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(PH061) Development of ELISAs and immunoaffinity columns for estrogens.
Goda, Yasuhiro1, Kobayashi, Ayako1, Hirobe, Masato1, Fujimoto, Shigeru1, Ike, Michihiko2, Fujita, Masanori2, Okayasu, Yuji3, Komori, Koya3, Tanaka, Hiroaki3, Rubio, Fernando4, 1 Japan EnviroChemicals Ltd., Osaka, Osaka, Japan2 Osaka Univ., Suita, Osaka, Japan3 Independent Administrative Institution Public Works Research Institute(PWRI), Tsukuba, Ibaraki, Japan4 Abraxis LLC, Warminster, Pennsylvania, USA
ABSTRACT- For the quantitative determination of 17beta-estradiol (E2), estrone (E1) and 17alfa-ethynylestradiol (EE2) in the environment, instrumental analysis, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS), and enzyme-linked immunosorbent assay (ELISA) are generally employed. ELISAs are quite useful for screening purposes because they are highly sensitive, rapid and easy to perform. However, sometimes they can give over estimated results due to cross-reactants and matrix interferences that might be present in environment samples. Therefore we have developed three kinds of ELISAs and pretreatment methods for E1, E2, and EE2 to reduce the over estimation of ELISAs. The lowest quantification limits of all 3 ELISAs were 0.05ng/ml (50ppt). The values obtained by the ELISAs and LC-MS/MS was compared using samples obtained from a wastewater treatment plant (WWTP). The samples were treated by two kinds of solid phase extraction (SPE) methods (method A and B). Method A comprised C18 and silica SPE columns and Method B comprised of C18, Florisil and aminopropyl SPE columns. Over estimations of E1 ELISA in effluent, E2 ELISA in influent and EE2 ELISA in influent and effluent samples were not observed. On the other hand, the over estimations of E1 ELISA in influent and E2 ELISA in effluent were observed, however, those over estimations were reduced by altering the pretreatment from method A to B (the plotted slope of ELISA / LC-MS/MS was reduced from 4.3 to 1.3 in E1 and from 3.3 to 1.5 in E2 measurement). It was also confirmed that aminopropyl SPE column could remove the cross-reactants of E1 ELISA such as E1-3-sulfate and E1-3-gulucuronide and those from E2 ELISA such as 16-keto E2 and E2-3-glucuronide. To simplify the pretreatment, method B, the method comprised of the C18 and only aminopropyl SPE columns was studied during this investigation. As another approach for a simpler pretreatment, an immunoaffinity column (IAC) which could bind E1, E2, and estriol (E3) was also developed by covalently coupling polyclonal antibodies to non-woven fabric disks. The evaluation of this IAC in distilled water and WWTP samples will be presented.
Key words: ELISA(Enzyme-linked Immunosorbent Assay), estrogen, LC-MS/MS, affinity column
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