nonfouling, regenerable, and highly reusable during continuous repetitive injections of ion solutions and binding proteins. In addition, the chip displayed good long-term reusability and storage stability. The individual binding studies of a polyHis-tagged glutathione-S-transferase and its native untagged form on various metal-charged chips revealed that Co2+, Cu2+, and Ni2+ ions each had different immobilization ability on the NTA surface, as well as their binding ability and selectivity with the tagged protein. As a result, the tagged protein immobilized on the Ni2+-charged chip can actively be bound with its antibody and substrate. Further, the quantitative analyses of the tagged protein in crude cell lysate with a single Ni2+-charged chip and of its substrate with a protein-coated chip were also successfully demonstrated. Therefore, this study initiates the possibilities of oriented, reversible, and universal immobilization of any polyHis-tagged protein and its functional study using a real-time PZ biosensor.     

Immobilization of metallothionein as a sensitive biosensor chip for the detection of metal ions by surface plasmon resonance

A biosensor based on mammalian metallothionein (MT) for the detection of metal ions was developed and characterized. MT was immobilized onto a carboxymethylated dextran matrix as a biosensor for the detection of metal ions by surface plasmon resonance (SPR). The optimal pH for the immobilization step was determined to be 4. The temperature for the analysis was also defined, and the highest interaction was observed at 30 degrees C. The MT sensor chip binds cadmium (Cd), zinc (Zn) or nickel (Ni), but not magnesium (Mg), manganese (Mn) and calcium (Ca). Calibration curves for the quantification of metal ions showed excellent linearity. The sensitivity for metal detection is at the micromolar level. The interaction between the metal ions and the sensor chip is influenced significantly by the presence of NaCl, Tween 20 and the pH of the reaction buffer. By decreasing the NaCl in the reaction buffer to 1 mM, the MT chip effectively differentiates cadmium from zinc and nickel. Kinetic parameters of the metal-MT interactions were also determined by using this chip. The binding affinity between the metal ions and the immobilized MT follows the order of cadmium > zinc > nickel, which is the same as that determined for MT in solution. Thus, the MT chip can be an effective biosensor for the detection and measurement of several metal ions.     

Structure and activity of lipid membrane biosensor surfaces studied with atomic force microscopy and a resonant mirror.

Three variants of the liposome fusion (coalescence) method to produce supported lipid bilayers, containing the ganglioside GM1 on silicon nitride surfaces, were studied. The first procedure involved attachment and fusion of liposomes containing DMPC, GM1 and a small amount of biotinylated lipid (Biotin-LC-DPPE) to a streptavidin coated surface. Direct fusion of liposomes composed of a mixture of DPPC, DPPG, DPPE, GM1 and cholesterol to the surface were the second variant. The final method utilised the second type of liposomes, fused onto a streptavidin layer with a small amount of exposed hydrophobic tails. The methods produced similar lipid layers, but with different ways of attachment to the surface. The binding of cholera toxin B-subunit (CTB) towards these sensor surfaces was measured in a resonant mirror biosensor instrument and the activity and longer-term stability of the layers were examined. The prepared surfaces were also imaged by atomic force microscopy (AFM) in liquid to characterise the topography of the lipid layers. The binding efficiency of CTB towards these surfaces was discussed in terms of lipid fluidity and surface roughness.     

A mouse/human chimeric anti-(ganglioside GD3) antibody with enhanced antitumor activities

Ganglioside GD3, which is one of the major gangliosides expressed on the cell surface of human tumors of neuroectodermal origin has been focused on as a target molecule for passive immunotherapy. We have cloned the cDNA encoding the immunoglobulin light and heavy chains of an anti-GD3 monoclonal antibody KM641 (murine IgG3, ), and constructed the chimeric genes by linking the cDNA fragments of the murine light and heavy variable regions to cDNA fragments of the human and 1 constant regions, respectively. The transfer of these cDNA constructs into SP2/0 mouse myeloma cells resulted in the production of the chimeric antibody, designated KM871, that retained specific binding activity to GD3. Indirect immunofluorescence revealed the same staining pattern for chimeric KM871 and the mouse counterpart KM641 on GD3-expressing melanoma cells. When human serum and human peripheral blood mononuclear cells were used as effectors in complement-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity respectively, the chimeric KM871 was more effective in killing GD3-expressing tumor cells than was the mouse counterpart KM641. Intravenous injection of chimeric KM871 markedly suppressed tumor growth in nude mice. The chimeric KM871, having enhanced antitumor activities and less immunogenicity than the mouse counterpart, would be a useful agent for passive immunotherapy of human cancer.     

