Linker-protein G mediated functionalization of polystyrene-encapsulated upconversion nanoparticles for rapid gene assay using convective PCR.

The authors report on a simplified approach to encapsulate upconversion nanoparticles (UCNPs) in polystyrene spheres by mini-emulsion polymerisation. The resulting particles (PS-UCNP) are hydrophilic, stable and suitable for biomolecular recognition and biosensing applications. Also, a strategy was developed for bioconjugation of antibodies onto the surface of the PS-UCNPs by using the bifunctional fusion protein linker-protein G (LPG). LPG mediates the functionalisation of PS-UCNPs with antibodies against digoxigenin allowing for specific labelling of convective PCR (cPCR) amplicons. Lambda DNA was amplified using cPCR on a heat block for 30 min using the digoxigenin labelled forward and biotin labelled reverse primers. The antibody functionalised PS-UCNPs bind to the digoxigenin end of the cPCR amplicons. Finally, the streptavidin labelled magnetic beads were used to selectively capture the PS-UCNP-labelled cPCR amplicons and the upconversion signal was detected at 537 nm under 980 nm excitation. This sandwich approach enables direct recognition of the target lambda DNA with a detection limit of 103 copies µL-1. The upconversion signal decreased proportionally to the concentration of the lambda DNA with a linear response between 107 and 103 copies of DNA. Graphical abstract Schematic representation of polystyrene-encapsulated upconversion nanoparticles (PS-UCNPs) prepared by mini-emulsion polymerisation. The PS-UCNPs were functionalised with anti-digoxigenin antibody using the fusion protein linker-protein G (LPG). Detection of digoxigenin-labelled amplicons is achieved (a) by using the antibody-functionalised LPG@PS-UCNP labels; (b) magnetic separation, and (c) 980 nm laser light for detection of the green upconversion luminescence peaking at 537 nm.

Rapid Detection of Drugs in Human Plasma Using a Small-Molecule-Linked Hybridization Chain Reaction.

Rapid detection and quantification of pharmaceutical drugs directly in human plasma is of major importance for the development of relevant point-of-care testing devices. Here, we report a method for detection and quantification of small molecules in human plasma. An assay employing a small molecule-linked hybridization chain reaction (HCR) has been devised for the detection of the pharmaceutically relevant drugs digoxin (Dig) and methotrexate (MTX). Double modification by small molecule ligands on the initiator strand act as sites to control the rate of the HCR. Upon protein binding to the modified initiator strand, the HCR is greatly inhibited. If the protein is preincubated with a sample containing the small molecule analyte, the protein binding site is occupied by the analyte and the initiator strand will initiate the HCR. This enables efficient detection and quantification of small-molecule analytes in nanomolar concentration even in 50% human plasma within 4 min. Thus, the rapidity and simplicity of this assay has potential for point-of-care testing.

Allosteric kissing complex-based electrochemical biosensor for sensitive, regenerative and versatile detection of proteins.

Herein, an allosteric kissing complex-based electrochemical biosensor was ingeniously proposed for the simple, sensitive, regenerative and versatile detection of proteins. Two hairpins (Hp1 and Hp2) were designed and the Hp1 was immobilized on the electrode surface, which could form a kissing complex with Hp2 through the apical loop-loop or kissing interaction of the RNA-RNA base sequences. The Hp2 possesses the appended single-stranded tails on each end, which hybridize with the recognition element-conjugated DNA strands to construct a protein responsive switch of Hp2 scaffold. After kissing complex formation between the Hp2 scaffold and the immobilized Hp1, the streptavidin-labeled alkaline phosphatase (SA-ALP) can be introduced onto the electrode surface for the generation of electrochemical signal. In the presence of target protein, its binding to the recognition elements linked onto the Hp2 scaffold endows the steric strain to open the Hp2 stem, propagated by the disruption of the kissing complex structure, resulting into a decreased electrochemical signal related with the protein quantification. Also, the Hp1 immobilized electrode can be directly regenerated after protein-induced kissing complex dissociation. The current kissing complex-based electrochemical biosensing strategy can be easily extended for the detection toward different protein targets of interest by simply changing the recognition elements conjugated onto the Hp2 scaffold. The sensitive and selective detection toward proteins could be achieved with the detection limits toward Anti-Dig antibody and thrombin of about 1ng/mL and 10pM, respectively. The developed kissing complex-based protein biosensing strategy should be a beneficial supplement in current biosensor field, providing a promising means for the applications in bioanalysis, disease diagnostics, and clinical biomedicine.

