Residual nanoparticle label immunosensor for wash-free C-reactive protein detection in blood.

Current diagnostic immunotechnologies are universally based on the measurement of the bound label-antibody fraction in direct binding or sandwich-assay type approaches with various detection techniques (e.g. enzyme-linked immunosorbent assay or ELISA) on solid stationary phase surface. Here an alternative reciprocal approach is presented based on the detection of the non-bound fraction of nanoparticle-labelled antibodies using microparticles as solid support. The advantage of detecting the non-bound fraction of the labelled antibody instead of the bound fraction is the high dynamics and the suggested increased flexibility in the selection of the detection mode. No actual washing steps are required as the bound and non-bound fractions of the detection nanoparticle label are separated using physical separation rather than consecutive washing repeats. The quantitative proof-of-concept set-up was demonstrated through blood-based detection of C-reactive protein (CRP). A blood sample containing CRP was diluted 1/50 and measured in 15-min resulting in a linear response at a range from 1 to 30µg/ml. The lowest limit of detection was below 0.03µg/ml and the assay coefficient of variation ranged from 0.3 to 9%. The nanoparticle-based residual label detection outperformed the corresponding molecular label method providing wider applicability with nearly an order of magnitude higher signal-to-background ratio for novel assay configurations in clinical diagnostics practices.

Quantitative detection of cell surface protein expression by time-resolved fluorimetry.

A method is introduced for quantitative detection of cell surface protein expression. The method is based on immunocytochemistry, the use of long decay time europium(III) chelate and platinum(II) porphyrin labels, and detection of photoluminescence emission from adhered cells by time-resolved fluorimetry. After immunocytochemistry, the assay wells are evaporated to dryness and measured in the dry state. This protocol allows repeated and postponed analysis and microscopy imaging. In order to investigate the performance of the method, we chose expression of intercellular adhesion molecule-1 (ICAM-1) of endothelial cell line EAhy926 as a research target. The expression of ICAM-1 on the cells was enhanced by introduction of a cytokine, tumour necrosis factor-alpha (TNFalpha). The method gave signal:background ratios (S:B) of 20 and 9 for europium and platinum labels, respectively, whereas prompt fluorescent FITC label gave a S:B of 3. Screening window coefficients (=Z'-factor) were >0.5 for all the three labels, thus indicating a score for an excellent screening assay. In conclusion, the method appears to be an appropriate choice for protein expression analysis, both in high-throughput screening applications, and for detailed sample investigation by fluorescent microscopy imaging.

Two-photon excitation in fluorescence polarization receptor-ligand binding assay.

Fluorescence polarization is one of the most commonly used homogeneous assay principles in drug discovery for screening of potential lead compounds. In this article, the fluorescence polarization technique is combined with 2-photon excitation of fluorescence. Theoretically, the use of 2-photon excitation of fluorescence increases the volumetric sensitivity and polarization contrast of fluorescence polarization assays. The work in this report demonstrates these predictions for an estrogen receptor ligand binding assay.