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Efficient gene transfer is necessary for advanced biotechnologies ranging from gene therapy to synthetic biology. Peptide nanoparticles provide suitable packaging systems promoting targeted gene expression or silencing. Though these systems have yet to match the transfection efficacy of viruses, they are typically devoid of drawbacks characteristic of virus-based vectors, including insertional mutagenesis, low packaging capacities, and strong immune responses. Given the promise nanoparticle formulations hold for gene delivery, methods of their preparation and accurate analysis of their physicochemical and biological properties become indispensable for progress toward systems that seek to outperform viral vectors. Herein, we report a comprehensive protocol for the preparation and characterization of archetypal peptide nanoparticles resulting from nonspecific and noncovalent complexation with RNA and DNA.

Efficient gene transfer is necessary for advanced biotechnologies ranging from gene therapy to synthetic biology. Peptide nanoparticles provide suitable packaging systems promoting targeted gene expression or silencing. Though these systems have yet to match the transfection efficacy of viruses, they are typically devoid of drawbacks characteristic of virus-based vectors, including insertional mutagenesis, low packaging capacities, and strong immune responses. Given the promise nanoparticle formulations hold for gene delivery, methods of their preparation and accurate analysis of their physicochemical and biological properties become indispensable for progress toward systems that seek to outperform viral vectors. Herein, we report a comprehensive protocol for the preparation and characterization of archetypal peptide nanoparticles resulting from nonspecific and noncovalent complexation with RNA and DNA.

DNA nanostructures constitute a rapidly advancing tool-set for exploring cell-membrane functions and intracellular sensing or advancing delivery of biomolecular cargo into cells. Chemical conjugation with lipid anchors can mediate binding of DNA nanostructures to synthetic lipid bilayers, yet how such structures interact with biological membranes and internalize cells has not been shown. Here, an archetypal 6-duplex nanobundle is used to investigate how lipid conjugation influences DNA cell binding and internalization kinetics. Cellular interactions of DNA nanobundles modified with one and three cholesterol anchors were assessed using flow cytometry and confocal microscopy. Nuclease digestion was used to distinguish surface-bound DNA, which is nuclease accessible, from internalized DNA. Three cholesterol anchors were found to enhance cellular association by up to 10-fold when compared with unmodified DNA. The bundles were endocytosed efficiently within 24 h. The results can help design controlled DNA binding and trafficking into cells.

A synthetic topology for everted viruses is reported. The topology is a single-stranded virion DNA assembled into a hollow cube with exterior decorated with HIV-Tat transduction domains. The cube incorporates a pH-responsive lid allowing for the controlled encapsulation of functional proteins and their transfer and release into live cells. Unlike viruses, which are protein shells with a [3,5]-fold rotational symmetry that encase nucleic acids, these cubes are [3, 4]-fold DNA boxes encapsulating proteins. Like viruses, such everted DNA-built viruses are monodisperse nanoscale assemblies that infect human cells with a specialist cargo. The design offers a bespoke bottom-up platform for engineering nonpolyhedral, nonprotein synthetic viruses.

A conceptual design for artificial antimicrobial viruses is described., A conceptual design for artificial antimicrobial viruses is described. The design emulates viral assembly and function to create self-assembling peptide capsules that promote efficient gene delivery and silencing in mammalian cells. Unlike viruses, however, the capsules are antimicrobial, which allows them to exhibit a dual biological function: gene transport and antimicrobial activity. Unlike other antimicrobials, the capsules act as pre-concentrated antimicrobial agents that elicit rapid and localised membrane-disrupting responses by converting into individual pores at their precise landing positions on membranes. The concept holds promise for engineering virus-like scaffolds with biologically tuneable properties.

