Electrochemical impedimetric immunosensor for insulin like growth factor-1 using specific monoclonal antibody-nanogold modified electrode

An electrochemical impedimetric immunosensor was developed for ultrasensitive determination of insulin-like growth factor-1 (IGF-1) based on immobilization of a specific monoclonal antibody on gold nanoparticles (GNPs) modified gold electrode. Self-assembly of colloidal gold nanoparticles on the gold electrode was conducted through the thiol groups of 1,6-hexanedithiol (HDT) monolayer as a cross linker. The redox reactions of [Fe(CN)(6)](4-)/[Fe(CN)(6)](3-) on the electrode surface was probed for studying the immobilization and determination processes, using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The interaction of antigen with grafted antibody recognition layer was carried out by soaking the modified electrode into antigen solution at 37°C for 3 h. The immunosensor showed linearity over 1.0-180.0 pg mL(-1) and the limit of detection was 0.15 pg mL(-1). The association constant between IGF-1 and immobilized antibody was calculated to be 9.17×10(11) M(-1). The proposed method is a useful tool for screening picogram amounts of IGF-1 in clinical laboratory as a diagnostic test.     

Application of electrochemical impedance spectroscopy for monitoring allergen-antibody reactions using gold nanoparticle-based biomolecular immobilization method

Gold nanoparticles were used to enhance the immobilization amount and retain the immunoactivity of recombinant dust mite allergen Der f2 immobilized on a glassy carbon electrode (GCE). The interaction between allergen and antibody was studied by electrochemical impedance spectroscopy (EIS). Self-assembled Au colloid layer (?=16nm) deposited on (3-mercaptopropyl)trimethoxysilane (MPTS)-modified GCE offered a basis to control the immobilization of allergen Der f2. The impedance measurements were based on the charge transfer kinetics of the [Fe(CN)(6)](3-/4-) redox pair, compared with bare GCE, the immobilization of allergen Der f2 and the allergen-antibody interaction that occurred on the electrode surface altered the interfacial electron transfer resistance and thereby slowed down the charge transfer kinetics by reducing the active area of the electrode or by preventing the redox species in electrolyte solution from approaching the electrode. The interactions of allergen with various concentrations of monoclonal antibody were also monitored through the change of impedance response. The results showed that the electron transfer resistance increased with increasing concentrations of monoclonal antibody.     

Atomic force spectroscopy-based study of antibody pesticide interactions for characterization of immunosensor surface

Development of immunobiosensor detector surfaces involves the immobilization of active antibodies on the capture surface without any significant loss of antigen binding activity. An atomic force microscope (AFM) was used to directly evaluate specific interactions between pesticides and antibodies on a biosensor surface. Oriented immobilization of antibodies against two herbicide molecules 2,4-dichlorophenoxyacetic acid (2,4-D) and atrazine, on gold, was carried out to create the active immunobiosensor surfaces. The adhesive forces between immobilized antibodies and their respective antigens were measured by force spectroscopy using hapten-carrier protein functionalized AFM cantilevers. Relative functional affinity (avidity) measurements of the antibodies carried out prior to immobilization, well correlated with subsequent AFM force measurement observations. Analysis showed that immobilization had not compromised the reactivity of the surface immobilized antibody molecules for antigen nor was there any change in their relative quality with respect to each other. The utility of the immunoreactive surface was further confirmed using a Surface Plasmon Resonance (SPR) based detection system. Our study indicates that AFM can be utilized as a convenient immunobiosensing tool for confirming the presence and also assessing the strength of antibody-hapten interactions on biosensor surfaces under development.     

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.     

Factors affecting the specific activity of immobilized antibodies and their biologically active fragments

Factors affecting the specific activity of immobilized antibodies and their biologically active fragments were studied with goat anti-mouse and goat anti-human immunoglobulin G. Antibodies were immobilized on HW 65 polymeric support matrix activated with carbonyldiimidazole, hydrazide and iodoacetic acid. The most significant factors influencing the specific activity of stochastic coupling of antibodies are multisite attachment, multiple orientations and steric hindrance imposed by crowding of antibody and the size of the antigen. In oriented immobilization the specific activity is affected only by steric hindrance. The specific activity of immunosorbents prepared by immobilization of F(ab′) fragments can be improved to almost 100% by limiting the amount of protein immobilization and the size of the antigen. The present study shows the protocols for optimizing immobilized antibody performance.     

