scholarly journals Sensitive protein detection using site-specifically oligonucleotide-conjugated nanobodies

2022 ◽  
Author(s):  
Rasel A. Al-Amin ◽  
Phathutshedzo M. Muthelo ◽  
Eldar Abdurakhmanov ◽  
Cecile Vincke ◽  
Serge Muyldermans ◽  
...  
Keyword(s):  
Polyclonal Antibodies ◽  
Protein Detection ◽  
Specific Protein ◽  
Protein Biomarkers ◽  
High Quality ◽  
Affinity Reagents ◽  
Protein Assay ◽  
Enzyme Coupling ◽  
Target Binding ◽  
Dna Strand

High-quality affinity probes are critical for sensitive and specific protein detection, in particular to detect protein biomarkers at early phases of disease development. Clonal affinity reagents can offer advantages over the commonly used polyclonal antibodies (pAbs) in terms of reproducibility and standardization of such assays. In particular, clonal reagents offer opportunities for site-directed attachment of exactly one modification per affinity reagent at a site designed not to interfere with target binding to help standardize assays. The proximity extension assays (PEA) is a widely used protein assay where pairs of protein-binding reagents are modified with oligonucleotides (oligos), so that their proximal binding to a target protein generates a reporter DNA strand for DNA-assisted readout. The assays have been used for high-throughput multiplexed protein detection of up to a few thousand different proteins in one or a few microliters of plasma. Here we explore nanobodies (Nb) as an alternative to polyclonal antibodies pAbs as affinity reagents for PEA. We describe an efficient site-specific approach for preparing high-quality oligo-conjugated Nb probes via Sortase A (SrtA) enzyme coupling. The procedure allows convenient removal of unconjugated affinity reagents after conjugation. The purified high-grade Nb probes were used in PEA and the reactions provided an efficient means to select optimal pairs of binding reagents from a group of affinity reagents. We demonstrate that Nb-based PEA for interleukin-6 (IL6) detection can augment assay performance, compared to the use of pAb probes. We identify and validate Nb combinations capable of binding in pairs without competition for IL6 antigen detection by PEA.

scholarly journals Directed Evolution of Aptamer Discovery Technologies

2021 ◽  
Author(s):  
Diana Wu ◽  
Chelsea Gordon ◽  
John Shin ◽  
Michael Eisenstein ◽  
Hyongsok Tom Soh
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Click Reaction ◽  
Clinical Diagnostics ◽  
High Quality ◽  
Affinity Reagents ◽  
Wide Range ◽  
Target Binding ◽  
Modified Nucleobases ◽  
Parallel Fashion

Although antibodies are a powerful tool for molecular biology and clinical diagnostics, there are many emerging applications for which nucleic acid-based aptamers can be advantageous. However, generating high-quality aptamers with sufficient affinity and specificity for biomedical applications is a challenging feat for most research laboratories. In this Account, we describe four techniques developed in our lab to accelerate the discovery of high quality aptamer reagents that can achieve robust binding even for challenging molecular targets. The first method is particle display, in which we convert solution-phase aptamers into aptamer particles that can be screened via fluorescence-activated cell sorting (FACS) to quantitatively isolate individual aptamer particles based on their affinity. This enables the efficient isolation of high-affinity aptamers in fewer selection rounds than conventional methods, thereby minimizing selection biases and reducing the emergence of artifacts in the final aptamer pool. We subsequently developed the multi-parametric particle display (MPPD) method, which employs two-color FACS to isolate aptamer particles based on both affinity and specificity, yielding aptamers that exhibit excellent target binding even in complex matrices like serum. The third method is a click chemistry-based particle display (click-PD) that enables the generation and high-throughput screening of non-nattural aptamers with a wide range of base modifications. We have shown that these base-modified aptamers can achieve robust affinity and specificity for targets that have proven challenging or inaccessible with natural nucleotide-based aptamer libraries. Lastly, we describe the non-natural aptamer array (N2A2) platform, in which a modified benchtop sequencing instrument is used to characterize base-modified aptamers in a massively parallel fashion, enabling the efficient identification of molecules with excellent affinity and specificity for their targets. This system first generates aptamer clusters on the flow-cell surface that incorporate alkyne-modified nucleobases, and then performs a click reaction to couple those nucleobases to an azide-modified chemical moiety. This yields a sequence-defined array of tens of millions of base-modified sequences, which can then be characterized in a high-throughput fashion. Collectively, we believe that these advancements are helping to make aptamer technology more accessible, efficient, and robust, thereby enabling the use of these affinity reagents for a wider range of molecular recognition and detection-based applications.

