Flotation Immunoassay: Masking the Signal from Free Reporters in Sandwich Immunoassays.
Ontology highlight
ABSTRACT:
SUBMITTER: Chen H
PROVIDER: S-EPMC4830940 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:We report a dual-readout, AuNP-based sandwich immunoassay for the device-free colorimetric and sensitive scanometric detection of disease biomarkers. An AuNP-antibody conjugate serves as a signal transduction and amplification agent by promoting the reduction and deposition of either platinum or gold onto its surface, generating corresponding colorimetric or light scattering (scanometric) signals, respectively. We apply the Pt-based colorimetric readout of this assay to the discovery of a novel monoclonal antibody (mAb) sandwich pair for the detection of an anthrax protective antigen (PA83). The identified antibody pair detects PA83 down to 1 nM in phosphate-buffered saline and 5 nM in human serum, which are physiologically relevant concentrations. Reducing gold rather than platinum onto the mAb-AuNP sandwich enables scanometric detection of subpicomolar PA83 concentrations, over 3 orders of magnitude more sensitive than the colorimetric readout.
| S-EPMC7418077 | biostudies-literature
Project description:Heterogeneous sandwich immunoassays are widely used for biomarker detection in bioanalysis and medical diagnostics. The high analyte sensitivity of the current "gold standard" enzyme-linked immunosorbent assay (ELISA) originates from the signal-generating enzymatic amplification step, yielding a high number of optically detectable reporter molecules. For future point-of-care testing (POCT) and point-of-need applications, there is an increasing interest in more simple detection strategies that circumvent time-consuming and temperature-dependent enzymatic reactions. A common concept to aim for detection limits comparable to those of enzymatic amplification reactions is the usage of polymer nanoparticles (NP) stained with a large number of chromophores. We explored different simple NP-based signal amplification strategies for heterogeneous sandwich immunoassays that rely on an extraction-triggered release step of different types of optically detectable reporters. Therefore, streptavidin-functionalized polystyrene particles (PSP) are utilized as carriers for (i) the fluorescent dye coumarin 153 (C153) and (ii) hemin (hem) molecules catalyzing the luminol reaction enabling chemiluminescence (CL) detection. Additionally, (iii) NP labeling with hemin-based microperoxidase MP11 was assessed. For each amplification approach, the PSP was first systematically optimized regarding size, loading concentration, and surface chemistry. Then, for an immunoassay for the inflammation marker C-reactive protein (CRP), the analyte sensitivity achievable with optimized PSP systems was compared with the established ELISA concept for photometric and CL detection. Careful optimization led to a limit of detection (LOD) of 0.1 ng/mL for MP11-labeled PSP and CL detection, performing similarly well to a photometric ELISA (0.13 ng/mL), which demonstrates the huge potential of our novel assay concept.
| S-EPMC10993196 | biostudies-literature
Project description:Immunoassays exploit the highly selective interaction between antibodies and antigens to provide a vital method for biomolecule detection at low concentrations. Developers and practitioners of immunoassays have long known that non-specific binding often restricts immunoassay limits of quantification (LOQs). Aside from non-specific binding, most efforts by analytical chemists to reduce the LOQ for these techniques have focused on improving the signal amplification methods and minimizing the limitations of the detection system. However, with detection technology now capable of sensing single-fluorescence molecules, this approach is unlikely to lead to dramatic improvements in the future. Here, fundamental interactions based on the law of mass action are analytically connected to signal generation, replacing the four- and five-parameter fittings commercially used to approximate sigmoidal immunoassay curves and allowing quantitative consideration of non-specific binding and statistical limitations in order to understand the ultimate detection capabilities of immunoassays. The restrictions imposed on limits of quantification by instrumental noise, non-specific binding, and counting statistics are discussed based on equilibrium relations for a sandwich immunoassay. Understanding the maximal capabilities of immunoassays for each of these regimes can greatly assist in the development and evaluation of immunoassay platforms. While many studies suggest that single molecule detection is possible through immunoassay techniques, here, it is demonstrated that the fundamental limit of quantification (precision of 10 % or better) for an immunoassay is approximately 131 molecules and this limit is based on fundamental and unavoidable statistical limitations.
| S-EPMC4636921 | biostudies-literature
Project description:State-of-the-art clinical detection methods typically involve standard immunoassay methods, requiring specialized equipment and trained personnel. This impedes their use in the Point-of-Care (PoC) environment, where ease of operation, portability, and cost efficiency are prioritized. Small, robust electrochemical biosensors provide a means with which to analyze biomarkers in biological fluids in PoC environments. Optimized sensing surfaces, immobilization strategies, and efficient reporter systems are key to improving biosensor detection systems. The signal transduction and general performance of electrochemical sensors are determined by surface properties that link the sensing element to the biological sample. We analyzed the surface characteristics of screen-printed and thin-film electrodes using scanning electron microscopy and atomic force microscopy. An enzyme-linked immunosorbent assay (ELISA) was adapted for use in an electrochemical sensor. The robustness and reproducibility of the developed electrochemical immunosensor were investigated by detecting Neutrophil Gelatinase-Associated Lipocalin (NGAL) in urine. The sensor showed a detection limit of 1 ng/mL, a linear range of 3.5-80 ng/mL, and a CV% of 8%. The results demonstrate that the developed platform technology is suitable for immunoassay-based sensors on either screen-printed or thin-film gold electrodes.