Novel approach to analyzing RNA aptamer-protein interactions: toward further applications of aptamers

Surface plasmon-resonance analysis using a Biacore biosensor is a powerful tool for the detailed study of biomolecular interactions. The authors examined the methods of immobilizing proteins on the surface of NTA, SA, and CM5 sensor chips to study RNA aptamer-protein interactions. RNA aptamers and their deletion variants were loaded onto a protein-immobilized sensor chip, and their binding affinities were analyzed. Immobilizing the protein on a CM5 sensor chip via an anti-His-tag antibody was the only strategy that clearly detected the kinetic parameters of the interactions. DeltaNEO-III-14U, one of the deletion variants of the NS3 aptamer, had the highest binding affinity for the deltaNS3 protein in this study (KD = 4 x 10(-8)). Moreover, the 29-amino-acid spacer fragment was essential for protein immobilization using this strategy. This novel method will be useful in comparing the affinity of various RNA aptamers and selecting the most suitable candidates for a given target, as well as facilitating the in vitro selection procedure itself.     

Affibody protein capture microarrays: synthesis and evaluation of random and directed immobilization of affibody molecules

Affibody molecules, 58-amino acid three-helix bundle proteins directed to different targets by combinatorial engineering of staphylococcal protein A, were used as capture ligands on protein microarrays. An evaluation of slide types and immobilization strategies was performed to find suitable conditions for microarray production. Two affibody molecules, Z(Taq) and Z(IgA), binding Taq DNA polymerase and human IgA, respectively, were synthesized by solid phase peptide synthesis using an orthogonal protection scheme, allowing incorporation of selective immobilization handles. The resulting affibody variants were used for random surface immobilization (through amino groups) or oriented surface immobilization (through cysteine or biotin coupled to the side chain of Lys58). Evaluation of the immobilization techniques was carried out using both a real-time surface plasmon resonance biosensor system and a microarray system using fluorescent detection of Cy3-labeled target protein. The results from the biosensor analyses showed that directed immobilization strategies significantly improved the specific binding activity of affibody molecules. However, in the microarray system, random immobilization onto carboxymethyl dextran slides and oriented immobilization onto thiol dextran slides resulted in equally good signal intensities, whereas biotin-mediated immobilization onto streptavidin-coated slides produced slides with lower signal intensities and higher background staining. For the best slides, the limit of detection was 3 pM for IgA and 30 pM for Taq DNA polymerase.     

一种葡聚糖芯片的再生方法、表征及其应用*

表面等离子体共振（surface plasmon resonance, SPR）生物传感器,作为一种适时快捷,无需标记的生物分子相互作用研究工具,已广泛应用于生物化学分析与研究。羧甲基化葡聚糖修饰的CM5传感芯片是Biacore 系列仪器应用最为普遍的核心部件,目前CM5芯片主要从法玛西亚公司购买,价格昂贵,且一旦共价交联的受体分子失活,就不能重复利用。阐述了一种简便、低成本、用于SPR生物传感器的葡聚糖修饰金膜芯片的再生方法及其表征和应用。用此方法再生的芯片能被循环伏安法和原子力显微镜很好地表征,并成功地用于抗前列腺特异性抗原(prostate-specific antigen,PSA)固定和PSA检测, 同时测定了PSA与其抗体之间的动力学和亲和常数。     

Quantitative analysis of EGFR affinity to immobilized glycolipids by surface plasmon resonance

EGF-induced activation of EGFR tyrosine kinase is known to be inhibited by ganglioside GM3, its dimer, and other mimetics. However, details of the interaction, such as kinetic properties, have not yet been clarified. The direct interaction is now defined by the surface plasmon resonance (SPR) technique. To determine the affinity of EGFR for lyso-GM3 or lyso-GM3 mimetic, these glycolipid ligands were covalently immobilized onto a sensor chip, and binding affinities were investigated. Results of these studies confirmed the direct interaction of lyso-GM3 or its mimetic with EGFR. A strong interaction between EGFR and lyso-GM3 or its mimetic was indicated by increased binding of EGFR to glycolipid-immobilized surface, in an EGFR dose-dependent manner.     

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.     

Controlled antibody immobilization onto immunoanalytical platforms by synthetic peptide

Antibody immobilization on a solid surface is inevitable in the preparation of immunochips/sensors. Antibody-binding proteins such as proteins A and G have been extensively employed to capture antibodies on sensor surfaces with right orientations, maintaining their full functionality. Because of their synthetic versatility and stability, in general, small molecules have more advantages than proteins. Nevertheless, no small molecule has been used for oriented and specific antibody immobilization. Here is described a novel strategy to immobilize an antibody on various sensor surfaces by using a small antibody-binding peptide. The peptide binds specifically to the Fc domain of immunoglobulin G (IgG) and, therefore, affords a properly oriented antibody surface. Surface plasmon resonance analysis indicated that a peptide linked to a gold chip surface through a hydrophilic linker efficiently captured human and rabbit IgGs. Moreover, antibodies captured by the peptide exhibited higher antigen binding capacity compared with randomly immobilized antibodies. Peptide-mediated antibody immobilization was successfully applied on the surfaces of biosensor substrates such as magnetic particles and glass slides. The antibody-binding peptide conjugate introduced in this work is the first small molecule linker that offers a highly stable and specific surface platform for antibody immobilization in immunoassays.     

