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A sensitive TSH assay in spot-coated microwells utilizing recombinant antibody fragments

2005; Elsevier BV; Volume: 306; Issue: 1-2 Linguagem: Inglês

10.1016/j.jim.2005.08.002

1872-7905

Johanna Ylikotila, Lasse Välimaa, Markus Vehniäinen, Harri Takalo, Timo Lövgren, Kim Pettersson,

Microfluidic and Capillary Electrophoresis Applications

We have developed a novel TSH immunoassay based on a simplified test protocol suitable for point-of-care testing yet providing 3rd generation TSH assay sensitivity. The antibody density and the functional solid phase capacity were improved up to six-fold by capturing the site-specifically biotinylated recombinant Fab fragment or single chain antibody fragment onto the surface of immobilized streptavidin. An important mechanism for obtaining a low limit of detection (0.003 mIU/l) was the reduction of the coated area to a size ("spot") more closely coinciding with the excitation beam. The reporter technology was based on time-resolved fluorometric detection of inherently fluorescent europium chelates of high quantum yield. The ready-to-use assay concept employed the all-in-one (Aio!) principle--holding all assay components in a dry form in the microtitration well--to provide a simple assay protocol. Direct signal measurement from the surface was done after the washing step without a separate development step. It is concluded that size reduction and site-specific labeling of the antibodies to create a surface with high functional capacity provides a rapid, highly sensitive immunoassay.