Monoclonal Antibody Generation And Immunoassays Development For The Determination Of Sodium Pentachlorophenolate

Sodium Pentachlorophenolate(PCP-Na)has been used as an efficient and cheap insecticide,fungicide and molluscicide world-widely.However,further study revealed that its stable physical and chemical properties cause its' POPs-like remains and slow degradation in the environment.It has been widely dissipated through water and soil circulation system,and could eventually accumulated in human or animal through the food chain.It could jeopardize liver and nervous system of humans and animals,and cause reproductive toxicity,carcinogenic,teratogenic and mutagenic effects.Even though PCP-Na has been banned for use in many countries,it has been frequently found and reported in agriproducts,animal-derived foods in particular.At present,instrumental based methods have been used as the authorizing methodology for the measurement of PCP-Na residues.These methods offer high accuracy and precise results of detections,however,it will take a long time to prepare and require professional,well-trained personal to operate the equipment.It is not suitable for rapid and large-scale screening of samples on site.Immunoassay,with characteristic of simple,cheap and high-throughput detection,which is suitable for on-site detection.However,high-quality antibody and signal delivery system play key roles in the development of immunoassay to achieve highly sensitive performance.In this study,a monoclonal antibody against PCP-Na with high affinity and high specificity was obtained through the synthesis of PCP-Na hapten and animal immunization.Thereafter,indirect competitive chemiluminescence enzyme immunoassay(ic-CLEIA),colloidal gold immunochromatography asssay(GICA)and dual-signal readout immunoassay based on poly dopamine-coated gold(Au@PDA)nanoparticle quenching quantum dots(QDs)fluorescence were developed for the detection of PCP-Na.It was expected to provide affordable,convenient,and sensitive,on-site detection approach for practical implementation of different scenarios adoption.The main results are as follows:(1)Preparation of monoclonal antibody of PCP-Na:Three different PCP-Na haptens were synthesized and then conjugated with proteins to prepare artificial antigens.After mouse immunization,cell fusion,subclonal screening,and hybridoma cell expansion,a monoclonal cell line No.6-6H-2F-10H that could stably produce antibody against PCP-Na was obtained.Then,monoclonal antibody against PCP-Na was obtained by preparing and purifying mouse ascites.The indirect competitive enzyme-linked immunosorbent assay(ic-ELISA)method was developed for PCP-Na detection,and the half-maximal inhibition concentration(IC50)of PCP-Na was 2.003 ng/mL.The IC50 of the pentachlorophenol(PCP)is 3.060 ng/mL,and there is no significant crossreactivities to other chlorophenols and analog.(2)Development of an indirect competitive chemiluminescence immunoassay:A series of parameters of conditions affecting the ic-CLEIA reaction system were optimized,such as chemiluminescence reaction time,antigen-antibody concentration,pH value,methanol content,etc.,and a highly sensitive ic-CLEIA method for the detection of PCP-Na was established.The IC50 could reach 0.11 ng/mL.(3)Development of colloidal gold immunochromatographic strip:Colloidal gold nanoparticles(AuNPs)were prepared by trisodium citrate reduction method,which was coupled with PCP-Na monoclonal antibody to form gold-labeled probes.After optimizing the pH of antibody,the amount of antibody,the amount of coating antigen,the amount of gold-labeled probe and other conditions,a colloidal gold immunochromatography method of PCP-Na was established.The limit of detection for PCP-Na was 31.3 ng/mL.(4)Development of the dual-signal immunochromatographic assay using quantum dots and polydopamine coated gold nanoparticles:PDA-based Au@PDA was synthesized and fluorescence of QDs was introduced as the signal output material,which provided a "turn on" positive signal readout of fluorescence mode and "turn off" negative signal readout of colorimetric mode at the same time.Thus,the detection sensitivity of the immunochromatography strip was improved by"turn on" mode.The limit of detection of the established Au@PDA and fluorescence quenching dual signal immunochromatographic test strip for PCP-Na reached 0.4 ng/mL under ultraviolet light,and 25 ng/mL under natural light with naked eye.The entire test could be finished within 10 min.