The batch-to-batch assay performance ‘activity’ of antibody conjugated particles is often variable, leading to poor reproducibility between different production batches. We therefore sought to quantify the properties of such particles using differential centrifugal sedimentation (DCS) to see how they influence assay performance, with the aim to improve the reproducibility of the conjugation reaction. The DCS high resolution size distributions of the antibody conjugated particles allowed us to examine the thickness of the antibody corona on the particles and to quantify the amount of ‘contaminating’ particle oligomers produced via carbodiimide chemistry. The DCS data was correlated with the assay response of the resulting conjugate using an interleukin 6 (IL6) lateral flow assay developed in house. We prepared a series of antibody conjugates using various carbodiimide reaction conditions and analysed the size, antibody corona and oligomerisation profile of the resulting particles using both dynamic light scattering (DLS) and DCS. Both the amount of antibody bound to the particle and the presence of higher order particle oligomers produced conjugates that when applied in an IL6 lateral flow assay were associated with an enhanced fluorescent signal. Both the amount of particle oligomers and antibody bound to the particle was found to be positively correlated with increased assay response in the lateral flow. The DCS estimation of protein corona thickness for each carbodiimide condition tested was found to correlate with the amount of antibody coupled to the particles, as assessed using the bicinchoninic acid (BCA) assay. We have shown the novel application of DCS for the analysis of antibody-particle conjugates. DCS analysis provides a quantitative method to characterise particles and provides a rationale for variable assay performance observed from batch-to-batch production.

Sequence-prescribed biomolecular assemblies find increasing use in the development of novel nanostructured materials. Critical requirements for emerging designs remain in matching form with function. Peptide assembly diversifies form and supports function, but lacks control over both. Herein we exploit length correlations in peptide nanoscale fibres (form) using a model helical template. We establish that different assembly patterns result from a synergistic interplay between peptide length, net charge and folding and supra-molecular cooperativity, while correlating with increases in cell proliferation (function) as a function of peptide length. The revealed correlations offer an efficient rationale for the programming of longitudinally finite and biologically active nanoscale fibres.

We present the first assay combining a dual aptamer sandwich format with detection by anodic stripping voltammetry with ionic silver amplification. This assay format lends itself to rapid point-of-care tests, where the use of aptamers could improve the overall stability of the assay. We have used human alpha-thrombin as a model system, and demonstrate a detection limit of 6.09 μg L−1. We present the optimization of the aptamer-silver colloid attachment chemistry and the final assay format to achieve sensitive analyte detection. The use of a sandwich assay coupled with magnetic separation and ionic silver amplification, generates an assay with similar sensitivity than those reported in the literature in a format that can be used in rapid, portable testing regimes.

A de novo topology of virus-like assembly is reported. The design is a trifaceted coiled-coil peptide helix, which self-assembles into ultrasmall, monodisperse, anionic virus-like shells that encapsulate and transfer both RNA and DNA into human cells. Unlike existing artificial systems, these shells share the same physical characteristics of viruses being anionic, nonaggregating, abundant, hollow, and uniform in size, while effectively mediating gene silencing and transgene expression. These are the smallest virus-like structures reported to date, both synthetic and native, with the ability to adapt and transfer small and large nucleic acids. The design thus offers a promising solution for engineering bespoke artificial viruses with desired functions.

RNAi is an indispensable research tool with a substantial therapeutic potential. However, the complete transition of the approach to an applied capability remains hampered due to poorly understood relationships between siRNA delivery and gene suppression. Here we propose that interfacial tertiary contacts between α-helices can regulate siRNA cytoplasmic delivery and RNAi. We introduce a rationale of helical amphipathic lockers that differentiates autonomously folded helices, which promote gene silencing, from helices folded with siRNA, which do not. Each of the helical designs can deliver siRNA into cells via energy-dependent endocytosis, while only autonomously folded helices with pre-locked hydrophobic interfaces were able to promote statistically appreciable gene silencing. We propose that it is the amphipathic locking of interfacing helices prior to binding to siRNA that enables RNAi. The rationale offers structurally balanced amphipathic scaffolds to advance the exploitation of functional RNAi.

