title: The development of an indirect competitive immunomagnetic-proximity ligation assay for small-molecule detection.
abstract: 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.
authors.affiliation: College of Life Sciences, Zhejiang University, Hangzhou, 310058, China.
authors.forename: Xuecheng
authors.initials: X
authors.lastname: Jiang
authors.forename: Zhenhong
authors.initials: Z
authors.lastname: Zhu
authors.forename: Zhihao
authors.initials: Z
authors.lastname: Sun
authors.forename: Luming
authors.initials: L
authors.lastname: Wang
authors.forename: Lixiao
authors.initials: L
authors.lastname: Zhou
authors.forename: Hanqiang
authors.initials: H
authors.lastname: Miao
authors.forename: Zhengting
authors.initials: Z
authors.lastname: Zhang
authors.forename: Feng
authors.initials: F
authors.lastname: Shi
authors.forename: Chenggang
authors.initials: C
authors.lastname: Zhu
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000378:metabolism; D015374:Biosensing Techniques; D007118:Immunoassay; D018189:Immunomagnetic Separation; D057230:Limit of Detection; D009132:Muscles / Q000737:chemistry; D010627:Phenethylamines / Q000097:blood* / Q000652:urine*; D019809:Streptavidin / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; D010627:Phenethylamines; C060897:ractopamine; D019809:Streptavidin