scholarly journals Dual-reporter SERS-based biomolecular assay with reduced false-positive signals

2017 ◽  
Vol 114 (34) ◽  
pp. 9056-9061 ◽  
Author(s):  
Tracy T Chuong ◽  
Alessia Pallaoro ◽  
Chelsea A. Chaves ◽  
Zhe Li ◽  
Joun Lee ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Hot Spot ◽  
Quantitative Measure ◽  
Protein Detection ◽  
Surface Enhanced Raman Spectroscopy ◽  
Affinity Reagents ◽  
Affinity Labels ◽  
Detection Assay ◽  
Surface Enhanced Raman ◽  
Target Binding

We present a sensitive and quantitative protein detection assay that can efficiently distinguish between specific and nonspecific target binding. Our technique combines dual affinity reagents with surface-enhanced Raman spectroscopy (SERS) and chemometric analysis. We link one Raman reporter-tagged affinity reagent to gold nanoparticles and another to a gold film, such that protein-binding events create a “hot spot” with strong SERS spectra from both Raman reporter molecules. Any signal generated in this context is indicative of recognition by both affinity labels, whereas signals generated by nonspecific binding lack one or the other label, enabling us to efficiently distinguish true from false positives. We show that the number of hot spots per unit area of our substrate offers a quantitative measure of analyte concentration and demonstrate that this dual-label, SERS-linked aptasensor assay can sensitively and selectively detect human α-thrombin in 1% human serum with a limit of detection of 86 pM.

scholarly journals Electrokinetic Formation of “Microbridges” for Protein Biomarkers as Sensors

2007 ◽  
Vol 12 (5) ◽  
pp. 311-317 ◽  
Author(s):  
Vindhya Kunduru ◽  
Shalini Prasad
Keyword(s):  
Electric Fields ◽  
Microelectrode Array ◽  
Protein Detection ◽  
High Specificity ◽  
Detection Methods ◽  
Label Free ◽  
Protein Biomarkers ◽  
Detection Scheme ◽  
Polystyrene Beads ◽  
Conducting Path

We demonstrate a technique to detect protein biomarkers contained in vulnerable coronary plaque using a platform-based microelectrode array (MEA). The detection scheme is based on the property of high specificity binding between antibody and antigen similar to most immunoassay techniques. Rapid clinical diagnosis can be achieved by detecting the amount of protein in blood by analyzing the protein's electrical signature. Polystyrene beads which act as transportation agents for the immobile proteins (antigen) are electrically aligned by application of homogenous electric fields. The principle of electrophoresis is used to produce calculated electrokinetic movement among the anti-C-reactive protein (CRP), or in other words antibody funtionalized polystyrene beads. The electrophoretic movement of antibody-functionalized polystyrene beads results in the formation of “Microbridges” between the two electrodes of interest which aid in the amplification of the antigen—antibody binding event. Sensitive electrical equipment is used for capturing the amplified signal from the “Microbridge” which essentially behaves as a conducting path between the two electrodes. The technique circumvents the disadvantages of conventional protein detection methods by being rapid, noninvasive, label-free, repeatable, and inexpensive. The same principle of detection can be applied for any receptor—ligand-based system because the technique is based only on the volume of the analyte of interest. Detection of the inflammatory coronary disease biomarker CRP is achieved at concentration levels spanning over the lower microgram/milliliter to higher order nanogram/milliliter ranges.