Here we describe scattering based signal suppression artifacts encountered while developing multiplex lateral flow (LF) immunoassay using surface enhanced Raman spectroscopy (SERS) "nanotags" as analyte labels. Using these SERS nanotags, we have produced a quantitative test for inflammation biomarkers that is transferable to the point of care (POC). The SERS assay shows similar performance when compared with a fluorescent nanoparticle POC test. Here, using cardiac and inflammation biomarkers, we highlight the need to carefully optimize the concentration of assay components when using SERS nanotags and a single-line multiplexing approach. We show that in certain circumstances the SERS signal may be suppressed, leading to a significant underestimation of the analyte concentrations. Using electron microscopy and optical spectroscopy, we demonstrate that the error in the measurement is associated with the light scattering properties of the nanotags. These findings will be applicable to other nanoparticle labels with high light scattering coefficients. Through careful modification of the assay to reduce the impact of light scattering, it is possible to produce quantitative assays, but potentially at the expense of multiplexing capability and assay sensitivity.

One of the main challenges in nanoecotoxicological investigations is in the selection of the most suitable measurement methods and protocols for nanoparticle characterisation. Several parameters have been identified as being important as they govern nanotoxicological activity, with some parameters being better defined than others. For example, as a parameter, there is some ambiguity as to how to measure dispersion stability in the context of ecotoxicological investigations; indeed, there is disagreement over which are the best methods to measure nanoparticle dispersion stability. The purpose of this article is to use various commercially available tools to measure dispersion stability and to understand the information given by each tool. In this study, CeO2 was dispersed in two different types of media: de-ionised water and electrolyte-containing fish medium. The DLS mean particle size of freshly dispersed sample in DI water was ~200 nm in diameter. A visual sedimentation experiment showed that nanoparticle dispersion made in the fish medium was less stable compared to corresponding dispersion in de-ionised water. Stability of these dispersions was monitored using various techniques, for a period of 3 days. Our findings have shown that dispersion stability can be suitably assessed by monitoring: (a) surface charge, (b) sedimentation events and (c) presence of agglomerates, through time. The majority of techniques employed here (zeta potential, particle size via DLS, fluorescence and UV–Vis spectroscopy and SEM) were shown to provide useful, complementary information on dispersion stability. Nanoparticle Tracking Analysis (NTA) provides useful, quantitative information on the concentration of nanoparticles in suspension, but is limited by its inability to accurately track the motion of large agglomerates found in the fish medium.

Synthetic polymers and colloids are increasingly being exploited in bioassays to help measure gene expression, sequence genomes, monitor metabolic disorders and detect the presence of disease. This can be attributed to their potential to reduce reaction scales, improve throughput, lower costs and improve the sensitivity, selectivity, stability and reproducibility of assays. This review highlights the newest application areas, including some of the strategies employed, as well as major technical challenges and future opportunities. The move away from conventional assay approaches is being driven by a desire to improve our basic understanding of human biology, to diagnose diseases earlier, and to manage healthcare resources more efficiently. These endeavors are important owing to a rising world population and an increasing average life span.

This paper describes the production and characteristics of the nanoparticle test materials prepared for common use in the collaborative research project NanoChOp (Chemical and optical characterisation of nanomaterials in biological systems), in casu suspensions of silica nanoparticles and CdSe/CdS/ZnS quantum dots. This paper is the first to illustrate how to assess whether nanoparticle test materials meet the requirements of a 'reference material' (ISO Guide 30:2015) or rather those of the recently defined category of 'representative test material' (ISO TS 16195:2013). The NanoChOp test materials were investigated with small-angle X-ray scattering (SAXS), dynamic light scattering (DLS) and centrifugal liquid sedimentation (CLS) to establish whether they complied with the required monomodal particle size distribution. The presence of impurities, aggregates, agglomerates and viable microorganisms in the suspensions was investigated with DLS, CLS, optical and electron microscopy and via plating on nutrient agar. Suitability of surface functionalization was investigated with attenuated total reflection Fourier transform infrared spectrometry (ATR-FTIR) and via the capacity of the nanoparticles to be fluorescently labeled or to bind antibodies. Between-unit homogeneity and stability were investigated in terms of particle size and zeta potential. This paper shows that only based on the outcome of a detailed characterization process one can raise the status of a test material to representative test material or reference material, and how this status depends on its intended use.

