Determination of polychlorinated biphenyls in soil and sediment by selective pressurized liquid extraction with immunochemical detection.

A selective pressurized liquid extraction (SPLE) method was developed for a streamlined sample preparation/cleanup to determine Aroclors and coplanar polychlorinated biphenyls (PCBs) in soil and sediment. The SPLE was coupled with an enzyme-linked immunosorbent assay (ELISA) for an effective analytical approach for environmental monitoring. Sediment or soil samples were extracted with alumina, 10% AgNO3 in silica, and sulfuric acid impregnated silica with dichloromethane at 100°C and 2000 psi. The SPLE offered simultaneous extraction and cleanup of the PCBs and Aroclors, eliminating the need for a post-extraction cleanup prior to ELISA. Two different ELISA methods: (1) an Aroclor ELISA and (2) a coplanar PCB ELISA were evaluated. The Aroclor ELISA utilized a polyclonal antibody (Ab) with Aroclor 1254 as the calibrant and the coplanar PCB ELISA kit used a rabbit coplanar PCB Ab with PCB-126 as the calibrant. Recoveries of Aroclor 1254 in two reference soil samples were 92±2% and 106±5% by off-line coupling of SPLE with ELISA. The average recovery of Aroclor 1254 in spiked soil and sediment samples was 92±17%. Quantitative recoveries of coplanar PCBs (107-117%) in spiked samples were obtained with the combined SPLE-ELISA. The estimated method detection limit was 10 ng g(-1) for Aroclor 1254 and 125 pg g(-1) for PCB-126. Estimated sample throughput for the SPLE-ELISA was about twice that of the stepwise extraction/cleanup needed for gas chromatography (GC) or GC/mass spectrometry (MS) detection. ELISA-derived uncorrected and corrected Aroclor 1254 levels correlated well (r=0.9973 and 0.9996) with the total Aroclor concentrations as measured by GC for samples from five different contaminated sites. ELISA-derived PCB-126 concentrations were higher than the sums of the 12 coplanar PCBs generated by GC/MS with a positive correlation (r=0.9441). Results indicate that the SPLE-ELISA approach can be used for quantitative or qualitative analysis of PCBs in soil and sediments. To our knowledge, this is the first report of an SPLE-ELISA approach not requiring a post-extraction cleanup step for detecting Aroclors and coplanar PCBs in soil and sediment.

Development of a multianalyte enzyme-linked immunosorbent assay for permethrin and aroclors and its implementation for analysis of soil/sediment and house dust extracts.

Development of a multianalyte enzyme-linked immunosorbent assay (ELISA) for detection of permethrin and aroclors 1248 or 1254 and implementation of the assay for analysis of soil/sediment samples are described. The feasibility of using the multianalyte ELISA to monitor aroclors 1254 and permethrin simultaneously was tested with permethrin and aroclor standards and with aroclor- and permethrin-containing soil/sediment and house dust samples. Comparison of the I50 and I20 values of the multianalyte with those of a single-analyte assay revealed similar results, and multianalyte ELISA determination of analyte amounts in soil/sediment dust samples yielded similar results to those of a single-analyte assay. A single-analyte assay of permethrin content in permethrin-containing dust samples showed that the ELISA can determine the analyte accurately in samples with dust matrix contents ranging from 6.25 to 100 mg as indicated by the good correlation between the results of the immunoassay and those of the gas chromatography analysis.

Development of an immunoassay and a sol-gel-based immunoaffinity cleanup method for coplanar polychlorinated biphenyls from soil and sediment samples.

Two polychlorinated biphenyls (PCB) enzyme linked immunosorbent assays (ELISAs) were developed using goat PCB purified immunoglobulin (IgG) antibodies (Abs). The IgGs exhibited the highest affinity toward PCB-77 (24 ng mL(-1)) with sensitivities in the range of 6-11 ng mL(-1). The Abs cross-reacted with PCB-126 and the heptachlorodibenzofuran 1,2,3,4,6,7,8-HpCDF but not with PCB-169, PCB-118, Aroclor 1232, 1248, 1260 or the hexachlorodibenzofuran 2,3,4,6,7,8-HxCDF. The IgGs were also used to develop a sol-gel-based immunoaffinity purification (IAP) method for cleanup of PCB-126. Recovery efficiencies depended on the sol-gel formats; a 1:12 format resulted in the highest binding capacity. Net binding capacity ranged from 112 to 257 ng, and 90% of the analyte could be eluted with only 2 mL of ethanol. The method was also very efficient in purifying PCB-126 from spiked soil and sediment samples from contaminated sites; and eliminating matrix interferences to a degree that enabled analysis of the purified samples by ELISA. The approaches developed in the course of the study form a basis for the development of additional IAP methods for other PCBs, and their implementation in high-throughput screening programs for PCB in food, soil, and other environmental and biological samples.