Accurate Electrochemistry Analysis of Circulating Methylated DNA from Clinical Plasma Based on Paired-End Tagging and Amplifications.

Circulating methylated DNA has been a new kind of cancer biomarker, yet its small fraction of trace total DNA from clinical samples impairs the accurate analysis. Though fluorescence methods based on quantitative methylation specific PCR (qMSP) have been adopted routinely, yet alternative electrochemistry assay of such DNA from clinical samples remains a great challenge. Herein, we report accurate electrochemistry analysis of circulating methylated DNA from clinical plasma samples based on a paired-end tagging and amplifications strategy. Two DNA primers each labeled with digoxigenin (Dig) and biotin are designed for the recognition and amplification of methylated DNA. Paired-end tagging amplicons and avidin-HRP molecules are successively captured on the electrode modified with Anti-Dig. Then HRP executes catalytic reaction to generate amplified signal. The design of paired-end tagging can readily integrate downstream electrochemical amplified reaction, and two heterogeneous amplifications enable high assay sensitivity. As little as 40 pg of methylated genomic DNA (∼10 genomic equivalents) is well identified, and our strategy can even distinguish as low as 1% methylation level. Tumor-specific methylated DNA is clearly detected in the plasma of 10 of 11 NSCLC patients. The high clinical sensitivity of 91% (10/11) indicates the good consistency with clinical diagnosis. Excellent spatial control of electrochemistry allows simpler detection of more methylation patterns compared to fluorescence methods. The developed electrochemical assay is a promising liquid biopsy tool for the analysis of tumor-specific circulating DNA.

Steric hindrance inhibition of strand displacement for homogeneous and signal-on fluorescence detection of human serum antibodies.

Based on a new steric hindrance inhibition of the DNA strand displacement strategy, we report the design of a robust fluorescence signal-on method for homogeneous and sensitive detection of antibodies from human serum samples. Such a steric hindrance effect leads to sensitive detection of the target antibodies with a detection limit of 5.6 nM. In addition, the developed sensing approach shows high selectivity against other interference proteins and the detection of the target antibodies in human sera by this method is also verified.

Quantifying Nanomolar Protein Concentrations Using Designed DNA Carriers and Solid-State Nanopores.

Designed "DNA carriers" have been proposed as a new method for nanopore based specific protein detection. In this system, target protein molecules bind to a long DNA strand at a defined position creating a second level transient current drop against the background DNA translocation. Here, we demonstrate the ability of this system to quantify protein concentrations in the nanomolar range. After incubation with target protein at different concentrations, the fraction of DNA translocations showing a secondary current spike allows for the quantification of the corresponding protein concentration. For our proof-of-principle experiments we use two standard binding systems, biotin-streptavidin and digoxigenin-antidigoxigenin, that allow for measurements of the concentration down to the low nanomolar range. The results demonstrate the potential for a novel quantitative and specific protein detection scheme using the DNA carrier method.

Platform switching from ELISA to Gyrolab™: a novel generic reagent omits the need to change critical reagents.

During development of biotherapeutics, availability of specific assay reagents is usually limited. The possibility to switch from one ligand binding assay technology to another, while using the same reagents, would be desirable. Here, we report on an Alexa647(®)-labeled monoclonal antibody against digoxigenin (mAb-Alexa647(®)) that enables the detection of digoxigenylated analyte-specific ELISA reagents by Gyrolab(™). In an analysis of non-monoclonal antibody (mAb) and mAb drugs, this approach maintained the dynamic range, accuracy and precision of the standard Gyrolab™ approach using analyte-specific Alexa647(®)-labeled Ab. In a rat PK study, results of our approach, standard Gyrolab™ and ELISA were comparable, with difference values within the incurred sample reanalysis acceptance criteria. Therefore, mAb-Alexa647(®) enables an easy switch between ELISA and Gyrolab™, providing an effective way to benefit from both platforms.

Acoustic force spectroscopy.

Force spectroscopy has become an indispensable tool to unravel the structural and mechanochemical properties of biomolecules. Here we extend the force spectroscopy toolbox with an acoustic manipulation device that can exert forces from subpiconewtons to hundreds of piconewtons on thousands of biomolecules in parallel, with submillisecond response time and inherent stability. This method can be readily integrated in lab-on-a-chip devices, allowing for cost-effective and massively parallel applications.

The selection performance of an antibody library displayed on filamentous phage coat proteins p9, p3 and truncated p3.