Optimizing immobilization on two-dimensional carboxyl surface: pH dependence of antibody orientation and antigen binding capacity

The performance of immunosensors is highly dependent on the amount of immobilized antibodies and their remaining antigen binding capacity. In this work, a method for immobilization of antibodies on a two-dimensional carboxyl surface has been optimized using quartz crystal microbalance biosensors. We show that successful immobilization is highly dependent on surface pK_a, antibody pI, and pH of immobilization buffer. By the use of EDC/sulfo-NHS (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride/N-hydroxysulfosuccinimide) activation reagents, the effect of the intrinsic surface pK_a is avoided and immobilization at very low pH is therefore possible, and this is important for immobilization of acidic proteins. Antigen binding capacity as a function of immobilization pH was studied. In most cases, the antigen binding capacity followed the immobilization response. However, the antigen-to-antibody binding ratio differed between the antibodies investigated, and for one of the antibodies the antigen binding capacity was significantly lower than expected from immobilization in a certain pH range. Tests with anti-Fc and anti-Fab₂ antibodies on different antibody surfaces indicated that the orientation of the antibodies on the surface had a profound effect on the antigen binding capacity of the immobilized antibodies.     

抗体固定化方法研究进展

在免疫分析和生物芯片中,抗原-抗体特异性结合被广泛应用,其中抗体的固定化是研发高效诊断和分离工具的关键环节。生物分子工程、材料化学与交联剂化学的进步极大地促进了抗体固定化技术的发展。 抗体可以通过物理吸附、共价偶联和亲和相互作用固定到不同类型的固相表面。 抗体固定化的目标是以一种正确的空间取向将抗体固定到固相表面,在完全保留抗体构象和活性的同时最大化抗原的结合能力,这对固相化抗体的分析性能至关重要。 对固定抗体到固相载体表面的各种最新方法进行了阐述,包括物理吸附法,通过羧基、氨基、巯基、糖基和点击化学的共价结合法以及基于生物亲和作用的固定法,并对固定化抗体的表征方法进行了归纳,最后对抗体固定化方法的发展方向进行了展望。     

Detection of polyclonal antibody against any area of the protein-antigen using immobilized protein-antigens: the critical role of the immobilization protocol

Antigens immobilized on solid supports may be used to detect or purify their corresponding antibodies (Ab) from serum. Direct immobilization of antigens on support surfaces (through short spacer arms) may promote interesting stabilizing effects on the immobilized antigen. However, the proximity of the support may prevent the interaction of some fractions of polyclonal Ab with some regions of the antigen (those placed in close contact with the support surface). Horseradish peroxidase (HRP) was immobilized on agarose by different protocols of multipoint covalent immobilization involving different regions of the antigen surface. Glyoxyl-agarose, BrCN-agarose, and glutaraldehyde-agarose were used as activated supports. Each HRP-immobilized preparation was much more stable than the soluble enzyme, but it was only able to adsorb up to 60-70% of a mixture of polyclonal anti-HRP antibodies. On the other hand, HRP was also immobilized on agarose through a very long, flexible, and hydrophilic spacer arm (dextran). This immobilized HRP was hardly stabilized, but it was able to adsorb 100% of the polyclonal anti-HRP. The absence of steric hindrances seems to play a critical role favoring the complete recognition of all classes of polyclonal Ab. Another solution to achieve a complete adsorption of polyclonal Ab on immobilized-stabilized antigens has been also reached by using a mixture of the differently immobilized and stabilized HRP-agarose preparations. In this case, an improved storage and operational stabilities of the immobilized antigens can be combined with the complete adsorption of any class of antibody.     