Abstract 14727: Meteorin-like Glial Cell Differentiation Regulator is a Novel Protein Biomarker of Cardiovascular Outcomes in Patients With Diabetes and Vascular Disease: A TECOS Substudy

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Lauren K Truby ◽  
Jessica A Regan ◽  
Maggie Nguyen ◽  
Stephani Giamberardino ◽  
Robert J Mentz ◽  
...  
Keyword(s):  
Heart Failure ◽  
Conditional Logistic Regression ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Specific Protein ◽  
Cardiovascular Outcomes ◽  
Diabetic Patients ◽  
Proteomic Profiling ◽  
Protein Biomarkers ◽  
Novel Protein

Introduction: To date, there are limited data on the potential role of proteomic biomarkers to predict future cardiovascular (CV) events among patients with type 2 diabetes mellitus (DM). Hypothesis: Specific protein biomarkers will be predictive of major adverse CV events (MACE) and incident heart failure hospitalization (HFH) among patients with DM. Methods: Using the Olink aptamer-based platform, we performed proteomic profiling (>700 proteins) on 440 paired cases and matched controls from placebo-assigned participants in the Trial Evaluating Cardiovascular Outcomes with Sitagliptin (TECOS). Cases were defined as having met the primary composite outcome of MACE or incident HFH and matched to controls on baseline prevalent heart failure, coronary artery disease, BMI, hemoglobin A1C, creatinine, low-density lipoprotein cholesterol, fasting status and ejection fraction. Conditional logistic regression was used to determine the association between log-transformed relative protein expression and incident MACE or HFH. False-discovery-rate (FDR) was used to adjust for multiple comparisons. Results: We identified three specific proteins that were significantly associated with prevalent MACE or HFH: METRNL, Notch 3, and ROR1 (OR 2.1, 1.6, 1.7 and q-value 0.01, 0.02, and 0.05 respectively) (Figure 1). METRNL, in particular, performed similarly to the established biomarker NT-proBNP (Figure 1). When MACE and HFH were analyzed separately, METRNL, in particular, remained strongly associated with both outcomes (OR 2.0, p<0.001 and OR 2.7, p=0.05). Conclusions: Three novel protein biomarkers, in particular METRNL (a circulating adipokine that regulates insulin-sensitivity), may identify diabetic patients at high risk for subsequent HF and MACE. Additional studies are needed to replicate these findings and uncover the biologic mechanism linking adipokine signaling and heart failure.

scholarly journals Streamlined circular proximity ligation assay provides high stringency and compatibility with low-affinity antibodies

2018 ◽  
Vol 115 (5) ◽  
pp. E925-E933 ◽  
Author(s):  
Roxana Jalili ◽  
Joe Horecka ◽  
James R. Swartz ◽  
Ronald W. Davis ◽  
Henrik H. J. Persson
Keyword(s):  
Limit Of Detection ◽  
Biological Fluids ◽  
Protein Detection ◽  
Ease Of Use ◽  
Proximity Ligation Assay ◽  
Quantitative Detection ◽  
High Signal ◽  
Affinity Reagents ◽  
Proximity Ligation ◽  
Assay Format

Proximity ligation assay (PLA) is a powerful tool for quantitative detection of protein biomarkers in biological fluids and tissues. Here, we present the circular proximity ligation assay (c-PLA), a highly specific protein detection method that outperforms traditional PLA in stringency, ease of use, and compatibility with low-affinity reagents. In c-PLA, two proximity probes bind to an analyte, providing a scaffolding that positions two free oligonucleotides such that they can be ligated into a circular DNA molecule. This assay format stabilizes antigen proximity probe complexes and enhances stringency by reducing the probability of random background ligation events. Circle formation also increases selectivity, since the uncircularized DNA can be removed enzymatically. We compare this method with traditional PLA on several biomarkers and show that the higher stringency for c-PLA improves reproducibility and enhances sensitivity in both buffer and human plasma. The limit of detection ranges from femtomolar to nanomolar concentrations for both methods. Kinetic analyses using surface plasmon resonance (SPR) and biolayer interferometry (BLI) reveal that the variation in limit of detection is due to the variation in antibody affinity and that c-PLA outperforms traditional PLA for low-affinity antibodies. The lower background signal can be used to increase proximity probe concentration while maintaining a high signal-to-noise ratio, thereby enabling the use of low-affinity reagents in a homogeneous assay format. We anticipate that the advantages of c-PLA will be useful in a variety of clinical protein detection applications where high-affinity reagents are lacking.

