Affinity between immobilised monoclonal and polyclonal antibodies and steroid-enzyme tracers increases sharply at high surface density

Giraudi, Gianfranco; Rosso, Ilaria; Baggiani, Claudio; Giovannoli, Cristina

doi:10.1016/s0003-2670(98)00749-1

Cited by 12 publications

(11 citation statements)

References 14 publications

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“…54 It is also known that increased density of the immobilized probe molecules on a surface can affect the affinity constant for probe-analyte interactions by, in most cases, increasing the affinity by slowing the dissociation rates. 55,56,57 We hypothesize that the above-mentioned factors all contribute to the difference in the antibody behavior observed in the two instances. Different surface densities of the antibodies can result in altered saturation thickness ( t max ) values for the proteins and affect the absolute reflectance values observed with our sensor, but the process of normalization to the saturation reflectance R max corrects for such changes, and hence the model itself is unaltered.…”

Section: Resultsmentioning

confidence: 94%

Validation of Arrayed Imaging Reflectometry Biosensor Response for Protein–Antibody Interactions: Cross-Correlation of Theory, Experiment, and Complementary Techniques

et al. 2011

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One of the critical steps in the development of an analytical technique is to confirm that its experimental response correlates with predictions derived from the theoretical framework on which it is based. This validates the technique quantitatively, and, in the case of a biosensor, facilitates a correlation of the sensor’s output signal to the concentration of the analyte being tested. Herein we report studies demonstrating that the quantitative response of Arrayed Imaging Reflectometry (AIR), a highly sensitive label-free biosensing method, is a predictable function of probe and analyte properties. We first incorporated a standard one-site Langmuir binding model describing probe-analyte interactions at the surface into the theoretical model for thickness-dependent reflectance in AIR. This established a hypothetical correlation between analyte concentration and the AIR response. Spectroscopic ellipsometry, surface plasmon resonance (SPR) and AIR were then used to validate this model for two biomedically important proteins, fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor (VEGF). While our studies demonstrated that the 1:1 one-site Langmuir model accurately described the observed response of macro spot AIR arrays, either a two-site Langmuir model or a Sips isotherm better described the behavior of AIR microarrays. These studies confirmed the quantitative performance of AIR across a range of probe-analyte affinities. Furthermore, the methodology developed here can be extended to other label-free biosensing platforms, thus facilitating a more accurate and quantitative interpretation of the sensor response.

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Section: Resultsmentioning

confidence: 94%

Validation of Arrayed Imaging Reflectometry Biosensor Response for Protein–Antibody Interactions: Cross-Correlation of Theory, Experiment, and Complementary Techniques

et al. 2011

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“…The cortisol-3-CMO-HRP conjugate was prepared according to a method previously reported in literature, 17 with minor modifications. Equimolar amounts of F-3-CMO, N,N'-diisopropylcarbodiimide and N-hydroxysuccinimide were mixed under vigorous stirring in dry cold 1,4-dioxane (about 5 mL/mmol of steroid).…”

Section: Preparation Of the Enzyme Tracermentioning

confidence: 99%

Development of a biomimetic enzyme-linked immunosorbent assay based on a molecularly imprinted polymer for the detection of cortisol in human saliva

et al. 2019

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“…As opposed to the p53 peptide, the HLA peptide had an estimated half maximal binding of 3.3 n m , suggesting that monoclonals may bind random sequence peptides with a dynamic range of several orders of magnitude, and the array allows detection of even relatively weak interactions. Although solution affinity of the random peptides for an antibody may be quite low, the apparent affinity for some peptides on the array is very high, presumably because of surface effects 33 .…”

Section: Resultsmentioning

confidence: 99%

Physical Characterization of the “Immunosignaturing Effect”

Stafford

Halperin

Legutki

et al. 2012

Molecular & Cellular Proteomics

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Identifying new, effective biomarkers for diseases is proving to be a challenging problem. We have proposed that antibodies may offer a solution to this problem. The physical features and abundance of antibodies make them ideal biomarkers. Additionally, antibodies are often elicited early in the ontogeny of different chronic and infectious diseases. We previously reported that antibodies from patients with infectious disease and separately those with Alzheimer's disease display a characteristic and reproducible “immunosignature” on a microarray of 10,000 random sequence peptides. Here we investigate the physical and chemical parameters underlying how immunosignaturing works. We first show that a variety of monoclonal and polyclonal antibodies raised against different classes of antigens produce distinct profiles on this microarray and the relative affinities are determined. A proposal for how antibodies bind the random sequences is tested. Sera from vaccinated mice and people suffering from a fugal infection are individually assayed to determine the complexity of signals that can be distinguished. Based on these results, we propose that this simple, general and inexpensive system could be optimized to generate a new class of antibody biomarkers for a wide variety of diseases.

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Affinity between immobilised monoclonal and polyclonal antibodies and steroid-enzyme tracers increases sharply at high surface density

Cited by 12 publications

References 14 publications

Validation of Arrayed Imaging Reflectometry Biosensor Response for Protein–Antibody Interactions: Cross-Correlation of Theory, Experiment, and Complementary Techniques

Validation of Arrayed Imaging Reflectometry Biosensor Response for Protein–Antibody Interactions: Cross-Correlation of Theory, Experiment, and Complementary Techniques

Development of a biomimetic enzyme-linked immunosorbent assay based on a molecularly imprinted polymer for the detection of cortisol in human saliva

Physical Characterization of the “Immunosignaturing Effect”

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