Optimization of methods for immobilizing antibodies against the carcino-embryonic antigen on insoluble matrices. Equilibrium parameters of the interaction of the immobilized antibodies with the antigen

To obtain highly efficient immunosorbents for solid-phase immunoassay and affinity chromatography, methods for immobilization of antibodies against the carcino-embryonic antigen (CEA) on insoluble matrices were optimized. The immunosorbents obtained were characterized by equilibrium parameters of the reaction between immobilized anti-CEA and CEA calculated from rather a simple kinetic model. This model describes the interaction of the monovalent antigen with two independent types of binding sites. The role of some amino acid residues of anti-CEA in the interaction with CEA was investigated. The effects of immobilization density and the spacer arm length on the functional properties of the immobilized antibodies were studied. The optimal immunosorbent was used to purify 125I-CEA by immunoaffinity chromatography.     

Immobilization of specific antibody on SAM functionalized gold electrode for rabies virus detection by electrochemical impedance spectroscopy

The rabies constitutes one of the most dangerous viruses causing many death cases every year. Each year approximately 55,000 people die of rabies, with high percentage of children. High percentages (99%) of the registered cases were in Asia and Africa. In order to fight this dangerous disease, many techniques are usually used for diagnostic but are usually complex, heavy, expensive, difficult to implement, requiring high-qualified personnel, and this is a necessity of developing new detection process. In this work, we describe the development of an immunological sensor based on functionalized gold electrode allowing rabies antigen detection. The biosensor is based on the immobilization of specific rabies antibodies onto functionalized gold microelectrode. The affinity interaction of the immobilized antibody with the specific antigen can be measured with low limit detection and with a good reproducibility with impedance spectroscopy. The non-specific interaction has been tested using the Newcastle antigen.     

Comparison of antibody functionality using different immobilization methods

This study investigates the influence of antibody immobilization methods on antigen capture. Adsorption and two surface chemistries, an aminosilane chemistry and a common heterobifunctional crosslinker (N-gamma-maleimidobutyryloxy-succinimide ester, GMBS), were compared and evaluated for their ability to immobilize antibodies and capture antigen. The role of protein A as an orienting protein scaffold component in each of these techniques was also evaluated. Through experimentation it was determined that the GMBS technique immobilized the highest amount of antibody and minimized nonspecific binding. For all techniques, the most functional antibodies were found to be those immobilized with protein A. Interestingly, the aminosilane technique demonstrated the highest antigen capture with antibody alone but also exhibited the highest level of nonspecific binding.     

In situ deprotection: a method for covalent immobilization of peptides with well-defined orientation for use in solid phase immunoassays such as enzyme-linked immunosorbent assay.

The orientation of an immobilized antigen is important for recognition by, e.g., an antibody. When noncovalent passive adsorption is used for immobilization, the number of ways that the antigen can attach to the surface is numerous and control of how the antigen orientates on the surface is limited. Covalent immobilization restricts the number of the ways that the antigen can be immobilized to the number of reactive groups on the antigen and, hence, the orientation of the immobilized antigen is more predictable. Peptide antigens were synthesized and purified with protection groups on the lysine and cysteine side chains. These peptides, which have only one good nucleophilic group (the N-terminal alpha-amino group), were immobilized covalently in microtiter plates supplied with tresyl groups on the surface and the protection groups were cleaved off in situ after immobilization. The controlled orientation of these peptides resulted in enhanced recognition by antibodies in general. An enzyme-linked immunosorbent assay for detection of antibodies against a peptide derived from outer surface protein C from Borrelia burgdorferi, found in Lyme borreliosis patients, was established using this strategy. Lyme borreliosis suspect patient sera showed up to a 10-fold increase in the signal when the orientation of the peptide antigen was controlled by the in situ deprotection strategy.     

