Sensitive Detection Of Small Molecules By Competitive Immunomagnetic-proximity Ligation Assay

Fast and accurate detection of small molecules such as pesticides, additives and veterinary drugs in food and biological samples will have great significance in our life. A novel detection method of small molecules, Competitive Immunomagnetic-Proximity Ligation Assay (CIPLA), was developed and described in this paper, which achieved excellent results.In our assay two small molecules, Clenbuterol (CLE) and Ractopamine (RAC), were selected as targets analytes for CIPLA. CLE and RAC were used as medicines firstly to treat respiratory system diseases in clinical practice. But then researchers found that they could redistribute energy in organism. They can strengthen fat decomposition and promote protein synthesis, thus increase the percentage of lean meat in economic animals and improve their meat quality, which can achieve a greater economic benefit. But the metabolism of CLE and RAC in organisms is very slow, long-term using will cause accumulation in animal tissues, and the tainted organs will lead to food poisoning in human beings when the content exceeds the body’s toxicity response dose. Therefore, it is of great importance that some fast and accurate approaches can be developed in the detection of residues in edible tissues of economic animals. Conventional detecting approaches such as high performance liquid chromatography (HPLC) and Enzyme-linked immunosorbent assay (ELISA) are not fittest when the concentration of residues is lower than 1.0 ng/ml. In this paper it is demonstrated that CIPLA could be successfully used in the trace detection of both CLE and RAC.CIPLA established by our laboratory met the above requirements. Through proximity effect caused by special proximity probes we prepared, small molecules can be detected using only one monoclonal antibody. CIPLA overcomes the obstacle that conventional Proximity Ligation Assay (PLA) can not be used in small molecular detection, as two antibodies are unable to combine to one small molecule due to its small molecular structure. It is the first time that PLA was applied in small molecular detection, and achieved satisfying results. The most commonly used probes in PLA were complexes of antibodies conjugated with oligonucleotides, called antibody-DNA for short. In this work, we employed both the antibody-DNA and antigen-DNA as proximity probes, and both achieved excellent results. Through constant optimization of the experiment parameters, the limit of detection (LOD) reached 0.01 ng·mL-1, which was 10-50 folds lower than ELISA. With five orders of magnitude of the dynamic range achieved (0.01 ng/ml; 0.1 ng/ml; 1 ng/ml; 10 ng/ml; 100 ng/ml; 1000 ng/ml), indicating the excellent sensitivity and broad dynamic range of CIPLA. It can be applied widely in the sensitive detection of many other small molecular materials such as pesticides, additives in food, drugs and biological samples, which have great significance in both theoretical and practical aspects.