PubMed · 16111205
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Kenichi Sekita. 2005. [Conglutinin binding test].. https://pubmed.ncbi.nlm.nih.gov/16111205/
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The sensitivity and specificity of a polyethylene glycol terminated alkanethiol mixed self-assembled monolayers (SAM) on surface plasmon resonance (SPR) immunosensor to detect Escherichia coli O157:H7 is demonstrated. Purified monoclonal (Mabs) or polyclonal antibodies (PAbs) against E. coli O157:H7 were immobilized on an activated sensor chip and direct and sandwich assays were carried to detect E. coli O157:H7. Effect of Protein G based detection and effect of concentrations of primary and secondary antibodies in sandwich assay were investigated. The sensor surface was observed under an optical microscope at various stages of the detection process. The sensor could detect as low as 10(3)CFU/ml of E. coli O157:H7 in a sandwich assay, with high specificity against Salmonella Enteritidis. The detection limit using direct assay and Protein G were 10(6)CFU/ml and 10(4)CFU/ml, respectively. Results indicate that an alkanethiol SAM based SPR biosensor has the potential for rapid and specific detection of E. coli O157:H7, using a sandwich assay.
In the present study, we describe the synthesis of highly luminescent uncoated water-soluble CdSe quantum dots (QDs) possessing the following characteristics: approximately 2 nm in diameter, with very good size distribution (in 95% homodispersed) accompanied by a broad-band photoluminescent spectrum. The synthetic procedure is simple, is conducted at room temperature, in the absence of the most popular coordinating ligands (as TOPO or HDA), and is highly reproducible. The obtained CdSe core QDs possessed a comparatively long fluorescence half-life (approximately 30-90 ns, depending on the emission wavelength) detected by time-resolved spectroscopy. These QDs were further conjugated with antibodies and applied in several biochemical analyses.
It is well documented that diffusion has generally a strong effect on the binding kinetics in the microtiter plate immunoassays. However, a systematic quantitative experimental evaluation of the microspot kinetics is still missing in the literature. Our work aims at filling this important gap of knowledge on the example of antigen binding to antibody microspots. A mathematical model was derived within the framework of two-compartment model and applied to the quantitative analysis of the experimental data obtained for typical antibody microspot assays. A strong mass-transport dependence of the antigen-antibody microspot kinetics was identified to be one of the main restrictions of this new technology. The binding reactions are slowed down in the microspot immunoassays by several orders of magnitude as compared with the corresponding well-stirred bulk reactions. The task to relax the mass-transport limitations should thus be one of the most important issues in designing the antibody microarrays. These limitations notwithstanding, the detection range of more than five orders of magnitude and the high sensitivity in the low femtomolar range were experimentally achieved in our study, demonstrating thus an enormous potential of this highly capable technology.