In Europe, a limit value of 10ng L21 was set by the European Commission for benzo[a]pyrene(B[a]P) in water intended for human consumption (Council Directive 98/83/EC) and, therefore,sensitive and reliable methods are needed to evaluate its presence. We report here on thedevelopment of a highly sensitive indirect competitive ELISA for the detection of B[a]P in potablewater. Fourteen monoclonal antibodies were generated in mice using novel B[a]P derivatives. Theimmunoassay with the least interference and the best sensitivity was optimized and characterized.As co-solvent, ten percent methanol (v/v) was determined as the optimum concentration for B[a]Psolubilization for use with the developed ELISA. With the purified antibody (clone 22F12) theaverage IC50 for B[a]P and corresponding detection limit at a signal:noise (S/N) ratio of 3 was65 ng L21 and 24 ng L21, respectively. From the 16 EPA-designated PAHs, only chrysene,indeno[1,2,3-cd]pyrene, and benzo[b]fluoranthene showed a cross-reactivity (CR) higher than20%. No CR was observed for two- and three-ringed aromatics as well as dibenz[ah]anthraceneand benzo[ghi]perylene. The effect of pH value (range 6.5-9.5), ionic strength (specific electricconductivity 1 mS cm21–2.5mS cm21), and inorganic ions (sodium, copper, iron, aluminium,manganese, chloride, sulfate, nitrate, and nitrite at maximum permissible levels according to theCouncil Directive) on both signal and sensitivity of the ELISA was studied. No significantinfluence of these parameters on the ELISA competition curve was found. We suggest that theoptimized ELISA can be used to monitor potable water samples without previous extraction fromthe samples. The assay should facilitate the cleanup of B[a]P contaminated sites where B[a]P levelsfall close to the limit value of the new drinking water directive.

A highly sensitive and specific indirect competitiveenzyme-linked immunosorbent assay (ELISA) for thedetermination of diclofenac in water samples was developed.With pure water, the limit of detection (LOD, S/N ) 3)and IC50 were found to be 6 ng/L and 60 ng/L, respectively.The analytical working range was about 20-400 ng/L.Highest cross-reactivity (CR) of 26 tested pharmaceuticals,metabolites, and pesticides was found for 5-hydroxydiclofenac(100%). Other estimated values were well below4% and, therefore, are negligible. The assay was appliedfor the determination of diclofenac in tap and surface watersamples as well as wastewater collected at 20 sewagetreatment plants (STPs) in Austria and Germany. Humicsubstances were identified as main interference in surfacewater. Wastewater samples which were only submittedto filtration and dilution yielded about 25% higher diclofenacconcentrations using the ELISA compared to GC-MS.However, the ELISA turned out to be a simple, inexpensive,and accurate method for the determination of diclofenacboth in influent and effluent wastewater after rather simplesample preparation, i.e., filtration, acidification, andreadjustment to neutral pH-value, and at least 10-folddilution with pure water.

An amperometric immunoassay was developed by coupling the enzyme-linked immunosorbent assay (ELISA) microtiterplatesystem with a polyaniline-perfluorosulfonated ionomer composite (PA/NF) electrode incorporated flowinjection analysis(FIA) system and used for the analysis of Tal 1 protein, found in leukemic T cell. Rabbit polyclonal antibody (pAb) against Tal1 and urease–pAb were used, respectively as the captured protein and enzyme labeled conjugate for sandwich immunoassay ofTal 1. Characteristics of the PA/NF electrode such as reproducibility, stability and sensitivity were studied. The detection limitsof the PA/NF electrode for NH4+ and urease were found to be 5_M and 0.05 nM, respectively. Assay conditions such as theamount of pAb needed for coating the plate, the concentration of urease–pAb conjugate appropriate for the immunoreactionand the incubation time for urea to react with the bound urease–pAb in the microtiter-wells were also studied. A detectionlimit as lower as 0.5 ng/ml and a dynamic range of 1.0-100 ng/ml were found for the immunoassay of Tal 1 protein with thedeveloped immunoassay system.

The high sensitive dynamic reaction cell inductively coupled plasma mass spectrometry (DRC ICP-MS)-based immunoassayusing ferrocene (Fc) tethered hydroxysuccinimide ester as label for the determination of 2,4-dichlorophenoxyacetic acid(2,4-D) was presented. Ferrocene tethered hydroxysuccinimide ester was directly or via horse radish peroxidase (HRP) as abridge coupled to monoclonal antibody (mAb) of 2,4-D. Competitive immunoreactions were completed in microtiter plateand the signal of 56Fe in the bound ferrocene labeled conjugate was detected by DRC ICP-MS. The potentially interfering40Ar16O+ at the iron mass m/z 56 was reduced in intensity significantly by using NH3 as the reaction gas. The optimizationprocess of the immunoassay was greatly simplified in the aid of enzyme linked immunosorbent assay (ELISA) procedure. The2,4-D was determined in the dynamic range of 0.1–1000 ng/ml and the detection limits of the assays using the two ferrocenelabeled conjugates were found to be 0.044 and 0.055 ng/ml, respectively. The relative standard deviation for six measurementswere within 5.5–15.3%.

To extend the application of a multichannel electrochemical detector (MED) for immunoassay, a MED system consisting of 8 sets of Pt electrodes in an arrangement fitted with the dimensions of a row of microtiter wells in a microtiter plate and a microcomputer-assisted 16-channel potentiostat was constructed. With this developed MED system, electroactive enzymatic products produced in eight microtiter wells can be analyzed simultaneously with a developed amperometric procedure. To demonstrate the applicability of the MED system for immunoassay, an immunosystem containing rabbit-IgG and alkaline phosphatase-conjugated goat anti rabbit-IgG was studied. From the dynamic range of 10-1000 ng/mL (0.064-6.4pM) and the detection limit of 1.0 ng/mL (6.4pM) obtained, the developed MED shows a tremendous improvement in sensitivity, detection limit, and efficiency compared with that obtained from conventional electrochemical immunoassay.