BACKGROUND: Filamentous phage display has become an ordinary tool to engineer antibody fragments. Several capsid proteins have been applied for displaying antibodies, of which gene III (p3) protein is used the most followed by experiments with gene IX (p9) protein. Despite the popularity, there are no library scale studies to objectively compare differences in the selection performance of the libraries, when displayed via different capsid proteins. RESULTS: In this study, an identical antibody repertoire was displayed as Fab fragments on p9, p3 and truncated p3 (p3Δ). In addition, the library clones were displayed as ScFv fragments on p3Δ and the Fab-p3 display valency was modulated by hyperphage and VCS-M13 superinfections. The selection performances of the libraries were followed in repeated parallel panning reactions against streptavidin (STR) and digoxigenin (DIG). Selection was successful with all display formats, but the enrichment of specific clones from Fab-p9 library was clearly less efficient than from the other libraries. The most diverse outputs were obtained from p3Δ display and the highest affinity anti-DIG antibodies from the ScFv repertoire. Unfortunately, the number of retrieved specific clones was too low for explicit analysis of the differences in the number of obtained unique clones from each library. However, severe reduction in sequence diversity was observed in p3-Fab libraries prior to panning, which in turn, materialized as a low number of unique specific clones. Oligovalent display by hyperphage resulted in a higher number of unique clones, but the same highest affinity anti-DIG Fab was recovered also by VCS-M13 superinfection. CONCLUSIONS: The compromised enrichment of the target-specific clones from the Fab repertoire as a fusion to p9 capsid protein in our experiments, the significant loss of functional diversity in Fab-p3 library after single phage packing cycle and the retrieval of higher affinity anti-digoxigenin clones as ScFv molecules than as Fab molecules from the same source repertoire indicate that the chosen display format may have a significant impact on the selection outcome. This study demonstrates that in addition to library content, also display related issues, should be taken into consideration when planning directed evolution experiments.

Cell surface display yields evolvable, clickable antibody fragments.

Non-canonical amino acids (ncAAs) provide powerful tools for engineering the chemical and physical properties of proteins. However, introducing ncAAs into proteins can affect protein properties in unpredictable ways, thus necessitating screening efforts to identify mutants with desirable properties. In this work, we describe an Escherichia coli cell surface display platform for the directed evolution of clickable antibody fragments. This platform enabled isolation of antibody fragments with improved digoxigenin binding and modest affinity maturation in several different ncAA contexts. Azide-functionalized fragments exhibited improved binding kinetics relative to their methionine counterparts, facile chemical modification through azide-alkyne cycloaddition, and retention of binding properties after modification. The results described here suggest new possibilities for protein engineering, including modulation of molecular recognition events by ncAAs and direct screening of libraries of chemically modified proteins.

Free analyte QC concept: a novel approach to prove correct quantification of free therapeutic protein drug/biomarker concentrations.

Quantification of free drug concentrations is highly challenging due to the dynamic drug-ligand equilibrium, which may result in incorrect results. Current QC concepts do not adequately cover all of the important influencing factors: the assay itself (format and procedure); the calibration concept; the sample preparation; and the sample storage. Here, we propose a 'free analyte QC concept' that enables quantitative testing of these four factors and, thus, provides best possible proof of correct free drug quantification. The principle of the free analyte QC concept and an example of its application for a free drug assay is described. A comparison of this novel approach with current approaches and how the new concept fits (or does not fit) with current regulatory guidelines is discussed.

Two ScFv antibody libraries derived from identical VL-VH framework with different binding site designs display distinct binding profiles.

In directed evolution experiments, a single randomization scheme of an antibody gene does not provide optimal diversity for recognition of all sizes of antigens. In this study, we have expanded the recognition potential of our universal library, termed ScFvP, with a second distinct diversification scheme. In the second library, termed ScFvM, diversity was designed closer to the center of the antigen binding site in the same antibody framework as earlier. Also, the CDR-H3 loop structures were redesigned to be shorter, 5-12 aa and mostly without the canonical salt bridge between Arg106H and Asp116H to increase the flexibility of the loop and to allow more space in the center of the paratope for binding smaller targets. Antibodies were selected from the two libraries against various antigens separately and as a mixture. The origin and characteristics of the retrieved antibodies indicate that complementary diversity results in complementary functionality widening the spectrum of targets amenable for selection.

Performing the Labeled microRNA pull-down (LAMP) assay system: an experimental approach for high-throughput identification of microRNA-target mRNAs.