| S-EPMC10216821 | biostudies-literature
Project description:Fluorescence correlation and cross-correlation spectroscopy (FCS/FCCS) have enabled biologists to study processes of transport, binding, and enzymatic reactions in living cells. However, applying FCS and FCCS to samples such as whole blood and plasma is complicated as the fluorescence bursts of diffusing labels can be swamped by strong autofluorescence. Here we present cross-correlation spectroscopy based on two upconversion nanoparticles emitting at different wavelengths on the anti-Stokes side of a single excitation laser. This upconversion cross-correlation spectroscopy (UCCS) approach allows us to completely remove all Stokes shifted autofluorescence background in biological material such as plasma. As a proof of concept, we evaluate the applicability of UCCS to a homogeneous sandwich immunoassay for thyroid stimulating hormone measured in buffer solution and in plasma.
| S-EPMC6055621 | biostudies-literature
Project description:Point-of-care (POC) diagnostics such as lateral flow and dipstick immunoassays use gold nanoparticle (NP)-antibody conjugates for visual readout. We investigated the effects of NP conjugation, surface chemistries, and antibody immobilization methods on dipstick performance. We compared orientational, covalent conjugation, electrostatic adsorption, and a commercial conjugation kit for dipstick assays to detect dengue virus NS1 protein. Assay performance depended significantly on their conjugate properties. We also tested arrangements of multiple test lines within strips. Results show that orientational, covalent conjugation with PEG shield could improve NS1 detection. These approaches can be used to optimize immunochromatographic detection for a range of biomarkers.
| S-EPMC6202116 | biostudies-literature
Project description:The detection and monitoring of biological markers as disease indicators in a simple manner is a subject of international interest. In this work, we report two simple and sensitive label-free impedimetric immunoassays for the detection of C-reactive protein (CRP). The gold electrode modified with boronic acid-terminated self-assembled monolayers afforded oriented immobilization of capture glycosylated antibody (antihuman CRP monoclonal antibody, mAb). This antibody-modified surface was able to capture human CRP protein, and the impedance signal showed linear dependence with CRP concentration. We confirmed the immobilization of anti-CRP mAb using surface sensitive X-ray photoelectron spectroscopy (XPS) and electrochemical impedance. The oriented covalent immobilization of mAb was achieved using glycosylated Fc (fragment, crystallizable) region specific to boronic acid. The direct immunoassay exhibited a linear curve for concentration range up to 100 ng ml-1. The limit of detection (LoD) of 2.9 ng ml-1, limit of quantification (LoQ) of 9.66 ng ml-1, and sensitivity of 0.585 kΩ ng-1 ml cm-2 were obtained. The sandwich immunoassay was carried out by capturing polyclonal anti-CRP antibody (pAb) onto the CRP antigen immunoreaction. The impedance signal after pAb capture also showed linear dependence with CRP antigen concentration and acted as a CRP antigen detection signal amplifier. The detection of the CRP antigen using sandwich pAb immunoassay improved LoD to 1.2 ng ml-1, LoQ to 3.97 ng ml-1, and enhanced the sensitivity to 0.885 kΩ ng-1 ml cm-2. The real sample analysis, using newborn calf serum, showed excellent selectivity and % recovery for the human CRP ranging from 91.2 to 96.5%. The method was reproducible to 4.5% for direct immunoassay and 2.3% for sandwich immunoassay.
| S-EPMC8207519 | biostudies-literature
Project description:Procalcitonin (PCT) is well established as a highly specific biomarker for the detection of bacterial infections and sepsis. However, the currently available diagnostic tests are not able to detect very low or very early increases of PCT or even baseline levels in healthy individuals or patients with non-bacterial infections. In order to be able to detect these very low concentrations of PCT, a sandwich immunoassay was developed using high sensitivity Single Molecule Array technology (Simoa). The assay was thoroughly validated and applied to analyze human cerebrospinal fluid (CSF) and serum samples from patients with bacterial or viral meningitis as well as CSF, serum, and K2 EDTA plasma from healthy control subjects. A 50-fold increase in sensitivity compared to the current gold standard assays was achieved, which was sensitive enough for the detection of baseline PCT levels. Both serum and CSF showed significantly elevated PCT levels in patients with bacterial meningitis compared to patients with viral meningitis and the healthy control group. Procalcitonin concentration levels for patients with viral meningitis and the control group could be measured, but were not significantly different. The determination of PCT in the low pg·mL-1 range could help to improve the monitoring of bacterial infectious diseases, as PCT level changes could be detected earlier.
| S-EPMC5748597 | biostudies-literature
Project description:A new class of bead-based microarray that uses electrogenerated chemiluminescence (ECL) as a readout mechanism to detect multiple antigens simultaneously is presented. This platform demonstrates the possibility of performing highly multiplexed assays using ECL because all the individual sensing beads in the array are simultaneously imaged and individually resolved by ECL. Duplex and triplex assay results are demonstrated as well as a cross reactivity study.
| S-EPMC2711039 | biostudies-literature
Project description:We investigated the effect of the protein corona on the function of nanoparticle (NP) antibody (Ab) conjugates in dipstick sandwich immunoassays. Ab specific for Zika virus nonstructural protein 1 (NS1) were conjugated to gold NPs, and another anti-NS1 Ab was immobilized onto the nitrocellulose membrane. Sandwich immunoassay formation was influenced by whether the strip was run in corona forming conditions, i.e., in human serum. Strips run in buffer or pure solutions of bovine serum albumin exhibited false positives, but those run in human serum did not. Serum pretreatment of the nitrocellulose also eliminated false positives. Corona formation around the NP-Ab in serum was faster than the immunoassay time scale. Langmuir binding analysis determined how the immobilized Ab affinity for the NP-Ab/NS1 was impacted by corona formation conditions, quantified as an effective dissociation constant, KDeff. Results show that corona formation mediates the specificity and sensitivity of the antibody-antigen interaction of Zika biomarkers in immunoassays, and plays a critical but beneficial role.
| S-EPMC5245486 | biostudies-literature