Fig. 2.

Ambient analyte theory: repetitive measurement of biotin-PE from same sample. The diagram shows the results for five sequentially performed analyses of the same protein from the same sample from the same well. A model assay using biotin-PE and a biotin-specific capture antibody was used. Anti-biotin antibody-coated magnetic beads were incubated with biotin-PE at five different concentrations (300, 80, 20, 5, and 1 pm) in triplicates. After a 30-min incubation period, the beads were removed from the sample, and new antibody-coupled magnetic beads were added. This procedure was repeated four times, and the assay was read out on a Luminex 100 reader. No analyte limitations were detectable over the five measurements. The data from the first measurement were used for a five-parametric logistic fit. Using the parameters of this fit, the raw assay data were transformed to analyte concentrations (see Table III). After five assays, an analyte decrease of 20% was observed in total, suggesting a captured amount of less than 5% per assay.