Whole blood screening of antibodies using label-free nanoparticle biophotonic array platform.

Journal ArticleResearch Support, Non-U.S. Gov'tCopyright © 2012 Published by Elsevier B.V.A gold nanoparticle, localised plasmon array biosensor using light scattering has been employed in the detection of allergen-specific antibodies in whole blood and sera. The array sensor was functionalized with four different allergens, cat dander (Fel d1), dust mite (Der p1), peanut allergen (Ara h1) and dog dander (Can f1) and immuno-kinetic assay was performed to detect their respective anti-allergen IgG antibodies. Specific positive responses to antibodies at a concentration of 25 nM were observed for Fel d1, Der p1, and Ara h1 allergens, while the Can f1 channel served as a reference control. The sensitivity was further enhanced using a secondary anti-IgG detection antibodies to give a limit of detection of 2 nM. The results indicate the potential for nanoparticle scattering multiplexed arrays to screen unprepared blood samples at point-of-care for assays of complex samples such as the whole blood.BBSR

Differential immuno-kinetic assays of allergen-specific binding for peanut allergy serum analysis.

Journal ArticleResearch Support, Non-U.S. Gov'tCopyright © Springer-Verlag 2012A label-free nanoparticle array platform has been used to detect total peanut allergen-specific binding from whole serum of patients suffering from peanut allergy. The serum from 10 patients was screened against a four-allergen panel of cat and dog dander, dust mite and peanut allergen protein Ara h1. The IgE and IgG contributions to the total specific-binding protein load to Ara h1 were identified using two secondary IgG- and IgE-specific antibodies and were found to contribute less than 50 % of the total specific protein load. The total mass of IgE, IgE and the unresolved specific-binding protein ΔsBP for Ara h1 provides a new serum profile for high-RAST-grade patients 5 and 6 with the IgG/IgE ratio of 4 ± 2 and ΔsBP/IgE ratio of 17 ± 11, neither of which is protective for the small patient cohort.BBSRCEPSR

Accuracy and Precision Analysis for a Biophotonic Assay of C-Reactive Protein

This is the author accepted manuscript. The final version is available from the Royal Society of Chemistry via the DOI in this recordA multiplexed biophotonic assay platform has been developed using the localised particle plasmon in gold nanoparticles assembled in an array and functionalised for two assays: total IgG and C-reactive protein (CRP). A protein A/G (PAG) assay, calibrated with a NIST reference material, shows a maximum surface coverage of max = 7.13 ± 0.19 mRIU, equivalent to 1.5 ng mm-2 of F(ab)-presenting antibody. The CRP capture antibody has an equivalent surface binding density of max = 2.95 ± 0.41 mRIU indicating a 41% capture antibody availability. Free PAG binding to the functionalised anti-CRP surface shows that only 47 ± 3 % of CRP capture antibodies are correctly presenting Fab regions for antigen capture. The accuracy and precision of the CRP sensor assay was assessed with 54 blood samples containing spiked CRP in the range 2 – 160 mg/L. The mean accuracy was 0.42 mg/L with Confidence Interval (CI) at 95% from -14.7 to 13.8 mg/L and the precision had a Coefficient of Variation (CV) of 10.6% with 95% CI 0.9% - 20.2%. These biophotonic platform performance metrics indicate a CRP assay with 2 - 160 mg/L dynamic range, performed in 8 minutes from 5 L of whole blood without sample preparation.Attomarker Ltd

Label-free Fab and Fc affinity/avidity profiling of the antibody complex half-life for polyclonal and monoclonal efficacy screening

The final publication is available at Springer via http://dx.doi.org/10.1007/s00216-015-8897-6A unified approach to affinity screening for Fab and Fc interactions of an antibody for its antigen and FcγR receptor has been developed. An antigen array is used for the Fab affinity and cross-reactivity screening and protein A/G proxy is the FcγR receptor. The affinities are derived using a simple 1:1 binding model with a consistent error analysis. The association and dissociation kinetics are measured over optimised times for accurate determination. The Fab/Fc affinities are derived for ten antibodies: mAb-actin (mouse), pAb-BSA (sheep), pAb-collagen V (rabbit), pAb-CRP (goat), mAb-F1 (mouse), mAbs (mouse) 7.3, 12.3, 29.3, 36.3 and 46.3 raised against LcrV in Yersinia pestis. The rate of the dissociation of antigen–antibody complexes relates directly to their immunological function as does the Fc-FcγR complex and a new half-life plot has been defined with a Fab/Fc half-life range of 17–470 min. The upper half-life value points to surface avidity. Two antibodies that are protective as an immunotherapy define a Fab half-life >250 min and an Fc half-life >50 min as characteristics of ideal interactions which can form the basis of an antibody screen for immunotherapy.Biotechnology and Biological Sciences Research Council (BBSRC

Measurement of the localised plasmon penetration depth for gold nanoparticles using a non-invasive bio-stacking method.