We developed a simple, direct, and cost-effective approach to search for the most likely target genes of a known miRNA in vitro. We term this method "Labeled microRNA (miRNA) pull-down assay system," or LAMP. Briefly, the pre-miRNA is labeled with digoxigenin (DIG), mixed with cell extracts, and immunoprecipitated by anti-DIG antiserum. We concluded that LAMP is an experimental approach for high-throughput identification of the target gene of known miRNAs from both Caenorhabditis elegans and zebrafish (Danio rerio), yielding fewer false-positive results than those produced by using only the bioinformatics approach.

Bispecific digoxigenin-binding antibodies for targeted payload delivery.

Bispecific antibodies that bind cell-surface targets as well as digoxigenin (Dig) were generated for targeted payload delivery. Targeting moieties are IgGs that bind the tumor antigens Her2, IGF1R, CD22, or LeY. A Dig-binding single-chain Fv was attached in disulfide-stabilized form to C termini of CH3 domains of targeting antibodies. Bispecific molecules were expressed in mammalian cells and purified in the same manner as unmodified IgGs. They are stable without aggregation propensity and retain binding specificity/affinity to cell-surface antigens and Dig. Digoxigeninylated payloads were generated that retain full functionality and can be complexed to bispecific antibodies in a defined 21 ratio. Payloads include small compounds (Dig-Cy5, Dig-Doxorubicin) and proteins (Dig-GFP). Complexed payloads are targeted by the bispecifics to cancer cells and because these complexes are stable in serum, they can be applied for targeted delivery. Because Dig bispecifics also effectively capture digoxigeninylated compounds under physiological conditions, separate administration of uncharged Dig bispecifics followed by application of Dig payload is sufficient to achieve antibody-mediated targeting in vitro and in vivo.

Solution ELISA as a platform of choice for development of robust, drug tolerant immunogenicity assays in support of drug development.

Humanized monoclonal antibody therapeutics are in many ways indistinguishable from the anti-therapeutic/anti-drug antibodies generated in humans. Therefore, immunogenicity assessments to such therapeutics pose unique challenges in clinical trials especially when significant drug interference is encountered. There are several technology platforms based on the bridging immunogenicity assay format, which have been successfully used for detection and quantification of anti-drug antibodies (ADA) in serum or plasma samples. Enzyme-Linked Immunosorbent Assay (ELISA) and Electrochemiluminescent (ECL) immunoassay formats are among the most popular technology platforms. Pretreatment of samples with acid can also be used to lower drug interference. While ECL technology platform offered many advantages over traditional solid-phase ELISA methods, reliance on a single (or limited) vendor source became a significant concern within the biopharmaceutical industry especially for immunogenicity assays that need to be implemented over a period of many years in support of a single drug development program. We describe herein a systematic evaluation of solid-phase ELISA, GYROS, AlphaLISA, ECL Immunoassay, and solution ELISA platforms for detection of anti-drug antibodies with the goal of selection and development of a robust technology platform that meets the desired performance characteristics for most immunogenicity assays and can be easily implemented in a typical immunoassay laboratory. As part of this effort the Design of Experiments (DOE) approach was utilized in optimization of sample acid treatment conditions in order to improve drug tolerance in the evaluated assay platforms. After the initial evaluation of various technology platforms, a solution ELISA format was chosen for further development to support clinical trials for a humanized therapeutic antibody. As part of the assay development, flexible use of digoxigenin and 6-(2,4-dinitrophenyl) aminohexanoic acid (DNP) for labeling antibodies was evaluated and is presented in this manuscript. In addition, simple methods for evaluation and qualification of streptavidin-coated plates and overcoming soluble target interference in solution ELISA have also been investigated and highlights of these investigations are discussed. The selection of the solution ELISA format was based on availability of generic reagents, achievement of optimal drug tolerance and robust assay performance on a platform that is readily available in many laboratories. This approach removed the heavy reliance on specialized equipment sourced from a single vendor and assay conditions described here are broadly applicable to other immunogenicity assays across many biologics both during clinical development setting and in the post-marketing arena.

Stretching single DNA molecules to demonstrate high-force capabilities of holographic optical tweezers.

The well calibrated force-extension behaviour of single double-stranded DNA molecules was used as a standard to investigate the performance of phase-only holographic optical tweezers at high forces. Specifically, the characteristic overstretch transition at 65 pN was found to appear where expected, demonstrating (1) that holographic optical trap calibration using thermal fluctuation methods is valid to high forces; (2) that the holographic optical traps are harmonic out to >250 nm of 2.1 mum particle displacement; and (3) that temporal modulations in traps induced by the spatial light modulator (SLM) do not affect the ability of optical traps to hold and steer particles against high forces. These studies demonstrate a new high-force capability for holographic optical traps achievable by SLM technologies.

