A sensitive TSH assay in spot-coated microwells utilizing recombinant antibody fragments.
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.
Ylikotila
J
J
University of Turku, Department of Biotechnology, Tykistökatu 6A, FIN-20520 Turku, Finland. johanna.ylikotila@utu.fi
Välimaa
L
L
Vehniäinen
M
M
Takalo
H
H
Lövgren
T
T
Pettersson
K
K
D000911:Antibodies, Monoclonal / Q000276:immunology*; D002614:Chelating Agents / Q000737:chemistry; D005063:Europium / Q000737:chemistry; D019085:Fluorescent Antibody Technique, Direct; D015200:Fluoroimmunoassay; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology*; D008832:Microchemistry / Q000379:methods; D008904:Miniaturization; D011994:Recombinant Proteins / Q000276:immunology; D012680:Sensitivity and Specificity; D013972:Thyrotropin / Q000032:analysis* / Q000737:chemistry / Q000276:immunology
D000911:Antibodies, Monoclonal; D002614:Chelating Agents; D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins; D005063:Europium; D013972:Thyrotropin