Simplified Mercury Extraction from Coal Fly Ash for Quantification of Total Mercury by ELISA-based Immunoassay.

A simplified two-step mercury extraction procedure enabled the selective and reproducible mercury recovery from actual coal fly ash (CFA). The optimized extraction procedure involving conventional enzyme-linked immunosorbent assay (ELISA)-based immunoassay allowed the ultra-sensitive quantification of total mercury content in CFA. The total mercury content of 41 CFA samples were successfully determined using the above-mentioned method, and the results were in agreement with those obtained by standard instrumental analysis (thermal decomposition atomic absorption spectrometry) within a 15% coefficient of variation. Our method for total mercury quantification is not only simple but suitable for management of the mercury content at coal-fired electric power plants and landfill sites, which deal with large amounts of waste CFA.

A novel approach for measuring sphingosine-1-phosphate and lysophosphatidic acid binding to carrier proteins using monoclonal antibodies and the Kinetic Exclusion Assay.

Sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) are bioactive signaling lysophospholipids that activate specific G protein-coupled receptors on the cell surface triggering numerous biological events. In circulation, S1P and LPA associate with specific carrier proteins or chaperones; serum albumin binds both S1P and LPA while HDL shuttles S1P via interactions with apoM. We used a series of kinetic exclusion assays in which monoclonal anti-S1P and anti-LPA antibodies competed with carrier protein for the lysophospholipid to measure the equilibrium dissociation constants (Kd) for these carrier proteins binding S1P and the major LPA species. Fatty acid-free (FAF)-BSA binds these lysophospholipids with the following Kd values: LPA(16:0), 68 nM; LPA(18:1), 130 nM; LPA(18:2), 350 nM; LPA(20:4), 2.2 µM; and S1P, 41 µM. FAF human serum albumin binds each lysophospholipid with comparable affinities. By measuring the apoM concentration and expanding the model to include endogenous ligand, we were able to resolve the Kd values for S1P binding apoM in the context of human HDL and LDL particles (21 nM and 2.4 nM, respectively). The novel competitive assay and analysis described herein enables measurement of Kd values of completely unmodified lysophospholipids binding unmodified carrier proteins in solution, and thus provide insights into S1P and LPA storage in the circulation system and may be useful in understanding chaperone-dependent receptor activation and signaling.

Confirmation of the validity of the current characterization of immunochemical reactions by kinetic exclusion assay.

Prior observations that questioned the validity of kinetic exclusion assays were based on the mistaken assumption that the assays quantified the fraction of those antibody molecules that had unoccupied binding sites. Instead, the standard KinExA assay quantifies the fraction of total antibody binding sites that are unoccupied, regardless of the number of unoccupied sites on each antibody molecule. Although the standard KinExA analysis assumes that there is only a small probability of antibody-site capture by the affinity matrix, the results of numerical simulations demonstrate the reliability of dissociation constants obtained by the standard KinExA analysis for capture probabilities as high as 30%. This finding further strengthens the potential of kinetic exclusion assays as the procedure of choice for the rapid and accurate characterization of immunochemical reactions that forms part of screening processes in the search for therapeutic antibodies.

Development and verification of a model for estimating the screening utility in the detection of PCBs in transformer oil.

A simple new model for estimating the screening performance (false positive and false negative rates) of a given test for a specific sample population is presented. The model is shown to give good results on a test population, and is used to estimate the performance on a sampled population. Using the model developed in conjunction with regulatory requirements and the relative costs of the confirmatory and screening tests allows evaluation of the screening test's utility in terms of cost savings. Testers can use the methods developed to estimate the utility of a screening program using available screening tests with their own sample populations.

New antibody and immunoassay pretreatment strategy to screen polychlorinated biphenyls in Korean transformer oil.

Development and modifications are described that expand the application of an immunoassay from the detection of Kanechlors (Japanese technical PCBs mixtures) to the detection of Aroclors (U. S. technical PCB mixtures, used in Korea) in contaminated Korean transformer oil. The first necessary modification was the development of a new antibody with a reactivity profile favorable for Aroclors. The second modification was the addition of a second column to the solid-phase extraction method to reduce assay interference caused by the Korean oil matrix. The matrix interference is suspected to be caused by the presence of synthetic oils (or similar materials) present as contaminants. The modified assay was validated by comparison to high-resolution gas chromatography/high-resolution mass spectrometry analysis, and was shown to be tolerant of up to 10% of several common synthetic insulating oils. Finally the screening performance of the modified assay was evaluated using 500 used transformer oil samples of Korean origin, and was shown to have good performance in terms of false positive and false negative rates. This report provides evidence for the first establishment of immunoassay screening for Aroclor based PCB contamination in Korean transformer oil.

Optimization of a commercial biosensor for polychlorinated biphenyls and evaluation of its utility for screening.