Optimization of methods of immobilization of anti-immunoglobulins. Equilibrium parameters of the interaction of immobilized antibodies with an antigen

Methods for immobilization of anti-immunoglobulins on insoluble supports were optimized, and the interaction of immunoadsorbents obtained with [125I]-labeled rabbit IgG was investigated. It was shown that this interaction can be adequately described by a rather simple equilibrium model which reflects the interaction of a monovalent antigen with two independent types of binding sites. Within the framework of this model the association constants as well as the concentrations of high affinity binding sites which influence the capacity and efficiency of the separation system were determined. Optimization of the immobilization methods implicated a study on the role of certain functional groups of the antibody involved in the formation of covalent bonds, on the effect of the spacer arm length on the properties of immobilized antibody as well as on the role of the degree of immobilization. It was found that immunoadsorbents obtained after antibody immobilization via lysine or tyrosine residues on matrices with a specific spacer group are the optimal ones.     

Comparison of surface and hydrogel-based protein microchips

Protein microchips are designed for high-throughput evaluation of the concentrations and activities of various proteins. The rapid advance in microchip technology and a wide variety of existing techniques pose the problem of unified approach to the assessment and comparison of different platforms. Here we compare the characteristics of protein microchips developed for quantitative immunoassay with those of antibodies immobilized on glass surfaces and in hemispherical gel pads. Spotting concentrations of antibodies used for manufacturing of microchips of both types and concentrations of antigen in analyte solution were identical. We compared the efficiency of antibody immobilization, the intensity of fluorescence signals for both direct and sandwich-type immunoassays, and the reaction-diffusion kinetics of the formation of antibody-antigen complexes for surface and gel-based microchips. Our results demonstrate higher capacity and sensitivity for the hydrogel-based protein microchips, while fluorescence saturation kinetics for the two types of microarrays was comparable.     

Polymer nanoparticles covered with phosphorylcholine groups and immobilized with antibody for high-affinity separation of proteins

Novel polymer nanoparticles were prepared for the selective capture of a specific protein from a mixture with high effectiveness. The nanoparticle surface was covered with hydrophilic phosphorylcholine groups and active ester groups for easy immobilization of antibodies. Phospholipid polymers (PMBN) composed of 2-methacryloyloxyethyl phosphorylcholine, n-butyl methacrylate, and p-nitrophenyloxycarbonyl polyethyleneglycol methacrylate, were synthesized for the surface modification of poly( l-lactic acid) nanoparticles. Surface analysis of the nanoparticles using laser-Doppler electrophoresis and X-ray photoelectron spectroscopy revealed that the surface of nanoparticles was covered with PMBN. Protein adsorption was evaluated with regard to the nonspecific adsorption on the nanoparticles that was effectively suppressed by the phosphorylcholine groups. The immobilization of antibodies on nanoparticles was carried out under physiological conditions to ensure specific binding of antigens. The antibody immobilized on the nanoparticles exhibited high activity and strong affinity for the antigen similar to that exhibited by an antibody in a solution. The selective binding of a specific protein as an antigen from a protein mixture was relatively high compared to that observed with conventional antibody-immobilized polymer nanoparticles. In conclusion, nanoparticles having both phosphorylcholine and active ester groups for antibody immobilization have strong potential for use in highly selective separation based on the biological affinities between biomolecules.     

蛋白质微阵列检测抗原-抗体相互作用

为了制备蛋白质微阵列和研究芯片表面抗原-抗体的相互作用,研究了如何在玻片表面固化蛋白质和用荧光染料（Cy3,Cy5）对蛋白质进行标记.结果表明,在醛基修饰的玻璃表面,通过共价偶联的方法将抗原或抗体固定到芯片表面,能使二者保持其特异性结合能力.同时,荧光标记后的抗原或抗体仍然具有特异性结合能力.蛋白质微阵列是通过机械手在玻片表面排阵制作的.芯片上的荧光信号获取采用了激光共焦荧光扫描系统.用不同浓度的抗原探针阵列,对其相应的抗体靶分子的特异性结合进行了分析和研究.此外,还通过在玻片表面固定兔IgG和固定鼠IgG,对羊抗兔和羊抗鼠抗体与其相应抗原的特异性相互作用进行了检测.     