Structure and molecular interactions of a unique antitumor antibody specific for N-glycolyl GM3

N-glycolyl GM3 ganglioside is an attractive target antigen for cancer immunotherapy, because this epitope is a molecular marker of certain tumor cells and not expressed in normal human tissues. The murine monoclonal antibody 14F7 specifically recognizes N-glycolyl GM3 and shows no cross-reactivity with the abundant N-acetyl GM3 ganglioside, a close structural homologue of N-glycolyl GM3. Here, we report the crystal structure of the 14F7 Fab fragment at 2.5 A resolution and its molecular model with the saccharide moiety of N-glycolyl GM3, NeuGcalpha3Galbeta4Glcbeta. Fab 14F7 contains a very long CDR H3 loop, which divides the antigen-binding site of this antibody into two subsites. In the docking model, the saccharide ligand is bound to one of these subsites, formed solely by heavy chain residues. The discriminative feature of N-glycolyl GM3 versus N-acetyl GM3, its hydroxymethyl group, is positioned in a hydrophilic cavity, forming hydrogen bonds with the carboxyl group of Asp H52, the indole NH of Trp H33 and the hydroxyl group of Tyr H50. For the hydrophobic methyl group of N-acetyl GM3, this environment would not be favorable, explaining why the antibody specifically recognizes N-glycolyl GM3, but not N-acetyl GM3. Mutation of Asp H52 to hydrophobic residues of similar size completely abolished binding. Our model of the antibodycarbohydrate complex is consistent with binding data for several tested glycolipids as well as for a variety of 14F7 mutants with replaced VL domains.     

Kinetic analysis of antibody-antigen interactions at a supported lipid monolayer

Modified phospholipids possessing carboxyl head groups synthesized from phosphatidylethanolamine were incorporated into supported lipid monolayers on top of a thin gold film. A monoclonal antibody was chemically coupled to the modified lipids in these monolayers and the kinetics of antigen binding were determined by surface plasmon resonance. The binding could be analyzed using a conventional 1:1 binding algorithm and the derived kinetic and affinity constants were almost identical to those reported for the same interaction on a dextran hydrogel-based sensor chip. When an antigen was chemically coupled to a modified lipid monolayer, the binding of a monoclonal antibody to this surface was biphasic. A two-step algorithm describing the formation of a 1:2 antibody:antigen complex was developed which accurately described the data and enabled differentiation of the two binding steps. The binding was assayed varying both the concentration of antibody in solution and the density of antigen on the surface. The affinities determined by Scatchard analysis of equilibrium binding levels were similar to those values obtained from an ELISA.     

An interleukin-6 ZnO/SiO(2)/Si surface acoustic wave biosensor

A novel high sensitivity ZnO/SiO(2)/Si Love mode surface acoustic wave (SAW) biosensor for the detection of interleukin-6 (IL-6), is reported. The biosensors operating at 747.7MHz and 1.586GHz were functionalized by immobilizing the monoclonal IL-6 antibody onto the ZnO biosensor surface both through direct surface adsorption and through covalent binding on gluteraldehyde. The morphology of the IL-6 antibody-protein complex was studied using scanning electron microscopy (SEM), and the mass of the IL-6 protein immobilized on the surface was measured from the frequency shift of the SAW resonator biosensor. The biosensor was shown to have extended linearity, which was observed to improve with higher sensor frequency and for IL-6 immobilization through the monoclonal antibody. Preliminary results of biosensor measurements of low levels of IL-6 in normal human serum are reported. The biosensor can be fully integrated with CMOS Si chips and developed as a portable real time detection system for the interleukin family of proteins in human serum.     

Development of a micro-planar amperometric bile acid biosensor for urinalysis

The determination of bile acid concentration in urine is useful for the screening and diagnosis of various hepatobiliary diseases. Currently, there is no concise method to determine bile acid concentration in urine. This study describes a bile acid biosensor fabricated by electrochemical technique for urinalysis. The micro-planar electrodes employed for the study consisted of a working electrode (platinum), a counter electrode (platinum) and a reference electrode (silver/silver chloride (Ag/AgCl)). The sensor chip was coated with Nafion using a spin-coater in order to both eliminate many interference species in urine and achieve long-term stability of the reference electrode. Nafion coating allowed the sensor chip to prevent the electrode reaction from interference species in urine, because it is charged negative strongly (Nafion contains sulfonic acid group). Three enzymes (bile acid sulfate sulfatase: BSS, beta-hydroxysteroid dehydrogenase: beta-HSD, and NADH oxidase: NHO) were immobilized by glutaraldehyde (GA: cross-linker) onto the sensor chip, because the immobilization of enzymes by GA is simple and commonly carried out. The sensor chip was able to detect bile acid in buffer solution. The optimum enzyme ratio immobilized onto the sensor chip was BSS:beta-HSD:NHO=4:4:20 U/1 chip. There was a relationship between the concentration of bile acid and the response current value. The dynamic range of the sensor chip was 2-100 microM for bile acid. Additionally, bile acid in the urine specimen could be detected using this bile acid biosensor. We present a simple and rapid bile acid biosensor with high sensitivity and high reproducibility.     