As a part of an International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) project to prepare a commutable reference material for cardiac troponin I (cTnI), a pilot study evaluated current cTnI assays for measurement equivalence and their standardization capability.cTnI-positive samples collected from 90 patients with suspected acute myocardial infarction were assessed for method comparison by 16 cTnI commercial assays according to predefined testing protocols. Seven serum pools prepared from these samples were also assessed.Each assay was assessed against median cTnI concentrations measured by 16 cTnI assays using Passing-Bablok regression analysis of 79 patient samples with values above each assay’s declared detection limit. We observed a 10-fold difference in cTnI concentrations for lowest to highest measurement results. After mathematical recalibration of assays, the between-assay variation for patient samples reduced on average from 40% to 22% at low cTnI concentration, 37%–20% at medium concentration, and 29%–14% at high concentration. The average reduction for pools was larger at 16%, 13% and 7% for low, medium and high cTnI concentrations, respectively. Overall, assays demonstrated negligible bias after recalibration (y-intercept: –1.4 to 0.3 ng/L); however, a few samples showed substantial positive and/or negative differences for individual cTnI assays.All of the 16 commercial cTnI assays evaluated in the study demonstrated a significantly higher degree of measurement equivalence after mathematical recalibration, indicating that measurement harmonization or standardization would be effective at reducing inter-assay bias. Pooled sera behaved similarly to individual samples in most assays.

The measurement of a solubilized protein concentration in solution is an important assay in biochemistry research and development labs for applications ranging from enzymatic studies to providing data for biopharmaceutical lot release. Spectrophotometric protein quantification assays are methods that use UV and visible spectroscopy to rapidly determine the concentration of protein, relative to a standard, or using an assigned extinction coefficient. Where multiple samples need measurement, and/or the sample volume and concentration is limited, preparations of the Coomassie dye commonly known as the Bradford assay can be used.

The measurement of a solubilized protein concentration in solution is an important assay in biochemistry research and development labs for applications ranging from enzymatic studies to providing data for biopharmaceutical lot release. Spectrophotometric protein quantification assays are methods that use UV and visible spectroscopy to rapidly determine the concentration of protein, relative to a standard, or using an assigned extinction coefficient. Where multiple samples need measurement, and/or the sample volume and concentration is limited, preparations of the Coomassie dye commonly known as the Bradford assay can be used.

In this study we describe a novel method of sampling and quantifying wound biomarkers for clinical settings. We believe the chosen format will allow rapid assessments of wound healing and provide biomarker evidence-based decision points for treatment of the wound at the time of presentation. The wound monitoring principle uses a proprietary sample collection tool (a thermally reversible hydrogel) to sample and isolate biomarkers within a wound environment without further sample extraction/preparation steps. We show how gel samples can be analysed in a lateral flow assay format utilising fluorescent microspheres with optically discrete emission characteristics and demonstrate quantitative detection of two analytes (duplexing) achieved in a single test line. As a model assay, the chronic wound biomarkers interleukin 6 (IL6) and tumour necrosis factor alpha (TNFα) are used. Limits of detection of 48.5 pg/mL and 55.5 pg/mL respectively in hydrogel samples and 7.15 pg/mL and 10.7 pg/mL respectively in plasma are reported. We believe this is the first literature example of quantitative detection of multiple analytes within a single test line using spectral separation to distinguish the analytes.

The detection of specific proteins as biomarkers of disease, health status, environmental monitoring, food quality, control of fermenters and civil defence purposes means that biosensors for these targets will become increasingly more important. Among the technologies used for building specific recognition properties, molecularly imprinted polymers (MIPs) are attracting much attention. In this critical review we describe many methods used for imprinting recognition for protein targets in polymers and their incorporation with a number of transducer platforms with the aim of identifying the most promising approaches for the preparation of MIP-based protein sensors (277 references).

