(R)evolution of the Standard Addition Procedure for Immunoassays.

A new method to transfer the standard addition procedure for concentration determination to immunoassays with non-linear calibration curves was developed. The new method was successfully applied to simulated data and benchmarked against a state-of-the-art algorithm, showing a significantly improved performance with improvement factors between 2 and 192. The logit function was used to transform the immunoassay signal response of test samples spiked with known analyte concentrations. The relationship between logit(signal) and log-transformed estimated total analyte concentration is linear if the estimated total analyte concentration is correct. Finally, the new method was validated experimentally using different assays in varying, relevant complex matrices, such as serum, saliva, and milk. Different concentrations of testosterone and amitriptyline between 0.05 and 3.0 µg L-1 were quantified using a binding inhibition assay in combination with reflectometric interference spectroscopy (RIfS) as the transduction principle. The sample concentration was calculated using a numerical method. Samples could be quantified with recoveries between 70 and 118%. The standard addition method accounts for individual matrix interference on the immunoassay by spiking the test sample itself. Although the experiments were carried out using RIfS, the method can be applied to any immunoassay that meets the analytical requirements.

Tools to compare antibody gold nanoparticle conjugates for a small molecule immunoassay.

Antibody gold nanoparticle conjugates as recognition elements are essential for the overall performance of lateral flow assays. When immobilizing antibodies on gold nanoparticles, the challenge is to prevent aggregation and to ensure that the antibodies are correctly oriented so that they remain functional and their paratopes remain accessible. There are many methods available, and it is difficult to decide which one to use. To help selecting the most appropriate conjugate production method, different synthetic routes of binding antibodies to gold nanoparticles are systematically investigated for the purpose of a quantitative lateral flow test for small molecules. The direct comparison of different conjugate syntheses shows how to select a suitable conjugate for a lateral flow assay. The syntheses examined are direct adsorption of antibody, direct adsorption of reduced antibody, covalent binding to polyethylene glycol linker, and binding via biotin-streptavidin interaction. The conjugates are characterized using UV-Vis spectroscopy and dynamic light scattering to determine their stability. Their performance on structured lateral flow test strips is examined using calibrations for different amitriptyline concentrations. It was shown that the best conjugate for quantification of amitriptyline was realized by direct adsorption of an UV-light irradiated antibody to gold nanoparticles. The methods employed can serve as a guide for selecting the most appropriate conjugate for an application and enhance the performance of lateral flow assays.

Parallelized label-free monitoring of cell adhesion on extracellular matrix proteins measured by single colour reflectometry.

The understanding of the initial cell adhesion to biomaterials is crucial for the survival of implants. The manifold possibilities to tailor an implant surface and the diverse requirements for different implant applications necessitate a timesaving and highly parallelized analytical methodology. Due to its intrinsic advantages (label-free, time-resolved, robust against temperature fluctuations, and particularly the multiplexing possibilities), single colour reflectometry (SCORE) is used for the first time to investigate cell adhesion to different extracellular matrix protein-coated surfaces. The excellent correlation between the novel SCORE technology and well-established reference methods proves that the results obtained by using this direct optical method are able to reflect the cell binding processes at the transducer surface. Additionally, the high time resolution of SCORE revealed the differences in the adhesion behaviour of the cells on the different extracellular matrix protein-coated glass slides during the initial adsorption phase and during the spreading of the cells on the surfaces. Therefore, we conclude that SCORE is a perfectly suited methodology for studying the entire cell adsorption process, including morphological changes, and shows great potential for other cell-based sensing applications.

Comparison of methods for quantitative biomolecular interaction analysis.