Therapeutic potential of chimeric anti-(ganglioside GD3) antibody KM871: antitumor activity in xenograft model of melanoma and effector function analysis

KM871 is a chimeric antibody recognizing ganglioside GD3, which is one of the major gangliosides expressed on the cell surface of human tumors of neuroectodermal origin. This study demonstrates the antitumor activity of KM871 against human melanoma xenografts in nude mice, and analyzes the effector function operating in mice. In a well-established tumor model, KM871 showed antitumor activity against H-15 and SK-MEL-28 human melanoma but not against H-187 and G361 human melanoma when administered intravenously 5 days/week for 2 weeks. The G361 tumor became sensitive when KM871 was first administered on the day of tumor inoculation. In this assay, it was observed that almost all the mice were tumor-free, but a few mice developed tumors. Therefore, we examined the amount and expression pattern of GD3 antigen on G361 tumors escaping from KM871 treatment, but no change was observed. Next we examined the optimal administration schedule for KM871 in mice, using H-15 melanoma. KM871 showed antitumor activity when administered intravenously either 5 days/week for 2 weeks or three biweekly doses. However, the effect of the former schedule was stronger than three biweekly doses. To compare the effector function in humans and mice, we studied the complement-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity and antibody-dependent macrophage-mediated cytotoxicity of KM871 using complement or effector cells prepared from humans and mice. It was found that the antibody-dependent cell-mediated cytotoxicity exerted by polymorphonuclear cells and antibody-dependent macrophage-mediated cytotoxicity were the only antitumor mechanism of KM871 in mice. However their action was very weak compared with that in humans, and complement-mediated cytotoxicity, which was strong in humans, was not observed in mice. Therefore, the antitumor activity of KM871 against human melanomas evaluated by the nude mouse model might be underestimated. These results indicate that KM871 shows good antitumor activity against GD3-positive human melanoma and the antitumor activity expected in humans might be superior to that of the nude mouse model. Received: 10 July 1999 / Accepted: 21 January 2000     

Non-labeled detection of waterborne pathogen Cryptosporidium parvum using a polydiacetylene-based fluorescence chip

A non-labeling fluorescence sensor system was developed using polydiacetylene (PDA) liposomes composed of 10,12-pentacosadiynoic acid (PCDA) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) at a 8:2 molar ratio. The PDA liposomes were immobilized onto an amine-coated glass surface using peptide bonding between the carboxyl group of the liposome and the amine group of the glass surface. The optimum ratio of the cross linker (NHS/EDC) to PDA liposome was determined to be 50% for strong immobilization of the liposomes. Residual carboxyl groups of the PDA liposomes were selectively biotinylated, followed by sequential binding of streptavidin and biotin-antibody (bioreceptor). Finally, the performance of the PDA liposome chip was tested for detecting Cryptosporidium parvum, and yielded a detection limit of 1 x 10(3) oocysts/mL. From these results, it is expected that the PDA liposome chip will have high application potential for the detection of waterborne pathogens including C. parvum.     

High-density immobilization of an antibody fragment to a carboxymethylated dextran-linked biosensor surface

There are numerous chemical methods published that enable protein coupling to carboxymethyl (CM) dextran. Here we have taken traditional amine coupling using N-hydroxysuccinimide (NHS) and N'-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and coupled an antibody fragment (scFv) to CM dextran at a very high density. Using an upgraded BIAlite from Biacore AB, more than 7000 RU of scFv was coupled to a CM dextran biosensor chip. In addition, scanning electron microscopy was performed on CM dextran biosensor chips following amine coupling of 30 nm gold anti-IgG particles. This showed that amine coupling was uniform across the biosensor chip surface. Calculations show that 7620 RU of an scFv coupled to such a surface results in a mean distance between binding sites of 8.8 nm. This equates to a packing volume of approximately 20% of the available space occupied by the antibody fragment. Comparisons made with densities of covalently coupled IgG show that a greater number of antibody fragment molecules can be coupled per unit area. This is most likely due to the smaller size of an antibody fragment (scFv), which has a volume of less than 20% of an IgG molecule. The significance of these findings is discussed.