Background. Immunoassays are biochemical tests applied to measure even very small amounts of substance using the highly specific binding between an antibody and its antigen. They have a wide range of applications. The measurement however, might be associated with substantial uncertainty; this can have significant consequences for any diagnosis, or clinical decision. An international comparability study was thus performed to assess the uncertainty involved in the estimation of a protein cytokine concentration using a fluorescent ELISA. Methods. In contrast to the original publication for this international comparability study, we reanalyse the data using Bayesian inference. This provides a statistically coherent approach to estimate ELISA concentrations and their associated uncertainties. Results. The Bayesian uncertainties of individual ELISAs and laboratory estimates are considerably larger than previously reported uncertainties. The average concentrations estimated here differ from the ones estimated by each study participant. In general, this leads to different conclusions about the study. In particular, the inter- and intra-laboratory consistency is increased, and repeatability problems occur for fewer laboratories. Conclusions. Decisions which are based on plausible ranges of measurements (such as credible intervals), are generally superior to those solely based on point estimates (such as the mean). Reliable uncertainties are thus vital - and not only in metrology. In this paper, a general method is developed to derive concentration estimates and valid uncertainties for ELISAs. Guidance on applying this Bayesian method is provided and the importance of reliable uncertainties associated with ELISAs is underlined. The applicability and virtues of the presented method are demonstrated in the context of an international comparability study.

In this study, the first steps in the development of a secondary reference measurement procedure (RMP) ‘higher metrological order measurement procedure’ to support the cardiac troponin I (cTnI) standardization initiative is described. The RMP should be used to assign values to serum-based secondary reference materials (RMs) without analytical artifacts causing bias. A multiplexed bead-based assay and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were used to identify the optimum monoclonal antibody pair (clones 560 and 19C7) for the RMP. Using these antibodies, an ELISA-based procedure was developed to accurately measure the main cTnI forms present in blood. The proposed RMP appears to show no bias when tested on samples containing various troponin complexes, phosphorylated and dephosphorylated forms, and heparin. The candidate assay displayed suitable linearity and sensitivity (limit of detection, 0.052 μg/L) for the measurement of the proposed cTnI secondary RMs. Preliminary comparison data on patient samples with a commercial cTnI assay are also provided to support the suitability of RMP for value assignment to RMs. Full validation and final assessment of the RMP will be performed through transferability and inter-comparison studies. Clin Chem Lab Med 2010;48:1603–10.

Colloidal metal particles, in particular gold, have found many biological applications often as probes in light and electron microscopy, and more recently since the 1980s in membrane-based rapid immunoaffinity tests. The surface plasmon resonance absorbance properties in the visible spectroscopy region of gold colloids make them useful tools in medical devices, as the colloids are directly visible to the naked eye. Despite the relative ease with which gold-protein conjugates can be prepared a major issue is the manufacture of poor-quality and poorly characterised bioconjugates that can result in the under performance of subsequent diagnostic tests. This paper describes the preparation of good-quality conjugates for use in immunoassays by optimising the adsorption of antibodies onto the surface of gold colloids, followed by their subsequent characterisation. The conjugates were characterized for size, aggregation and quality using a range of techniques: UV–visible (UV/Vis) absorption spectroscopy, transmission electron microscopy (TEM) and dynamic light scattering (DLS). The biological activities of the conjugated products were also assessed using an immunoassay format and electrochemical measurements. By utilising a number of measurement techniques we aimed to gain a better understanding of the extent of particle aggregation, and the resulting stability and activity of the biological molecule on the surfaces of nanoparticles. The tools developed will enable researchers and companies to ensure the sensitivity, quality and reproducibility of batches of nanoparticle bio-conjugates.