Relevance of urinary specific protein assay in the diagnosis of kidney diseases

2018 ◽  
Vol 76 (3) ◽  
pp. 259-269
Author(s):  
Adrien Flahault ◽  
Jean-François Chassé ◽  
Eric Thervet ◽  
Alexandre Karras ◽  
Nicolas Pallet
Keyword(s):  
Kidney Diseases ◽  
Specific Protein ◽  
Protein Assay

Possibility of using recombinant monoclonal antibodies in the treatment of the coronavirus infection

2021 ◽  
pp. 8-13
Author(s):  
Vladimir Anatolievich Klimov
Keyword(s):  
Monoclonal Antibodies ◽  
Plasma Cells ◽  
Polyclonal Antibodies ◽  
Fatal Outcome ◽  
Humoral Response ◽  
Specific Protein ◽  
Pathological Process ◽  
Human Immune System ◽  
Risk Of Progression ◽  
Severe Degree

Monoclonal antibodies are successfully used to treat a number of viral diseases; they are produced by the plasma cells of the human immune system and are the result of a natural humoral response to the ingress of a certain antigen. Antibodies designed to target only one specific protein — in this case, the protein of the virus that causes COVID-19 — are called «monoclonal». Their purpose is to block the COVID-19 virus and prevent its penetration and replication in human cells, and their ultimate focus is on combating infectious manifestations. They are believed to cause fewer undesirable effects than convalescent plasma, which is essentially a complex of polyclonal antibodies and circulating immune complexes. Monoclonal antibodies can be synthesized in the laboratory using the blood serum of patients who have undergone this disease as a starting material. The use of monoclonal antibodies is indicated for patients with a mild and moderate form of the disease at a high risk of progression of the pathological process to a severe degree, which implies the need for hospitalization and a high probability of a fatal outcome.

scholarly journals Modular protein-oligonucleotide signal exchange

2020 ◽  
Vol 48 (12) ◽  
pp. 6431-6444
Author(s):  
Deepak K Agrawal ◽  
Rebecca Schulman
Keyword(s):  
Specific Protein ◽  
Vascular Endothelial ◽  
Complementary Dna ◽  
Exchange Processes ◽  
Dna Strand Displacement ◽  
Specific Proteins ◽  
Dna Strand ◽  
Endothelial Growth Factor ◽  
Signal Exchange

Abstract While many methods are available to measure the concentrations of proteins in solution, the development of a method to quantitatively report both increases and decreases in different protein concentrations in real-time using changes in the concentrations of other molecules, such as DNA outputs, has remained a challenge. Here, we present a biomolecular reaction process that reports the concentration of an input protein in situ as the concentration of an output DNA oligonucleotide strand. This method uses DNA oligonucleotide aptamers that bind either to a specific protein selectively or to a complementary DNA oligonucleotide reversibly using toehold-mediated DNA strand-displacement. It is possible to choose the sequence of output strand almost independent of the sensing protein. Using this strategy, we implemented four different exchange processes to report the concentrations of clinically relevant human α-thrombin and vascular endothelial growth factor using changes in concentrations of DNA oligonucleotide outputs. These exchange processes can operate in tandem such that the same or different output signals can indicate changes in concentration of distinct or identical input proteins. The simplicity of our approach suggests a pathway to build devices that can direct diverse output responses in response to changes in concentrations of specific proteins.

Surface optimization of gold nanoparticle mass tags for the sensitive detection of protein biomarkers via immuno-capture LI-MS

The Analyst ◽  
2020 ◽  
Vol 145 (19) ◽  
pp. 6237-6242
Author(s):  
Toby Siu-Chung Tam ◽  
Yu-Hong Cheng ◽  
Chun-Nam Lok ◽  
Ho Yu Au-Yeung ◽  
Wen-Xiu Ni ◽  
...  
Keyword(s):  
Gold Nanoparticle ◽  
Surface Functionalization ◽  
Protein Detection ◽  
Detection Sensitivity ◽  
Sensitive Detection ◽  
Specific Interactions ◽  
Protein Biomarkers ◽  
Optimal Surface ◽  
Surface Optimization

Optimal surface functionalization of antibody-conjugated AuNPs for modulating specific versus non-specific interactions to enhance protein detection sensitivity.

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