In order to perform good kinetic experiments, not only the experimental conditions have to be optimized, but the evaluation procedure as well. The focus of this work is the in-depth comparison of different approaches and algorithms to determine kinetic rate constants for biomolecular interaction analysis (BIA). The different algorithms are applied not only to flawless simulated data, but also to real-world measurements. We compare five mathematical approaches for the evaluation of binding curves following pseudo-first-order kinetics with different noise levels. In addition, reflectometric interference spectroscopy (RIfS) measurements of two antibodies are evaluated to determine their binding kinetics. The advantages and disadvantages of the individual approach will be investigated and discussed in detail. In summary, we will raise awareness on how to evaluate and judge results from BIA by using different approaches rather than having to rely on "black box" closed (commercial) software packages.

Diagnostic performance study of an antigen microarray for the detection of antiphospholipid antibodies in human serum.

BACKGROUND: The parallelization of clinically relevant antigens in a microarray format is of growing importance due to the ability to measure multiple antigen-antibody interactions. With the development of a microarray for the detection of antiphospholipid antibodies we focussed on one important autoimmune disease that is still diagnostically challenging. Reasons are the heterogeneity of the autoantibodies and the unspecific clinical symptoms. METHODS: For the covalent immobilization of antigenic structures, glass transducers were coated with 11-aminoundecyltrimethoxysilane (11-AUTMS). In total 35 antiphospholipid syndrome (APS) patients, six patients with lupus erythematosus and 24 healthy controls were investigated on a microarray format using polarized imaging reflectometric interference spectroscopy. RESULTS: The novel surface modification based on the short derivative 11-AUTMS resulted in a selective biosensor allowing a clear differentiation of patient and control samples. It combined proteinogenic as well as phospholipid-derived antigens, namely ß2-glycoprotein I (ß2-GPI), prothrombin, cardiolipin (CL) and a ß2-GPI/CL complex. With optimized regeneration conditions, up to 20 consecutive measurements could be performed on one chip. Sensitivity was determined to be 0.800-0.929, specificity was between 0.733 and 0.969, depending on the respective antigen. CONCLUSIONS: Multiplexed determination of serological parameters has a great potential. We have shown that our biosensor is capable of detecting four different APS relevant antibodies in parallel exhibiting a sensitivity and specificity comparable to existing ELISA methods.

Size does matter! Label-free detection of small molecule-protein interaction.

This review is focused on methods for detecting small molecules and, in particular, the characterisation of their interaction with natural proteins (e.g. receptors, ion channels). Because there are intrinsic advantages to using label-free methods over labelled methods (e.g. fluorescence, radioactivity), this review only covers label-free techniques. We briefly discuss available techniques and their advantages and disadvantages, especially as related to investigating the interaction between small molecules and proteins. The reviewed techniques include well-known and widely used standard analytical methods (e.g. HPLC-MS, NMR, calorimetry, and X-ray diffraction), newer and more specialised analytical methods (e.g. biosensors), biological systems (e.g. cell lines and animal models), and in-silico approaches.

Development of a new parallelized, optical biosensor platform for label-free detection of autoimmunity-related antibodies.

Autoimmune diseases are characterized by the presence of autoantibodies in serum of affected patients. The heterogeneity of autoimmune relevant antigens creates a variety of different antibodies, which requires a simultaneous detection mode. For this reason, we developed a tool for parallelized, label-free, optical detection that accomplishes the characterization of multiple antigen-antibody interactions within a single measurement on a timescale of minutes. Using 11-aminoundecyltrimethoxysilane, we were able to immobilize proteinogenic antigens as well as an amino-functionalized cardiolipin on a glass surface. Assay conditions were optimized for serum measurements with a single spot antigen chip on a single spot 1-λ detection system. Minimized background signal allows a differentiation between patients and healthy controls with a good sensitivity and specificity. Applying polarized imaging reflectometric interference spectroscopy, we evaluated samples from three APS patients and three control subjects for this proof-of-principle and already obtained good results for ß2-glycoprotein I and cardiolipin.

A robust sensor platform for label-free detection of anti-Salmonella antibodies using undiluted animal sera.

