The development of an indirect competitive immunomagnetic-proximity ligation assay for small-molecule detection

The development of an indirect competitive immunomagnetic-proximity ligation assay (ICIPLA), which is a novel method for detecting small molecules, is described in this report. Free small molecules in samples can be detected using a proximity ligation assay (PLA); the detection is based on the proximity effect caused by a high concentration of small molecule-BSA conjugates bound to streptavidin magnetic beads. As an indirect format competitive immunoassay, the ICIPLA method has the advantage in that the quantity of monoclonal antibody (mAb) used for small-molecule detection is 8-fold lower than that required for the competitive immunomagnetic-proximity ligation assay (CIPLA) described in our previous work. Small molecules can be detected using a single monoclonal antibody, and the PLA method can be used to amplify high-performance signals. In this work, the small molecular compound ractopamine (RAC) was selected as a target for ICIPLA. The limit of detection (LOD) was 0.01 ng ml(-1), and the method exhibited a broad dynamic range of up to six orders of magnitude. We also employed the ICIPLA method to detect RAC in serum, urine, and muscle extracts; the results indicated that the LOD and dynamic range were not altered. The cross-reactivity studies showed that the cross-reactivity values for all RAC analogs were below 0.01%. These results suggest that ICIPLA is a sensitive, specific and practical method for small-molecule detection. This is the first report of the improved PLA technology for small-molecule detection by indirect competitive formats in the biological samples.