Monitoring of progestins: development of immunochemical methods for purification and detection of levonorgestrel.

A polyclonal antibody (Ab) for the progestin levonorgestrel (LNG) was generated, and immunochemical assays for its detection, clean-up and concentration were developed. A highly specific microplate diagnostic assay for the detection of LNG was developed that used the enzyme linked immunosorbent assay (ELISA) method. The LNG ELISA developed was sensitive and reproducible; it exhibited I(50) and I(20) values of 3.3+/-1.8 ng mL(-1) and 0.6+/-0.4 ng mL(-1), respectively, and the Abs did not cross react with any of the tested steroid hormones. The above Abs were used to develop a sol-gel-based immunoaffinity purification (IAP) method for concentration and clean-up of LNG that is compatible with subsequent immunochemical or instrumental chemical analytical procedures, such as liquid chromatography followed by mass spectrometry (LC-MS/MS). Development of the columns included successful entrapment of Abs within a tetramethoxysilane (TMOS)-based SiO(2) polymer network. The Abs could bind the free analyte from solution, and the bound analyte could be easily eluted from the sol-gel matrix at high recoveries. The Ab selectivity towards the antigen was high, in both ELISA and the sol-gel columns, but the entrapped Abs cross-reacted with two other steroid hormones--ethynylestradiol (EE2) and nortestosterone (NT) - which share similar epitopes with LNG, despite the lack of cross reactivity in the ELISA. The validity of the method was investigated by LC-MS/MS and a good analytical correlation was obtained.

PBAN receptor: employment of anti-receptor antibodies for its characterization and for development of a microplate binding assay.

This study describes generation of an anti-PBAN receptor (PBAN-R) antiserum and its employment for the characterization of the PK/PBAN-R(s). The antiserum recognized, in a specific and dose-dependent manner, the presence of PBAN-R in pheromone gland membrane preparations of three female moths: Heliothis peltigera, Helicoverpa armigera and Spodoptera littoralis. It also reacted specifically with the S. littoralis larval receptor in vivo, most likely by competing with the ligand on the binding site and consequently inhibiting cuticular melanization. Despite its ability to react with the receptor of H. peltigera in dot blot experiments, the antiserum did not react with the receptor in vivo and failed to inhibit sex pheromone biosynthesis. The antiserum was also used to develop two microplate binding assays. The Ab described in this study is the first raised against an insect neuropeptide (Np) receptor to be used in vivo, and its employment for characterization of the PK/PBAN-R(s) may thus provide important information on the mode of action of this Np family. The present study adds important information on the difference between the receptors in the two moth species, hints at the possible existence of receptor subtypes, and provides a platform for the development of a high-throughput assay (HTA) for screening of PK/PBAN agonists and antagonists.

Enzyme-linked immunosorbent assay (ELISA) and sol-gel-based immunoaffinity purification (IAP) of the pyrethroid bioallethrin in food and environmental samples.

Enzyme-linked immunosorbent assay (ELISA) and sol-gel-based immunoaffinity purification (IAP) methods for the pyrethroid bioallethrin were developed and applied for monitoring bioallethrin in spiked food, soil, and dust samples. Attempts to determine bioallethrin content in fruit and vegetable extracts revealed high variability between sample preparations and marked interferences with the assay. Sol-gel IAP followed by solid-phase sample concentration was effective in removing the interfering components and resulted in high recovery of bioallethrin from spiked crude acetonic extracts of fruits and vegetables, even in the presence of high extract concentrations (28%). Solid-phase treatment alone failed to remove the interfering components from the spiked sample. Gas chromatography-mass spectrometry analysis of the IAP samples revealed bioallethrin as a doublet unsolved peak because of the cis and trans isomer present in the standard with confirmation of its mass. Unlike fruit and vegetable extracts, soil and dust samples did not interfere with the ELISA, and the bioallethrin content in those samples could be determined with high precision without the need of any further purification.