Noncompetitive On-Chip Immunoassays for Detection of Nonlabeled Antibodies Based on the Excluded Volume Effect of the Target Itself

We propose a novel, noncompetitive, label-free, and on-chip immunoassay format based on the excluded volume effect by a target antibody that is itself bound to the corresponding epitope on a biochip. The advantage of the present method is that it only requires a biotin-tethering peptide epitope (capture agent) and fluorophore-labeled-avidin (a signal-generating agent) pair for signal readout. The method is based on the interaction between a nonlabeled target antibody and the corresponding peptide epitope of the antigen decelerating the association rate of the fluorophore-labeled avidin to the biotin moiety at the end of the peptide epitope due to the excluded volume effect of the target antibody. This causes significant changes in signal intensity. Without the target antibody, there is no interference in the binding of the signal-generating agent. The use of other specific interactions would allow fine-tuning of the excluded volume and binding rate to the capture agent to improve sensitivity and versatility.