Antibody orientation on biosensor surfaces: a minireview

Trilling, Anke K.; Beekwilder, Jules; Zuilhof, Han

doi:10.1039/c2an36787d

Cited by 377 publications

(322 citation statements)

References 83 publications

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“…18 The demand for a higher binding capacity for analytes and the need for decreased sample consumption while performing precise analyte quantification are leading to the application of these functionalized glass slides in single-cell microarrays. 22,23 In this study, we evaluated the feasibility of protein-G-coated glass surfaces in terms of their antibody-binding capacity and resulting sensitivity by employing these surfaces to obtain single-cell secretory profiles of cytokines and by comparing these results with those obtained from experiments with epoxy-coated glass slides. We hypothesized that the use of protein-G-coated slides would be advantageous because of their affinity for the fragment crystallizable (F c ) portion of antibodies.…”

Section: Introductionmentioning

confidence: 99%

Enhanced detection of single-cell-secreted proteins using a fluorescent immunoassay on the protein-G-terminated glass substrate

Jeong¹,

Lee²,

Park³

et al. 2015

IJN

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We present an evaluation of protein-G-terminated glass slides that may contain a suitable substrate for aligning the orientation of antibodies to obtain better binding moiety to the target antigen. The results of the protein-G-terminated slides were compared with those obtained with epoxy-based slides to evaluate signal enhancement for human immunoglobulin G (IgG) targets, and an increase in the average fluorescence intensity was observed for the lowest measurable amount of IgG target in the assay using protein-G-terminated slides. Applying this strategy for signal amplification to single-cell assays improves the limits of detection for human IgG protein and cytokines (interleukin-2 and interferon-γ) captured from hybridomas. Our data indicate that protein-G-terminated slides have a higher binding capacity for antigens and have better spot-to-spot consistency than that of traditional epoxy-based slides. These properties would be beneficial in the detection of fine amounts of single-cell-secreted proteins, which may provide key insights into cell–cell communication and immune responses.

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Section: Introductionmentioning

confidence: 99%

Enhanced detection of single-cell-secreted proteins using a fluorescent immunoassay on the protein-G-terminated glass substrate

Jeong¹,

Lee²,

Park³

et al. 2015

IJN

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“…168 Additionally, camelid antibodies with one single domain for antigen binding (known as VHH) have attracted attention due to their high solubility and stability, and may provide an opportunity for incorporation into sensors. 8 Also, peptides and aptamers provide a highly customizable method for producing covalent attachment of antibodies, or fragments thereof, either by active targeting of the protein, or the substrate, or both. In the future, it may be seen that metallic thin films are used to produce homogeneous substrates that can be targeted simply by endogenous antibody epitopes or material binding peptides coupled to Fc specific aptamers or peptides.…”

Section: Discussionmentioning

confidence: 99%

“…Disulfide-bridged cysteines, such as those present in the hinge region of antibodies, can also be targeted by reducing agents such as tris(2-carboxyethyl)phosphine (TCEP) or 2-mercaptoethylamine (2-MEA) to form reactive thiols, 8,53 which may subsequently react with maleimide or iodoacetyl activated surfaces. However, as the cysteines are internal to the antibody tertiary structure, covalent attachment via this method can disrupt the conformation of the antibody 54 while steric hindrance may limit antigen binding.…”

Section: Thiol Groupsmentioning

confidence: 99%

“…Further, sites for targeted immobilization (including natural or non-natural amino acids) can be introduced recombinantly to antibodies. [8][9][10] Physical adsorption of antibodies onto traditional immunoassay solid supports, such as polystyrene, occurs via hydrophobic and electrostatic interactions. 11 While this method offers the simplest attachment pathway, it is uncontrollable, and antibodies can be immobilized in a randomly oriented manner, denatured, or displaced in later steps by washing.…”

