| abstract |
The present invention relates to methods for detecting and quantifying an analyte in a sample, principally in proximity probe assays, and in particular to an improvement in such methods to extend the dynamic range of detection, which is particularly advantageous for the detection and quantification of an analyte where the concentration range of the analyte in said sample is unknown and/or the range is likely to be broad, said method comprising: (i) contacting said sample with at least a pair of proximity probes each comprising a proteinaceous target-binding domain coupled to a nucleic acid domain such that said nucleic acid domains may be allowed to interact directly or indirectly when the proximity probes have bound in proximity to their respective target, said target being either the analyte or a binding partner for the analyte; (ii) further contacting said sample with at least one set of markers which function to extend the dynamic range of detection of the method, wherein said set comprises at least two markers, wherein each marker is a nucleic acid molecule comprising a binding domain and a reporter domain which gives rise to a detectable signal, and each marker: (a) is capable of interacting either with said nucleic acid domains to form a nucleic acid molecule from which a detectable signal is generated, or with a nucleic acid molecule generated by interaction of said domains; (b) cannot interact with said nucleic acid domains simultaneously with another marker in the set; (c) generates a signal that is distinguishable from the signal of another marker in the set; and (d) is present in an amount capable of detecting the analyte at a range of concentrations that differs from the range of concentrations detectable by other markers; (ii) allowing said markers to interact with said nucleic acid domains or said generated nucleic acid molecule; and (iii) detecting said signal. |