Journal ArticleCopyright © 2013 Royal Society of ChemistryWe have used the formation of a bio-probe stack with up to 24 steps on gold nanoparticle and continuous gold surfaces to characterize the penetration depth of the plasmon field in a non-invasive manner by only involving biomolecules from standard bio-assays. An alternating anti-goat rabbit IgG and anti-rabbit IgG bio-probe stack is polymerized on protein A/G functionalized gold surfaces. The change in plasmon excitation angle or light scattering decreases exponentially with each stacking step although the bio-integrity of the antibody epitope is maintained. The exponential decay in the derived kinetic parameters is attributed to the change in the penetration depth and the step size is calibrated using a commercial continuous gold surface plasmon resonance surface to be 17.5 ± 0.8 nm, consistent with the expected dimension of the antibody. The penetration depth of the gold spherical nanoparticles of diameter 90 ± 13 nm is determined to be 93 ± 10 nm.BBSR

Glycosylation characterization of human and porcine fibrinogen proteins by lectin-binding biophotonic microarray imaging.

Journal ArticleResearch Support, Non-U.S. Gov'tCopyright © 2013 American Chemical SocietyLectin binding has been studied using the particle plasmon light-scattering properties of gold nanoparticles printed into an array format. Performance of the kinetic assay is evaluated from a detailed analysis of the binding of concanavalin A (ConA) and wheat germ agglutinin (WGA) to their target monosaccharides indicating affinity constants in the order of KD ∼10 nM for the lectin-monosaccharide interaction. The detection limits for the lectins following a 200 s injection time were determined as 10 ng/mL or 0.23 nM and 100 ng/mL or 0.93 nM, respectively. Subsequently, a nine-lectin screen was performed on the porcine and human fibrinogen glycoproteins. The observed spectra of lectin-protein specific binding rates result in characteristic patterns that evidently correlate with the structure of the glycans and allow one to distinguish between glycosylation of the porcine and human fibrinogens. The array technology has the potential to perform a multilectin screen of large numbers of proteins providing information on protein glycosylation and their microheterogeneity.Royal SocietyEuropean Commission’s Marie Curie programEPSRCBBSR

Label-free Fab and Fc affinity/avidity profiling of the antibody complex half-life for polyclonal and monoclonal efficacy screening

The final publication is available at Springer via http://dx.doi.org/10.1007/s00216-015-8897-6A unified approach to affinity screening for Fab and Fc interactions of an antibody for its antigen and FcγR receptor has been developed. An antigen array is used for the Fab affinity and cross-reactivity screening and protein A/G proxy is the FcγR receptor. The affinities are derived using a simple 1:1 binding model with a consistent error analysis. The association and dissociation kinetics are measured over optimised times for accurate determination. The Fab/Fc affinities are derived for ten antibodies: mAb-actin (mouse), pAb-BSA (sheep), pAb-collagen V (rabbit), pAb-CRP (goat), mAb-F1 (mouse), mAbs (mouse) 7.3, 12.3, 29.3, 36.3 and 46.3 raised against LcrV in Yersinia pestis. The rate of the dissociation of antigen–antibody complexes relates directly to their immunological function as does the Fc-FcγR complex and a new half-life plot has been defined with a Fab/Fc half-life range of 17–470 min. The upper half-life value points to surface avidity. Two antibodies that are protective as an immunotherapy define a Fab half-life >250 min and an Fc half-life >50 min as characteristics of ideal interactions which can form the basis of an antibody screen for immunotherapy.Biotechnology and Biological Sciences Research Council (BBSRC

Utilising a multiplexed gold nanoparticle-based sensor for antibody and antigen detection

This is the final versionUsing a multiplexed sensor, based on a gold nanoparticle array, to quantify SARS-CoV-2 antibodies in serum and SARS-CoV-2 antigens in saliva