Automated water analyser computer supported system (AWACSS) Part I: Project objectives, basic technology, immunoassay development, software design and networking.

A novel analytical system AWACSS (automated water analyser computer-supported system) based on immunochemical technology has been developed that can measure several organic pollutants at low nanogram per litre level in a single few-minutes analysis without any prior sample pre-concentration nor pre-treatment steps. Having in mind actual needs of water-sector managers related to the implementation of the Drinking Water Directive (DWD) (98/83/EC, 1998) and Water Framework Directive WFD (2000/60/EC, 2000), drinking, ground, surface, and waste waters were major media used for the evaluation of the system performance. The instrument was equipped with remote control and surveillance facilities. The system's software allows for the internet-based networking between the measurement and control stations, global management, trend analysis, and early-warning applications. The experience of water laboratories has been utilised at the design of the instrument's hardware and software in order to make the system rugged and user-friendly. Several market surveys were conducted during the project to assess the applicability of the final system. A web-based AWACSS database was created for automated evaluation and storage of the obtained data in a format compatible with major databases of environmental organic pollutants in Europe. This first part article gives the reader an overview of the aims and scope of the AWACSS project as well as details about basic technology, immunoassays, software, and networking developed and utilised within the research project. The second part article reports on the system performance, first real sample measurements, and an international collaborative trial (inter-laboratory tests) to compare the biosensor with conventional anayltical methods.

Automated Water Analyser Computer Supported System (AWACSS) Part II: Intelligent, remote-controlled, cost-effective, on-line, water-monitoring measurement system.

A novel analytical system AWACSS (Automated Water Analyser Computer Supported System) based on immunochemical technology has been evaluated that can measure several organic pollutants at low nanogram per litre level in a single few-minutes analysis without any prior sample pre-concentration or pre-treatment steps. Having in mind actual needs of water-sector managers related to the implementation of the Drinking Water Directive (DWD) [98/83/EC, 1998. Council Directive (98/83/EC) of 3 November 1998 relating to the quality of water intended for human consumption. Off. J. Eur. Commun. L330, 32-54] and Water Framework Directive (WFD) [2000/60/EC, 2000. Directive 2000/60/EC of the European Parliament and of the Council of 23 October 2000 establishing a framework for Community action in the field of water policy. Off. J. Eur. Commun. L327, 1-72], drinking, ground, surface, and waste waters were major media used for the evaluation of the system performance. The first part article gave the reader an overview of the aims and scope of the AWACSS project as well as details about basic technology, immunoassays, software, and networking developed and utilised within the research project. The second part reports on the system performance, first real sample measurements, and an international collaborative trial (inter-laboratory tests) to compare the biosensor with conventional anayltical methods. The systems' capability for analysing a wide range of environmental organic micro-pollutants, such as modern pesticides, endocrine disrupting compounds and pharmaceuticals in surface, ground, drinking and waste water is shown. In addition, a protocol using reconstitution of extracts of solid samples, developed and applied for analysis of river sediments and food samples, is presented. Finally, the overall performance of the AWACSS system in comparison to the conventional analytical techniques, which included liquid and gas chromatographic systems with diode-array UV and mass spectrometric detectors, was successfully tested in an inter-laboratory collaborative trial among six project partners.

Antibody-antigen-adjuvant conjugates enable co-delivery of antigen and adjuvant to dendritic cells in cis but only have partial targeting specificity.

Antibody-antigen conjugates, which promote antigen-presentation by dendritic cells (DC) by means of targeted delivery of antigen to particular DC subsets, represent a powerful vaccination approach. To ensure immunity rather than tolerance induction the co-administration of a suitable adjuvant is paramount. However, co-administration of unlinked adjuvant cannot ensure that all cells targeted by the antibody conjugates are appropriately activated. Furthermore, antigen-presenting cells (APC) that do not present the desired antigen are equally strongly activated and could prime undesired responses against self-antigens. We, therefore, were interested in exploring targeted co-delivery of antigen and adjuvant in cis in form of antibody-antigen-adjuvant conjugates for the induction of anti-tumour immunity. In this study, we report on the assembly and characterization of conjugates consisting of DEC205-specific antibody, the model antigen ovalbumin (OVA) and CpG oligodeoxynucleotides (ODN). We show that such conjugates are more potent at inducing cytotoxic T lymphocyte (CTL) responses than control conjugates mixed with soluble CpG. However, our study also reveals that the nucleic acid moiety of such antibody-antigen-adjuvant conjugates alters their binding and uptake and allows delivery of the antigen and the adjuvant to cells partially independently of DEC205. Nevertheless, antibody-antigen-adjuvant conjugates are superior to antibody-free antigen-adjuvant conjugates in priming CTL responses and efficiently induce anti-tumour immunity in the murine B16 pseudo-metastasis model. A better understanding of the role of the antibody moiety is required to inform future conjugate vaccination strategies for efficient induction of anti-tumour responses.

Optimisation of the detection of bacterial proteases using adsorbed immunoglobulins as universal substrates.

Bacterial proteases, Type XXIV from Bacillus licheniformens and Type XIV from Streptomyces griseus, were used to investigate the utility and optimisation of a solid phase assay for proteases, using immunoglobulin proteins as substrates. Immunoglobulins IgA and IgG were adsorbed on to surfaces of ELISA plates and exposed to various levels of the bacterial proteases which led to digestion and desorption of proportional amounts of the immunoglobulins. The assay signal was developed by measuring the remaining proteins on the polystyrene surface with appropriate enzyme-labelled anti-immunoglobulin reagents. The assay was fully optimised in terms of substrate levels employing ELISA techniques to titrate levels of adsorbed substrates and protease analytes. The critical factor which influences assay sensitivity was found to be the substrate concentration, the levels of adsorbed immunoglobulins. The estimated detection limits for protease XXIV and XIV were 10micro units/test and 9micro units/test using IgA as a substrate. EC(50) values were calculated as 213 and 48micro units/test for each protease respectively. Using IgG as a substrate, the estimated detection limits were 104micro units/test for protease XXIV and 9micro units/test for protease XIV. EC(50) values were calculated at 529micro units/test and 28micro units/test for protease XXIV and XIV respectively. The solid phase protease assay required no modification of the substrates and the adsorption step is merely simple addition of immunoglobulins to ELISA plates. Adsorption of the immunoglobulins to polystyrene enabled straightforward separation of reaction mixtures prior to development of assay signal. The assay exploits the advantages of the technical facilities of ELISA technology and commercially available reagents enabling the detection and measurement of a wide range of proteases. However, the key issue was found to be that in order to achieve the potential performance of the simple assay, optimisation of the method was essential.