We present a portable and easy-to-use biosensor platform, allowing for label-free detection of diagnostic markers in undiluted animal serum. Exemplarily, this is shown for the detection of anti-Salmonella antibodies. 1-lambda-Reflectometry was used as detection method, making the new biosensor platform portable, cheap, and robust. As recognition elements, lipopolysaccharides (LPSs) from Salmonella typhimurium bacteria were immobilized as sensitive layer on the transducer to carry out serological tests via a direct assay format. For this purpose, a new surface preparation protocol has been worked out allowing for immobilization of the LPS via hydrophobic interactions. It has been shown that results obtained by 1-lambda-Reflectometry are equivalent to those obtained by the non-portable Reflectometric Interference Spectroscopy setup. The new sensor platform was calibrated in both matrices, buffer and undiluted serum. Good sensitivity, selectivity and intra chip reproducibility have been observed. Furthermore, inter chip reproducibility was examined and recovery rates were found to be between 99 and 117% in undiluted serum.

A novel analytical tool for quantification of estrogenicity in river water based on fluorescence labelled estrogen receptor alpha.

A novel combined procedure for estrogen-affinity purification and labelling of estrogen receptor alpha ligand-binding domain with Cy 5.5 cystein reactive dye was established. By using this procedure, mainly functional proteins are recovered. It can be easily adapted to a large variety of other proteins for which ligand-coated affinity materials are available. The labelled receptor was used in a total internal reflection fluorescence-based binding inhibition assay for determination of the impact of pollutants in river water on the receptor. The great advantage compared to conventional methods is that the total effect on the receptor is measured instead of concentrations of single compounds and that even currently unknown ligands are found as well. Therefore, the obtained signal is related to the response of the organism, which is exposed to the water. The limit of detection was found to be 0.139 nM of estradiol equivalents. The assay also provides a highly sensitive tool for pharmaceutical research and can be adapted to diagnostic applications.

An advanced biosensor for the prediction of estrogenic effects of endocrine-disrupting chemicals on the estrogen receptor alpha.

A label-free and time-resolved biosensor based on reflectometric interference spectroscopy (RIfS) has been developed to evaluate the agonistic or antagonistic effects of potential ligands with unknown behavior. The biosensor utilizes the specific interaction between the estrogen receptor alpha (ER alpha) and short specific peptides. The unique feature of these peptides allows the investigation of the behavior of ligands and the discrimination between the agonistic and antagonistic effects caused by conformational changes of the receptor. Thus, this developed biosensor allows not only the differentiation between ligands and nonligands of a receptor, but also the potential of these ligands to influence conformational changes in the receptor, leading to activation or inhibition of the receptor-dependent pathways. Owing to the robustness of the direct optical detection principle used, the biosensor is applicable to complex biological matrices, even crude cell extracts. Moreover, the reliability of the biosensor, including regeneration steps when performing subsequent measurements, has been verified.

Direct optical detection in fragment-based screening.

Label-free biosensors based on direct optical detection principles are widely used in many different fields of research. Currently the higher level of automation and the increasing throughput of this technology are stimulating the interest of pharmaceutical companies. The information gained with label-free biosensors can be extremely valuable during the drug design process, particularly in combination with complementary techniques, including NMR, mass spectrometry and X-ray crystallography. In this article we focus on the advantages of direct optical biosensors especially in the field of fragment-based drug design, which is a widely used and extremely promising concept. Furthermore, we present optical biosensors as versatile tools for fragment-based screening and the future drug design process.

New and versatile optical-immunoassay instrumentation for water monitoring.

This article is a review of new and versatile optical-immunoassay instrumentation for water monitoring developed through two European Union projects, RIver ANAlyser (RIANA) and Automated Water Analyser Computer Supported System (AWACSS). Both projects utilise immunoassay techniques to isolate the analytes and Total Internal Reflection Fluorescence (TIRF) to quantify them. Completed in 1999, the RIANA project developed a sensitive and cost-effective analytical system capable of simultaneous detection of multiple-analytes in real-world water samples. The AWACSS project has been in progress since 2001 and is developing rugged-but-sensitive instrumentation that will detect up to 30 analytes simultaneously, will operate unattended, and will have networking capability.