We previously described our systematic progress that eventually resulted in a commercially available immunoassay based biosensor (PCB biosensor) for detecting PCBs in oil. However, IC50 of the commercialized PCB biosensor was approximately 2 ppb for PCBs, and did not achieve the theoretical detection limit (TDL) which would represent an IC50 of approximately 0.5 ppb. In this study, we characterize the effects of the antibody concentration, flow volume and flow rate on the PCB biosensor's response. Using the optimum operating conditions, the PCB biosensor achieved the TDL and its performance as a screening test was improved. Working at the stringent maximum residue limit specified by Japanese law (0.5 ppm total PCBs), the optimized biosensor exhibited excellent performance (0% false negatives and 7% false positives) in the screening of 110 samples of used Japanese transformer oil. The general approach for optimization described here is expected to benefit immunoassay researchers attempting to achieve optimum performance.

Least detectable concentration and dynamic range of three immunoassay systems using the same antibody.

The least detectable concentration (LDC) and dynamic range (DR) of three immunoassay systems are compared using four distinct antibodies (all specific for estradiol but with different affinities) on each system. The systems evaluated include the industry standard, ELISA, and two biosensors, surface plasmon resonance and kinetic exclusion. In all cases, the measurements of inhibition curves (response vs estradiol concentration) were contracted to outside experts (the biosensor manufacturers themselves in the case of the biosensors), each of whom was supplied with the same blind samples. Each biosensor manufacturer also reported an estimate of the equilibrium dissociation constant (Kd) for each of the antibodies. The LDC and DR observed for the kinetic exclusion biosensor are consistent with an interpretation of Kd limited detection while that from the other biosensor and ELISA show limits of detection somewhat above those expected for Kd limited performance. The determined LDC and DR of each biosensor is self-consistent in the sense that none of the inhibition data contradicts theoretical limits associated with the Kd as measured on that system; however, some contradictions are apparent across platforms. The use of multiple antibodies of differing Kd improves confidence that the observed differences in performance are associated with the immunoassay system rather than the particular analyte.

Improving an immunoassay response to related polychlorinated biphenyl analytes by mixing antibodies.

Immunoassays for detection of a class of closely related antigens, e.g., PCBs, have often been too specific (responding strongly to some members of the class and missing others) and no general method for adjusting the response has been described. In this paper, the difference in the response of a model immunoassay to different Kanechlors (Japanese commercial mixtures of PCBs, analogous to Aroclors in the United States) is reduced from 20- or 50-fold (depending on which antibody is used) to 3-fold when the antibodies are mixed at the proper ratio. A mathematical model based on competitive binding of two antibodies for up to four antigens has been developed and used to describe the assay performance and to predict optimum mix ratios for the antibodies used. The model (based on separate measurement of each antibody's effective Kd for each Kanechlor) provides an excellent fit to the measured mixed antibody assay response. The model is also successful in identifying cases where mixing monoclonal antibodies will not improve the response. It is thought the method described will have applicability in a variety of cases where the analytical goal is semiquantitative screening based on the total quantity of an unknown mixture of related compounds.

Simple immunoassay for detection of PCBs in transformer oil.

A rapid and inexpensive procedure to detect polychlorinated biphenyls (PCBs) in transformer oil is needed to facilitate identification and removal of PCB contaminated transformers. Here we describe a simple two-step liquid-liquid extraction using acidic dimethyl sulfoxide in conjunction with an immunoassay for detecting PCBs in transformer oil. The process described is faster and simpler than any previous immunoassay while maintaining comparable detection limit and false negative rate. Cross reactivity data, characterizing the immunoassay response to the four Kanechlor technical mixtures of PCBs in oil, are presented. Forty-five used transformer oil samples were analyzed by gas chromatography-high-resolution mass spectrometry and were also evaluated using the immunoassay protocol developed. Results presented show zero false negatives at a 1.4 ppm nominal cutoff for the transformer oils analyzed.

Measurement of the functional affinity constant of a monoclonal antibody for cell surface receptors using kinetic exclusion fluorescence immunoassay.

Measuring a protein-ligand interaction in solution, away from the ligand's cellular environment, may not provide an affinity value applicable in vivo. Here, we present a simple, accurate and highly sensitive method for determining the antibody affinity to cell surface receptor, hIGFR, and compare this data to affinity determined for the soluble receptor. Measurements were performed on both full-length bivalent IgG and the monovalent Fab fragments to assess possible differences in apparent affinity introduced by avidity of the bivalent IgG. Affinities determined for soluble hIGFR were 4 x 10(-12) M for the bivalent IgG and monovalent Fab. Comparable affinities of 6 x 10(-12) M and 1 x 10(-11) M for the bivalent IgG and Fab, respectively, were also determined for full-length hIGFR on cell surface. The method described allows estimation of reactant concentrations (anti-IGFR antibody) relative to one known reference concentration (the concentration of soluble hIGFR in our case) allowing us to estimate the average receptor density on the cell surface. Taken together, we believe these data can provide valuable insight into antibody behavior in vivo, especially in the case of insoluble or difficult to purify transmembrane receptors.

