Molar composition of generated conjugates.

<p>The table depicts the composition of the generated conjugates regarding the molar ratios of the antibody, the antigen (either SIINFEKL peptide or OVA protein) and the nucleic acid adjuvant (CpG or GpC ODN) moieties. Where several batches of conjugates were generated, mean values +/− SD are displayed.</p>*<p>Only one batch of these conjugates was generated.</p><p>N/A - not applicable.</p>∇<p>These preparations of DEC-Fus-CpG and OVA-CpG were used for direct comparison of these conjugates allowing for the application of similar amount of adjuvant when normalizing for the antigen dose.</p

CTL induction by antigen fusion antibody-adjuvant conjugate is promoted by DEC205-mediated targeting.

<p>C57BL/6 mice were immunised into the footpad with 8 µg of ovalbumin fusion antibody either containing DEC205-specific (DEC-Fus) or isotype control antibody (iso-Fus) in combination with 10 µg soluble CpG ODN. Alternatively, mice were immunised with 8 µg of CpG-containing conjugates generated with Dec205-specific (DEC-OVA-CpG) or isotype control antigen fusion antibody (iso-Fus-CpG). Control mice received PBS injections. At day 5 after immunisation, in vivo CTL assays were performed by injecting target cells intravenously and by analysing antigen-specific killing the following day. The depicted data are pooled from 3–5 independent experiments with three mice per group for each experiment. Each dot represents the CTL response of an individual mouse. The average antigen-specific killing for each group is depicted as a bar.</p

DEC205-independent CTL induction by antibody-antigen-adjuvant conjugates.

<p>(A–C) C57BL/6 mice were immunised into the footpad with 8 µg of DEC-OVA-CpG, iso-OVA-CpG or DEC-OVA conjugate. In (C) immunisations took place in the presence or absence of 200 µg of anti-DEC205 antibody and DEC-OVA was co-injected with 10 µg soluble CpG ODN. Control mice received PBS injections. At day 5 or 6 after immunisation, target cells were injected intravenously. The following day, the in vivo CTL assay was analysed by flow cytometry. Data in (A) are pooled from 7 experiments while the graphs in (B and C) contain data from 2 independent experiments. In (C) a one-tailed Student’s t-test was used for statistical analysis.</p

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

Contains fulltext : 109503.pdf (publisher's version ) (Open Access)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

Biosensors for unattended cost-effective and continuous monitoring of environmental pollution: Automated water analyser computer supported system (AWACSS) and river analyser (RIANA)

This work describes our recent progress and achievements in the field of fully automated biosensors (Automated Water Analyser Computer Supported System (AWACSS) and River Analyser (RIANA)) for unattended, cost-effective and continuous monitoring of environmental pollution. We report on ultra-sensitive immunoassays for the hormones progesterone, testosterone and estrone and the pesticides propanil and isoproturon as examples of the outstanding progress made on biosensors in the field of environmental monitoring and water analysis. Most of the bio-active organic pollutants (estrone, progesterone, propanil and isoproturon) were detected at levels as low as 1.0pgm/L or even below. In fact, the reported limits of detection (LOD) were between 0.2 and 6.0pgm/L. For the first time, commercially available derivatives and antibodies were incorporated into immunoassays (progesterone and testosterone) for fully automated biosensors. To verify the assay performance for quantifying testosterone, progesterone, and isoproturon in real-world samples using our immunosensors, we spiked river and drinking water at six different levels from 0.9pgm/L to 90ngm/L. Nearly all recovery rates could be obtained between 70 and 120% as the AOAC International recommends it chiefly for water analysis