A low budget luminometer for sensitive chemiluminescent immunoassays

We have designed a simple luminometer based on a reasonably priced Peltier-cooled charge-coupled device (CCD) camera, housed in a light-tight box, with straightforward lens imaging and a simple platform for a microtitre or other assay format. The quantitative readout of the CCD image is recorded on a PC using customised software. The instrument can be assembled in a standard university workshop for under £3000, compared with the cheapest commercial instruments retailing at £10,000 and above. Consistent with the general view on chemiluminescent assays, the sensitivity is 10–100 times greater than that obtained with parallel ELISA's using a chromogenic substrate. A unique feature of the CCD format is that it enables assays to be carried out on arrays of minidots and even nanodots of antigen on the floor of each microtitre well. This permits direct comparison and standardisation of reactivity of a single sample against several antigens and economy in the use of reagents, test sample and technician time; finger-prick samples of blood can be analysed. The instrument should have widespread applicability in developing countries and, indeed, in any laboratories with hard-pressed budgets

Rewiring of sIgM-mediated intracellular signaling through the CD180 toll-like receptor

Chronic Lymphocytic Leukaemia (CLL) development and progression is thought to be driven by unknown antigens/autoantigens through the B cell receptor (BCR), and environmental signals for survival and expansion including Toll-like receptor (TLR) ligands. CD180/RP105, a membrane-associated orphan receptor of the TLR family, induces normal B cell activation and proliferation and is expressed by approximately 60% of CLL samples. Half of these respond to ligation with anti-CD180 antibody by increased activation/phosphorylation of protein kinases associated with BCR signaling. Hence CLL cells expressing both CD180 and the BCR could receive signals via both receptors. Here we investigated cross-talk between BCR and CD180-mediated signaling on CLL cell survival and apoptosis. Our data indicate that ligation of CD180 on responsive CLL cells leads to activation of either pro-survival BTK/PI3K/AKT-mediated, or pro-apoptotic p38MAPK-mediated signaling pathways, whilst sIgM ligation predominantly engages the BTK/PI3K/AKT pathway. Furthermore, pre-treatment of CLL cells with anti-CD180 redirects IgM-mediated signaling from the pro-survival BTK/PI3K/AKT towards the pro-apoptotic p38MAPK pathway. Thus pre-engaging CD180 could prevent further pro-survival signaling mediated via the BCR and, instead, induce CLL cell apoptosis, opening the door to therapeutic profiling and new strategies for the treatment of a substantial cohort of CLL patients