The measurement of protein concentration in an aqueous sample is an important assay in biochemistry research and development labs for applications ranging from enzymatic studies to providing data for biopharmaceutical lot release. Spectrophotometric protein quantitation assays are methods that use UV and visible spectroscopy to rapidly determine the concentration of protein, relative to a standard, or using an assigned extinction coefficient. Methods are described to provide information on how to analyze protein concentration using UV protein spectroscopy measurements, traditional dye-based absorbance measurements; BCA, Lowry, and Bradford assays and the fluorescent dye-based assays; amine derivatization and detergent partition assays. The observation that no single assay dominates the market is due to specific limitations of certain methods that investigators need to consider before selecting the most appropriate assay for their sample. Many of the dye-based assays have unique chemical mechanisms that are prone to interference from chemicals prevalent in many biological buffer preparations. A discussion of which assays are prone to interference and the selection of alternative methods is included.

The measurement of protein concentration in an aqueous sample is an important assay in biochemistry research and development labs for applications ranging from enzymatic studies to providing data for biopharmaceutical lot release. Spectrophotometric protein quantitation assays are methods that use UV and visible spectroscopy to rapidly determine the concentration of protein, relative to a standard, or using an assigned extinction coefficient. Methods are described to provide information on how to analyze protein concentration using UV protein spectroscopy measurements, traditional dye-based absorbance measurements; BCA, Lowry, and Bradford assays and the fluorescent dye-based assays; amine derivatization and detergent partition assays. The observation that no single assay dominates the market is due to specific limitations of certain methods that investigators need to consider before selecting the most appropriate assay for their sample. Many of the dye-based assays have unique chemical mechanisms that are prone to interference from chemicals prevalent in many biological buffer preparations. A discussion of which assays are prone to interference and the selection of alternative methods is included.

Background: Immunoassays allow the specific detec- a tion and quantitation of biological molecules in la complex samples at physiologically relevant concen- in trations. However, there are concerns over the gl comparability of such techniques when the same sa assay is performed by different operators or labora- st tories. An international intercomparison study was ar performed to assess the uncertainty involved in the ol estimation of a protein cytokine concentration using a fluorescent ELISA. Methods: The intercomparison study method was based on a non-competitive sandwich immunoassay with an enhancement step to generate a fluorescent readout. The intercomparison was performed in two phases, with the uncertainty of the instrument determined separately from that of the assay. The 11 laboratories participating in the study represented national metrology institutes or nominated expert laboratories. Results: Participants were asked to determine an undisclosed concentration of interferon using a supplied standard. The mean participant estimate and experimental standard deviation of the mean was 3.54±0.22 mg/L, with the spread of data ranging around ±35% of the mean. The quantitation range of the ELISA and of participants' instruments displayed large variation that contributed to the overall uncertainty. Conclusions: Identified sources of uncertainty within the ELISA methodology included pipetting, data fitting, model selection and instrument/plate variation.

Gene expression technology offers great potentials to generate new insights into human disease pathogenesis; however, the data quality remains a major obstacle for realizing its potentials. In the present study 60-mers oligonucleotide target immobilized on coated glass slides were utilized as a model system to investigate parameters, such as target concentration, retention, signal linearity, and fluorescence properties of fluorophores, which likely affect the quality of microarray results. An array calibration slide was used to calibrate an Axon GenePix 4000A scanner and ensure the dynamic range of the instrument. The work is a first step toward our goal of quantitative gene expression measurements.

In order to rationally select and design probes for real-time PCR, we have determined the influence of the overhang region of the complementary strand on the resulting fluorescence from a hybridising probe. A series of target oligonucleotides, each with a unique 3' overhang (4 bases), was hybridised to either 5' fluorescein (FAM)- or Alexa-488-labelled probes, and the changes in fluorescence properties were monitored. We found that the number of guanine bases in the overhang region of the target oligonucleotides was proportional to the amount of fluorescence quenching observed for both the FAM and Alexa-488 dyes. FAM appeared to be more sensitive to guanine-induced quenching with three and four guanine bases resulting in greater than a twofold decrease in the quantum yield of the fluorophore compared to the no-overhang target. In addition, we found that adenine bases caused fluorescence quenching of the Alexa-488-labelled probe, whereas the FAM-labelled probe appeared insensitive. The quenching data, generated with the steady-state fluorescence measurements, displayed a linear correlation with that obtained using a fluorescent thermal cycler, suggesting the applicability to real-time PCR measurements. Anisotropy data from the series of duplexes correlated with the fluorescence quantum yield, suggesting that quenching was accompanied by increased dye mobility.

