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Development Of Immunoassais For The Determination Of Ketamine

Posted on:2011-11-18Degree:MasterType:Thesis
Country:ChinaCandidate:W D ZhangFull Text:PDF
GTID:2144360305485167Subject:Chemistry
Abstract/Summary:PDF Full Text Request
During the past decade, the abuse of ketamine abuse has dramatically increased worldwide and emerges as a public health concern. Ketamine, including its salts or prepareations, has been classified as a Class One psychotropic drug by State Food and Drug Administration in 2004. Therefore, a rapid, sensitive and reliable method should be developed not only for the analysis of ketamine but also for the screening of suspected ketamine abusers.In this study, complete antigen of ketamine was prepared and immunoassais for the determination of ketamine were developed using obtained polyclonal antibody and monoclonal antibody against ketamine.Enzyme-linked immunosorbent assay (ELISA) was developed under optimized immunoassay conditions using polyclonal antibody. The linear range of ketamine was 1-10000 ng/mL with recoveries from 86% to 116% in spiked sample analysis. The detection limit of ketamine was 0.61 ng/mL, the cross-reactivity of norketamine, morphine and cocaine were 35.73%,2.42% and 1.09%, respectively. Biotin-avidin amplified enzyme-linked immunosorbent assay was developed under optimized immunoassay conditions using polyclonal antibody. The linear range of ketamine was 0.1-1000 ng/mL with recoveries from 84% to 116% in spiked sample analysis. The detection limit of ketamine was 0.03 ng/mL, the cross-reactivity of norketamine, morphine and cocaine were 35.13%,1.09% and 0.48%, respectively. Compared with the ELISA, the BA-ELISA has lower detection limit and can be applied to the determination of ketamine at lower levels.Based on indirect enzyme linked immunosorbent assay, a rapid and effective fluorescence immunoassay was developed to determine ketamine with the probe of fluorescein isothiocyanate. Different parameters affecting the sensitivity of assay were optimized experimentally. The linear range of the presented method was from 1 to 1000 ng/mL, and the limit of detection was found to be 0.48 ng/mL. The recoveries of ketamine spiked in human serum and urine were between 89% and 113%.Enzyme-linked immunosorbent assay (ELISA) was developed under optimized immunoassay conditions using monoclonal antibody. The linear range of ketamine was 1-10000 ng/mL with recoveries from 90% to 122% in spiked sample analysis. The detection limit of ketamine was 0.18 ng/mL, the cross-reactivity of norketamine, morphine and cocaine were 10.50%,0.49% and 1.57%, respectively. Biotin-avidin amplified enzyme-linked immunosorbent assay was developed under optimized immunoassay conditions using monoclonal antibody. The linear range of ketamine was 0.1-1000 ng/mL with recoveries from 91% to 112% in spiked sample analysis. The detection limit of ketamine was 0.03 ng/mL, the cross-reactivity of norketamine, morphine and cocaine were 10.02%,0.13% and 0.15%, respectively. Compared with the ELISA, the BA-ELISA has lower detection limit and can be applied to the determination of ketamine at lower levels. Compared with the polyclonal antibody, the cross-reactivities of the method using monoclonal antibody are lower. Therefore, monoclonal antibody is more specific for the determination of ketamine.
Keywords/Search Tags:ketamine, polyclonal antibody, monoclonal antibody, enzyme-linked immunosorbent assay, biotin, avidin, fluorescence immunoassay
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