Development of a candidate secondary reference procedure (immunoassay based measurement procedure of higher metrological order) for cardiac troponin I: I. Antibody characterization and preliminary validation.

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.

Preparation and quality control of silver nanoparticle-antibody conjugate for use in electrochemical immunoassays.

Metal nanoparticle-antibody conjugates are often used as optical or electrochemical markers in applications like immunohistochemistry, lateral flow tests, biosensors and immunoassays. In order to serve that role, an antibody needs to be immobilized on the surface of the nanoparticle. This is easily done, as proteins bind to gold and silver nanoparticles spontaneously. However, this immobilization process might result in nanoparticle aggregation or the loss of the bioactivity of the conjugated antibodies. In this work the optimization of antibody immobilization on silver colloid in order to obtain conjugates with the best possible activity is investigated. The parameters investigated were the type of immobilization buffer, its molarity and pH, the nanoparticle/antibody ratio and also blocking and washing protocols to reduce non-specific binding. The functionality of the obtained conjugates was tested with electrochemical immunoassay. It was found out that the optimum environment for immobilization of an anti-myoglobin antibody on silver nanoparticles was 0.2M boric acid pH 6.5 with 10 µg of antibody loading per 1 mL of silver colloid. For an anti-troponin antibody it was 0.1M boric acid pH 7.5 also with 10 µg/mL of antibody loading. The main problem for silver conjugation was the tendency of silver nanoparticles to aggregate during the immobilization process, but by choosing the optimum conditions the aggregation problem was completely removed. Here it is demonstrated that by using the conjugates prepared with an optimized protocol an increase in the sensitivity of the assay 10 times can be achieved. The electrochemical immunoassay described here can be used as a test for quality control of conjugates and for the estimation of batch-to-batch variability.

Bioconjugation and characterisation of gold colloid-labelled proteins.

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.

Standardisation of cardiac troponin I measurement: past and present.

The laboratory measurement of cardiac troponin (cTn) concentration is a critical tool in the diagnosis of acute myocardial infarction (MI). Current cTnI assays produce different absolute troponin numbers and use different clinical cut-off values; hence cTnI values cannot be interchanged, with consequent confusion for clinicians. A recent Australian study compared patient results for seven cTnI assays and showed that between-method variation was approximately 2- to 5-fold. A major reason for poor method agreement is the lack of a suitable common reference material for the calibration of cTnI assays by manufacturers. Purified complexed troponin material lacks adequate commutability for all assays; hence a serum-based secondary reference material is required for cTnI with value assignment by a higher order reference measurement procedure. There is considerable debate about how best to achieve comparability of results for heterogeneous analytes such as cTnI, whether it should be via the harmonisation or the standardisation process. Whereas harmonisation depends upon consensus value assignment and uses those commercial methods which give the closest agreement at the time, standardisation comes closer to the true value through a reference measurement system that is based upon long-term calibration traceability. The current paper describes standardisation efforts by the International Federation of Clinical Chemistry and Laboratory Medicine Working Group on Standardization of cTnI (IFCC WG-TNI) to establish a reference immunoassay measurement procedure for cTnI of a higher order than current commercial immunoassay methods and a commutable secondary reference material for cTnI to which companies can reference their calibration materials.

Standardization of troponin I measurements: an update.

Standardization of cardiac troponin I (cTnI) measurement is important because of the central role for diagnosis of myocardial infarction. In blood, cTnI is present as a heterogeneous mixture of different molecular species. The analytical problem caused by this heterogeneity may be circumvented by recognition of a unique, invariant part of the molecule that is common to all components of the mixture. Antibodies used for the development of cTnI assays should selectively recognize epitopes within this invariant part, leading to a consequential increase in the homogeneity of immunoassay reactivity. This should be associated with the use of a reference material that represents the natural and major antigen in blood after tissue release, i.e., the troponin complex. Although a primary reference material for cTnI is available, studies indicate that cTnI assays remain without harmony after recalibration using this material. To achieve closer comparability of cTnI values between assays, the use of a secondary reference material, consisting of a panel of human serum pools, is proposed for use by manufacturers to calibrate their assays. To assign true cTnI concentration values to this secondary reference material, establishment of a reference measurement procedure for cTnI is required. A practical approach to the development of a reference procedure could be to design an immunochemical assay with well-characterized specificity to the invariant part of the cTnI molecule and calibrated using the primary reference material.

An international comparability study to determine the sources of uncertainty associated with a non-competitive sandwich fluorescent ELISA.

BACKGROUND: Immunoassays allow the specific detection and quantitation of biological molecules in complex samples at physiologically relevant concentrations. However, there are concerns over the comparability of such techniques when the same assay is performed by different operators or laboratories. An international intercomparison study was performed to assess the uncertainty involved in the 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.