Competitive homogeneous digoxigenin immunoassay based on fluorescence quenching by gold nanoparticles.

We report on a competitive, homogeneous immunoassay for the detection of the hapten digoxigenin. The assay is based on competitive fluorescence quenching by gold nanoparticles. Digoxigenin is indirectly labeled with the fluorophore Cy3B through bovine serum albumin and used as a marker. Gold nanoparticles functionalized with anti-digoxigenin antibodies serve as fluorescence quenchers. Free digoxigenin molecules in the analyte solution compete with the labeled markers for antibodies on the gold nanoparticles. The fluorescence signal depends linearly on the free digoxigenin concentration within a range of concentration from 0.5 to 3 ng mL(-1). The limit of detection is estimated as 0.2 ng mL(-1) and the limit of quantitation is estimated as 0.6 ng mL(-1). The method can be used to detect digoxin, a drug used to cure cardiac arrhythmia.

Dual-allele dipstick assay for genotyping single nucleotide polymorphisms by primer extension reaction.

We have developed a dry-reagent dipstick test for simultaneous visual detection of two alleles in single nucleotide polymorphisms (SNPs). The strip comprises two test zones and a control zone. Oligonucleotide-functionalized gold nanoparticles are used as reporters. PCR-amplified DNA that spans the interrogated sequence is subjected to primer extension (PEXT) reactions using allele-specific primers. Digoxigenin-dUTP and biotin-dUTP are incorporated in the extended fragments. The primers contain an oligo(dA) segment at the 5' end. The PEXT products are applied to the sample area of the strip, which is then immersed in the appropriate buffer. As the buffer migrates along the strip by capillary action, the extension products of the two alleles are captured at the test zones from immobilized anti-digoxigenin and streptavidin, whereas the oligo(dA) segment of the primers hybridizes with oligo(dT) strands attached to gold nanoparticles, thus generating characteristic red lines. The excess nanoparticles are captured from immobilized oligo(dA) strands at the control zone of the strip. The test was applied to the genotyping of two SNPs of the Toll-like receptor 4 gene (Asp299Gly and Thr399Ile), one SNP of CYP2C19 gene (CYP2C19(*)3) and one SNP of the TPMT gene (TPMT(*)2). Contrary to most genotyping methods, the dipstick test does not require costly specialized equipment for detection of PEXT products. The PCR product is pipetted directly into the PEXT reaction mixture without prior purification. The high sensitivity of the strip allows completion of PEXT reaction in three cycles only (7 min). The visual detection of both alleles is complete in 15 min.

Shaping emission spectra of fluorescent molecules with single plasmonic nanoresonators.

We show that plasmonic nanoresonators composed of two gold nanoparticles change not only the intensity but also the spectral shape of the emission of fluorescent molecules. The plasmonic resonance frequency can be tuned by varying the distance between the nanoparticles, which allows us to selectively favor transitions of a fluorescent molecule to a specific vibrational ground state. Experimental data from correlated scattering and fluorescence microscopy agree well with calculations in the framework of generalized Mie theory. Our results show that the widely used description of a dye molecule near a metal surface as a mere two-level system is inadequate.

Production of anti-digoxigenin antibody HRP conjugate for PCR-ELISA DIG detection system.

There are several methods used to visualize the end product of polymerase chain reactions. One of these methods is an ELISA-based detection system (PCR-ELISA) which is very sensitive and can be used to measure the PCR products quantitatively by a colorimetric method. According to this technique, copies of DNA segments from genomic DNA are amplified by PCR with incorporation of digoxigenin-11-dUTP. Samples are analyzed in a microtiter plate format by alkaline denaturation and are hybridized to biotinylated allele-specific capture probes bound to streptavidin coated plates. Use of the produced anti-digoxigenin antibody horseradish peroxidase conjugate and the substrate 2,2'-azino-di-3-ethylbenzthiazolinsulfonate (ABTS) detected the hybridized DNA. One of the key components in this procedure is the anti-digoxigenin antibody HRP conjugate. Described here is the preparation, purification, and characterization of anti-digoxigenin antibody HRP conjugate for use in the PCR-ELISA DIG detection system. Several biochemical protocols and modifications were applied to increase the sensitivity and specificity of this conjugate for an efficient and cost-effective product.