Section: Introductionmentioning

confidence: 99%

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Orientation and characterization of immobilized antibodies for improved immunoassays (Review)

et al. 2017

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Orientation of surface immobilized capture proteins, such as antibodies, plays a critical role in the performance of immunoassays. The sensitivity of immunodiagnostic procedures is dependent on presentation of the antibody, with optimum performance requiring the antigen binding sites be directed toward the solution phase. This review describes the most recent methods for oriented antibody immobilization and the characterization techniques employed for investigation of the antibody state. The introduction describes the importance of oriented antibodies for maximizing biosensor capabilities. Methods for improving antibody binding are discussed, including surface modification and design (with sections on surface treatments, three-dimensional substrates, self-assembled monolayers, and molecular imprinting), covalent attachment (including targeting amine, carboxyl, thiol and carbohydrates, as well as “click” chemistries), and (bio)affinity techniques (with sections on material binding peptides, biotin-streptavidin interaction, DNA directed immobilization, Protein A and G, Fc binding peptides, aptamers, and metal affinity). Characterization techniques for investigating antibody orientation are discussed, including x-ray photoelectron spectroscopy, spectroscopic ellipsometry, dual polarization interferometry, neutron reflectometry, atomic force microscopy, and time-of-flight secondary-ion mass spectrometry. Future perspectives and recommendations are offered in conclusion.

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“…Biosensors are analytical devices that measure binding of analytes to surfaces, a binding biomolecules (e.g., DNA, protein, enzyme, and antibody) induce a signal in a physichochemical transducer [1][2][3][4]. Several developments in biosensors have been reported, such as fluidic design [5][6][7], sensor surface immobilization [8][9][10][11], advances in detection methods [12][13][14][15], and data analysis [15][16].…”

Section: Introductionmentioning

confidence: 99%

Effect of alkanethiol molecular structure on sensitivity of surface plasmon resonance sensor

Suherman

Morita

Kawaguchi

2015

Sensors and Actuators B: Chemical

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This work aims to find the key factor determining the detection limit for an indirect competitive inhibition immunoassay using surface plasmon resonance (SPR) sensing. In our previous work, the thiol solution concentration used in a self-assembly process highly affected the alkanethiol monolayer structure on the sensor surface [Suherman et al., 2014]. It was noticed that the monolayer structure determined the immunoassay sensitivity due to the orientation and the surface concentration of antigen in domain structure of monolayer. To study the effect of orientation of antigen, we examined here three types of alkanethiol compounds: dithiobis(succinimidyl undecanoate) (DSU; straight-chain alkanethiol), carboxy-EG 6 -undecanethiol (CEG 6 ; flexible-chain alkanethiol), and(C 2 -NTA; three-branched alkanethiol). By electrochemical reductive desorption, the surface concentration of DSU, CEG 6 , and C 2 -NTA were estimated to be 6.6 × 10 -10 , 8.5 × 10 -10 , 4.6 × 10 -10 mol cm -2 , respectively. Subsequently, SPR suggested that the ratios of immobilized antigen (clenbuterol) per thiol were 0.13, 0.16, and 0.15 for DSU, CEG 6 , and C 2 -NTA, respectively. This suggests that the amount of immobilized clenbuterol was defined by the molecular size of clenbuterol. Sensor surface structures on a molecular scale were evaluated using high-resolution scanning tunneling microscopy (STM), which showed tilted clenbuterol with θ DSU > θ CEG6 > θ C2-NTA . For SPR sensing of clenbuterol, C 2 -NTA showed the highest sensitivity (LOD = 10 ppt) among the examined alkanethiols, because clenbuterol formed a laid-on structure. Based on kinetics, the key factor for sensing performance is discussed.2

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Antibody orientation on biosensor surfaces: a minireview

Cited by 377 publications

References 83 publications

Enhanced detection of single-cell-secreted proteins using a fluorescent immunoassay on the protein-G-terminated glass substrate

Enhanced detection of single-cell-secreted proteins using a fluorescent immunoassay on the protein-G-terminated glass substrate

Orientation and characterization of immobilized antibodies for improved immunoassays (Review)

Effect of alkanethiol molecular structure on sensitivity of surface plasmon resonance sensor

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