Development And Application Of An Immunoassay For Detection Of Cd Based On Speciifc Nanobody Against Cd-chelate

With the rapid development of global industry, the present situation of heavymetals pollution of our country and even the world becomes more and more serious,this has aroused the great attention of scientists around the world, the problem isimminent. Current analytical methods for detection of heavy metals include micro-spectral analysis(MS)，ultraviolet visible spectrophotometry^UV), atomic sbsorptionspectrometry(AAS), atomic fluorescence spectrometry(AFS), X fluorescencespectroscopy(XRF), and inductively coupled plasma mass spectrometry(ICP-MS), etc.which are sensitive and reliable. However, these methods are expensive and requirehighly specialized apparatus and trained operators, time-consuming and costly todetect, which makes them unsuitable for onsite detection. Based on theseconsiderations, the build of a simple, rapid, specific and sensitive detection methodhas become a hot topic. Immunological detection methods for heavy metal detectionprovides a new strategy and approach, which is a highly specific and sensitiveanalytical methods, and the simple，inexpensive，short detection time (approximately3?5min)，suitable for large scale screening site. At present, domestic immunoassayfor heavy metals cadmium focused on cadmium(Cd), lead(Pb)，mercury(Hg),chromium(Cr) and other heavy metal with high toxicity. Here we select the heavymetal cadmium(Cd) for the study, using Phage-Nanobody display technologysuccessfully demonstrated a novel technique specific for Cd-DTPA non-traditionalmonoclonal antibody? Nanobody(Nb), initially established the indirect competitiveELISA immunoassays, and for the detection of cadmium ion in water samples.In this study, p-SCN-Bn-DTPA was chosen as a bifunctional chelator to facilitatethe synthesis of antigens for Cd, and gained the complete antigenCd-DTPA-BSA/OVA successfully. The phage antibodies against Cd-DTPA:Cd-DTPA-VHH4-18and Cd-DTPA-VHH6-15were produced through phage displaytechnology successfully and Cd-DTPA-VHH4-18is the best，the relative aiffnity is5><.95108L/mol，there is no cross-reaetivity exists between the antibody and the carrierprotein BSA/OVA and also the non-metallic antigen DTPA-BSA/OVA，and almost nocross reaction with other metal ions.The initially established indirect competitive ELISA immunoassay methoddetection for the detection of Cd with the prepared phage antibody was met thenational standard of water quality monitoring. The lowest detection limit for Cd using the phage antibody Villi4-18was20ng/I.. and the spiked results showed that themoan recover〉was more than90%. The topic combined the heavy metalimmunoassay with phage display technology successfully; laying the foundation forheavy metal immunoassay later.