Validation of accuracy of enzyme-linked immunosorbent assay in hybridoma screening and proposal of an improved screening method.

The 96-well plate format of enzyme-linked immunosorbent assay (ELISA) is the de facto standard in screening hybridomas for active antibody. Despite its widespread use, there have been few or no systematic attempts to validate its accuracy and answer the fundamental question, is it finding all the positives? We report here on a comparison between ELISA and a semiautomated flow-based kinetic exclusion assay (KinExA), both used in screening the same hybridoma cell line. Our finding is that ELISA is both overreporting (false positives) and underreporting (false negatives) compared to the KinExA system. The large number of hybridoma cells (e.g., cultured in six 96-well plates) that must be checked is daunting in considering any method other than ELISA for routine screening. To overcome this, we devised a sampling strategy in which wells are combined in a specified pattern, allowing a significant reduction in the total number of measurements required.

Evaluation of a compact bench top immunoassay analyzer for automatic and near continuous monitoring of a sample for environmental contaminants.

A compact bench top immunoassay analyzer is evaluated and shown to possess sufficient automation to allow continuous unattended sampling and measuring while still achieving the theoretical (antibody affinity based) detection limit for analyte. The system is comprised of antigen coated particles in a disposable flow cell held at the focus of a filter fluorometer. Capture of fluorescently labeled antibody from the flow stream is inhibited by analyte in the sample, allowing analyte concentrations to be determined from the fluorescent intensity. The disposable cell was designed to allow easy end user changing of test specificity, e.g. for selection of any member of a panel of environmental contaminants. Standard curves are shown for six analytes of environmental interest, dioxin F114 (2,3,4,7,8-PeCDF), the pesticide Fenitrothion, three coplanar PCBs, including the most toxic, PCB 126, and estradiol. In each case the curves are constructed using antibody concentrations at or below the Kd of the antibody, assuring that the sensitivity shown is limited by the antibody itself rather than the analyzer. The dynamic range for the six analytes investigated ranged from a low of 5 to 340 pM for fenitrothion to a high of 0.8 to 59 nM for dioxin F114, and is correlated to the antibody Kd in every case. Data is also shown for 17 consecutive samples, including both high and low values, measured completely automatically over a period of hours. With further development and characterization, the bench top analyzer is expected to fill an important niche in environmental testing.

A combination of labeled and unlabeled antibody enables self-calibration and reduction of sample matrix effects in immunoassay.

Sample matrices interfering with analyte determinations, termed matrix effects, are one of the factors limiting the more widespread use of environmental immunoassays. Previous attempts to reduce matrix effects have focused on particular assays in specific matrices rather than on general methods. Here we describe a novel method to eliminate one class of matrix effects in immunoassay, independent of the particular matrix or analyte. The method is demonstrated with a model system detecting estradiol in either a 10% methanol or a 5% dimethyl sulfoxide matrix. Fluorescently labeled antiestradiol antibody is introduced as the detecting antibody and excess unlabeled antiestradiol antibody is included as a reference antibody. The binding of the excess reference antibody to the sample analyte artificially creates a sample containing no free analyte to bind to the detecting antibody. This allows estimation of the fluorescent signal for "zero" analyte in the actual sample matrix. The solvents employed as model systems reduce the affinity of the detecting antibody and cause false positive results at low estradiol concentrations and false negative results at high concentrations. The proposed reference method, including addition of the reference antibody, resulted in a self-calibrating assay in which the matrix effects, both positive and negative, were completely eliminated.

Use of excess solid-phase capacity in immunoassays: advantages for semicontinuous, near-real-time measurements and for analysis of matrix effects.

A flow-based immunoassay system using solid-phase particles with high binding capacity was used for semicontinuous, near-real-time, measurement of 17beta-estradiol (E2). The high binding capacity of the solid phase was exploited to enable (i) a quantitative determination of E2 concentration, based on rate of accumulation of fluorescently labeled anti-E2 antibody on the solid phase, and (ii) the use of a single solid phase for more than a dozen competitive binding measurements. The high binding capacity of the solid phase also permitted the immobilization of a second capture antigen. Biotin was immobilized as a second antigen and used to evaluate a biotin anti-biotin system as a control for matrix effects in the E2 immunoassay. In phosphate-buffered saline, E2 could be quantified (in the range of 10-1000 pM) by using either the summation or ratio of the signals from the labeled anti-E2 and anti-biotin antibody in the presence of biotin at a constant concentration. The same referencing system was applied to estimate the matrix effects in selected environmental samples. Matrix effects that inhibited the binding of the anti-E2 antibody to the solid phase led to false positive responses, but these matrix effects could be identified and partially corrected using the response from the anti-biotin antibody.