A homogeneous microplate assay for the serine/threonine protein phosphatases PP1 and PP2A, employing fluorescent-labeled phosphopeptides, has been developed. Phosphopeptides derived from a phosphoacceptor site in myelin basic protein were designed with a cysteine adjacent to the phosphoresidue, allowing site-selective labeling with dyes. The fluorescence emission from the environmentally sensitive fluorophore 7-fluorobenz-2-oxa-1,3-diazole-4-sulfonamide was found to be sensitive to the phosphorylation status of an adjacent threonine residue. Upon complete dephosphorylation of the dye-labeled phosphopeptide, a 56% decrease in fluorescence intensity was observed. The change in fluorescence was correlated with the release of inorganic phosphate from the phosphopeptide as measured using the malachite green assay. Conjugation of the fluorophore to the phosphopeptide was found to have no adverse effect on catalysis. A series of four phosphopeptide substrates were developed and characterized to probe PP1 and PP2A activity. The optimum phosphopeptides were then used to determine inhibition parameters for three natural protein phosphatase inhibitors. The use of a peptide-based approach has introduced a degree of specificity not observed with many conventional phosphatase substrates, while retaining the advantages of a real-time homogeneous fluorescence-based format, making the assay ideal for high-density screening.

The CCQM study P58.1 assessed the equivalence of immunoassay measurements between participating NMIs. The aim of P58.1 was to demonstrate the equivalence of immunoassay measurements to determine the mass concentration of the clinically-relevant protein human cardiac troponin I (cTnI) present at low concentration relative to the protein concentration of the sample matrix. The measurement equivalence was assessed using traceability to a common certified reference material. To quantify cTnI, participants used a homogeneous sandwich-based immunoassay with an enzymatic amplification step. The antibody format consisted of a single capture and single detection antibody (referred to as 1 + 1), both were supplied to study participants. In the previous P58 study, ELISA measurement results were compared between laboratories which all used common ELISA reagents (including 96-well plates), samples, a standard for the production of calibrants, and a detailed ELISA protocol, which were supplied by a single laboratory. The P58.1 study only utilized common samples, a standard of the production of calibrants, and a set of monoclonal antibodies (mAbs). Because much of the experimental procedure for the P58 study was essentially standardized across participating labs, the study primarily highlighted between-laboratory differences in plate sampling designs and in plate reader response. As the participants in the P58.1 study had to produce most of their own analytical reagents and develop their own measurement procedure, the study provides a better evaluation of the equivalence of ELISA measurements between the participating laboratories.Main text. To reach the main text of this paper, click on Final ReportThe final report has been peer-reviewed and approved for publication by CCQM.

Dye-based protein determination assays are widely used to estimate protein concentration, however various reports suggest that the response is dependent on the composition and sequence of the protein, limiting confidence in the resulting concentration estimates. In this study a diverse set of model proteins representing various sizes of protein and covalent modifications, some typical of biopharmaceuticals have been used to assess the utility of dye-based protein concentration assays. The protein concentration assays (Bicinchoninic acid (BCA), Bradford, 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA™), DC, Fluorescamine and Quant-iT™) were compared to the ‘gold standard’ assay, quantitative amino acid analysis (AAA). The assays that displayed the lowest variability between proteins, BCA and DC, also generated improved estimates when BSA was used as a standard, when compared to AAA derived concentrations. Assays read out by absorbance tended to display enhanced robustness and repeatability, whereas the fluorescence based assays had wider quantitation ranges and lower limits of detection. Protein modification, in the form of glycosylation and PEGylation, and the addition of excipients, were found to affect the estimation of protein concentration for some of the assays when compared to the unmodified protein. We discuss the suitability and limitations of the selected assays for the estimation of protein concentration in biopharmaceutical applications.