Site-directed antibody immobilization using a protein A–gold binding domain fusion protein for enhanced SPR immunosensing

Juan-Franco, Elena de; Caruz, Antonio; Pedrajas, José Rafael; Lechuga, Laura M.

doi:10.1039/c3an36498d

Cited by 71 publications

(53 citation statements)

References 30 publications

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“…Alternatively, antibody-binding proteins (ABPs, such as proteins A and G of bacterial origin) rapidly bind to the Fc region of antibodies, ensuring their correct orientation [28]. Therefore, SBPs have been fused genetically to ABPs [29,30] to avoid the specific modification of antibodies and to increase versatility. The SBP directs the immobilisation onto specific nanomaterials, while the ABP couples to antibodies and retains their biological activity.…”

Section: Antibodiesmentioning

confidence: 99%

See 1 more Smart Citation

Solid-binding peptides: smart tools for nanobiotechnology

Care

Bergquist

Sunna

2015

Trends in Biotechnology

153

131

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Section: Antibodiesmentioning

confidence: 99%

“…The SBP directs the immobilisation onto specific nanomaterials, while the ABP couples to antibodies and retains their biological activity. This approach has been shown to improve significantly the density and orientation of antibodies immobilised on material surfaces when compared to covalent methods [29].…”

Section: Antibodiesmentioning

confidence: 99%

Solid-binding peptides: smart tools for nanobiotechnology

Care

Bergquist

Sunna

2015

Trends in Biotechnology

153

131

View full text Add to dashboard Cite

“…To date, there have been a few SPR-based assays developed to detect hGH [11][12][13][14] . One particular strategy, known as the isoform method 15 , relies on the detection of the ratio of 22 kDa hGH to total hGH, as non-22-kDa endogenous levels drop after exogenous rhGH administration.…”

Section: Introductionmentioning

confidence: 99%

Comparative Analysis of Human Growth Hormone in Serum Using SPRi, Nano-SPRi and ELISA Assays

Vance

Zeidan

Henrich

et al. 2016

JoVE

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Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml −1 and minimum levels of 0.03 ng ml −1 ) in circulating blood are essential as variable levels may indicate altered physiology. For example, growth disorders occurring in childhood can be diagnosed by measuring levels of hGH in blood. Also, the misuse of recombinant hGH in sports not only poses an ethical issue it also presents serious health threats to the abuser. One popular strategy for measuring hGH misuse, relies on the detection of the ratio of 22 kDa hGH to total hGH, as non-22 kDa endogenous levels drop after exogenous recombinant hGH (rhGH) administration. Surface plasmon resonance imaging (SPRi) is an analytical tool that allows direct (label-free) monitoring and visualization of biomolecular interactions by recording changes of the refractive index adjacent to the sensor surface in real time. In contrast, the most frequently used colorimetric method, enzyme-linked immunosorbent assay (ELISA) uses enzyme labeled detection antibodies to indirectly measure analyte concentration after the addition of a substrate that induces a color change. To increase detection sensitivity, amplified SPRi uses a sandwich assay format and near infrared quantum dots (QDs) to increase signal strength. After direct SPRi detection of recombinant rhGH in spiked human serum, the SPRi signal is amplified by the sequential injection of detection antibody coated with near-infrared QDs (Nano-SPRi). In this study, the diagnostic potential of direct and amplified SPRi was assessed for measuring rhGH spiked in human serum and compared directly with the capabilities of a commercially available ELISA kit.

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“…The current standard methods of immobilizing Ab include physical/electrostatic adsorption, covalent coupling via amine moieties, or affinity binding using affinity tags [4][5][6]. Due to their simplicity of execution, these methods are widely reported and commonly used.…”

Section: Introductionmentioning

confidence: 99%

“…Based on the characteristic mentioned above, numerous methods have been described for achieving oriented immobilization of Ab. The most common one is achieved by protein G or protein A [4,8,9]. These proteins bind specifically to the Fc region of Ab, leaving the Ab available to bind their target Ag and producing well-aligned immobilized Ab with minimal steric hindrance.…”

Section: Introductionmentioning

confidence: 99%

Oriented immobilization of antibody through carbodiimide reaction and controlling electric field

Sun

Feng

et al. 2015

J Solid State Electrochem

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A novel and simple approach for oriented immobilization of antibody (Ab) was proposed through carbodiimide reaction and controlling electric field (CEF). The electrode was modified by self-assembled monolayer of cysteine (CYS SAM) with amino group stretching outside, and Fc region of Ab was activated via carbodiimide reaction. By adjusting pH of solution and applying a proper electric field, Abs were manipulated owing to their charged nature and immobilized on the electrode via the activated Fc region. The stepwise modified electrodes were characterized with cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The results proved that Abs could be successfully immobilized on the surface of electrode. Under optimal experimental parameters, the immunosensor could detect antigen (Ag) in a range from 0.04 to 400 ng/mL. Furthermore, the immunosensors fabricated with and without CEF were comparatively studied, and the results showed that the former had better detection properties than the later. Finally, the effect of CEF on Ab was studied by polarization modulation infrared reflection-absorption spectroscopy (PM-IRRAS) and scanning electron microscope (SEM); the results indicated that Abs were arranged in better order under electric field, this may be the reason for the better properties of the immunosenor. In a word, Ab immobilization through carbodiimide reaction and CEF was very simple, fast, and easy to perform, and it would find wide application in the immune-based assay systems.

show abstract

Site-directed antibody immobilization using a protein A–gold binding domain fusion protein for enhanced SPR immunosensing

Cited by 71 publications

References 30 publications

Solid-binding peptides: smart tools for nanobiotechnology

Solid-binding peptides: smart tools for nanobiotechnology

Comparative Analysis of Human Growth Hormone in Serum Using SPRi, Nano-SPRi and ELISA Assays

Oriented immobilization of antibody through carbodiimide reaction and controlling electric field

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