Highly sensitive detection of cocaine using a piezoelectric immunosensor

This paper describes the development of a highly sensitive competitive immunoassay with the piezoelectric sensor. The immobilized derivative of cocaine was benzoylecgonine-1,8-diamino-3,4-dioxaoctane (BZE-DADOO). For the immobilization of BZE-DADOO, the conjugate BZE-DADOO with 11-mercaptomonoundecanoic acid (MUA) was synthesized via 2-(5-norbornen-2,3-dicarboximide)-1,1,3,3-tetramethyluronium-tetrafluoroborate (TNTU), followed by the creation of the conjugate monolayer on the piezosensor electrodes. For the optimization of the competitive assay we used electrodes with rough or smooth gold areas and for the interaction with immobilized antigen different anti-cocaine sheep polyclonal (pAb, either whole IgG or Fab fragment) and mouse monoclonal (mAb, whole IgG) antibodies. The assay of cocaine developed achieved a detection limit (LOD) of 100 pmol/l (34 ng/l) using the sheep antibody (IgG) and piezoelectric sensors with a smooth gold surface. The total time of one analysis was 15 min and the measuring area of the sensor could be used more than 40 times without losing its sensitivity.     

Study on orientation of immunoglobulin G on protein G layer

A comparative study of immunoglobulin G (IgG) immobilization was performed, both on a thiolated protein G layer, where this immobilization was due to affinity binding with an Fc fragment of IgG, and on 11-mercaptoundecanoic acid (11-MUA), where the immobilization was due to chemical bonding. The change of IgG layer formation on the two base layers as a function of the IgG concentration was investigated by surface plasmon resonance (SPR), atomic force microscopy (AFM) in a non-contact mode, and spectroscopic ellipsometry (SE). It was observed that the IgG layer was immobilized more evenly on the thiolated protein G layer than on the 11-MUA layer, based on the SPR measurements. The surface topology analysis by AFM indicated that the IgG layer was immobilized on the protein G layer according to the envelope profile of the base layer. Based on the SE analysis, it was determined that the IgG layer thickness on the thiolated protein G layer increased with increasing IgG concentration. Based on the above analyses, the scheme for orientation of IgG immobilized on the thiolated protein G layer was proposed.     

The use of Fab-masking antigens to enhance the activity of immobilized antibodies

This study demonstrates that masking the Feb regions of a monoclonal antibody (Mab) with synthetic antigens prior to covalent immobilization efficiency. Water-soluble adducts of poly(2-methyloxazoline) polymers and a syntheticpeptide epitope for the Mab were constructed. These synthetic antigens are referred to as Fab-masking antigents (FMAs). The antibody used in this study is a Ca(2+)-dependent murine monoclonal lgG directed against the plasma protein, human protein C (hPC). The FMAs were pre-equilibrated with Mab in the presence of calcium prior to immobilization and were then removed by EDTA, which destabilized the FMA-Mab complexes. The antigen binding efficiency and accessibility of the Fab domain of the immobilized antibody was significantly increased for Mab immobilized in the presence of FMA relative to those Mab immobilized without FMA. The increase in binding efficiency was most pronounced for the largest FMA employed. No appreciable differences were detected in the avidity of hPC-Mab complexes formed by immunosorbents produced by either masked or unmaked antibody. These results provide evidence that orientgation may play an important role in the binding activity of immobilized antibodies.     

3D antibody immobilization on a planar matrix surface

The amount of active capture antibodies immobilized per unit square is crucial to developing effective antibody chips, biosensors, immunoassays and other molecular recognition technologies. In this study, we present a novel yet simple method for oriented antibody immobilization at high density based on the formation of an orderly, organized aggregation of immunoglobulin G (IgG) and staphylococcal protein A (SPA). Following the chelation of His-tag with Ni(2+), antibodies were immobilized on a solid surface in a three-dimensional (3D) manner and exposed the analyte receptor sites well, which resulted in a substantial enhancement of the analytical signal with more than 64-fold increase in detection sensitivity. Pull-down assays confirmed that IgG antibody can only bind to Ni(2+) matrix indirectly via a SPA linkage, where the His-tag is responsible for binding Ni(2+) and homologous domains are responsible for binding IgG Fc. The immobilization approach proposed here may be an attractive strategy for the construction of high performance antibody arrays and biosensors as long as the antibody probe is of mammalian IgG.