In vivo immune activation of splenocytes following exposure to tar from Asian sand dust.

Air pollution, especially that initiated by particulate matter (PM), has been implicated as a risk factor for several inflammatory diseases. Previously, it was reported that PM enhances immune responses. PM includes the tar fraction that contains polycyclic aromatic hydrocarbons (PAHs), which produce adverse health effects in exposed individuals. However, the influence of the tar fraction (as a component of PM) on splenocytes is not fully understood. The aim of this study was to determine the effects of the tar fraction extracted from PM collected from the atmosphere in Fukuoka, Japan, on mouse splenocytes. ICR mice were administered tar (1 or 5 µg/mouse) intratracheally 4 times at 2-week intervals, and splenocytes from the tar-treated mice were extracted and examined. The parameters determined were proliferation, cytokine concentrations and transcription factors activation. Following tar treatment, splenocyte proliferation increased relative to controls. Concanavalin A (ConA)-induced interleukin (IL)-2 formation and ConA- or lipopolysaccharide (LPS)-induced interferon-γ production were elevated in splenocytes from tar-exposed mice. However, the production of tumor necrosis factor-α and IL-6 induced by LPS was not markedly changed following tar treatment. Further, nuclear factor of activated T cells, but not nuclear factor-κB, was enhanced in splenocytes of tar-exposed mice. Data indicate that tar-activated splenocytes and PM-bound PAHs might contribute to T cell activation in the spleen.

Evaluation of a time efficient immunization strategy for anti-PAH antibody development.

The development of monoclonal antibodies (mAb) with affinity to small molecules can be a time-consuming process. To evaluate shortening the time for mAb production, we examined mouse antisera at different time points post-immunization to measure titer and to evaluate the affinity to the immunogen PBA (pyrene butyric acid). Fusions were also conducted temporally to evaluate antibody production success at various time periods. We produced anti-PBA antibodies 7 weeks post-immunization and selected for anti-PAH reactivity during the hybridoma screening process. Moreover, there were no obvious sensitivity differences relative to antibodies screened from a more traditional 18-week schedule. Our results demonstrate a more time efficient immunization strategy for anti-PAH antibody development that may be applied to other small molecules.

Tissue-specific metabolism of benzo[a]pyrene in rainbow trout (Oncorhynchus mykiss): a comparison between the liver and immune organs.

Polycyclic aromatic hydrocarbons (PAHs) are immunotoxicants in fish. In mammals, phase I metabolites are believed to be critically involved in the immunotoxicity of PAHs. This mechanism has been suggested for fish as well. The present study investigates the capacity of immune organs (head kidney, spleen) of rainbow trout, Oncorhynchus mykiss, to metabolize the prototypic PAH, benzo[a]pyrene (BaP). To this end, we analyzed 1) the induction of enzymatic capacity measured as 7-ethoxyresorufin-O-deethylase (EROD) activity in immune organs compared with liver, 2) the organ profiles of BaP metabolites generated in vivo, and 3) rates of microsomal BaP metabolite production in vitro. All measurements were done for control fish and for fish treated with an intraperitoneal injection of 15 mg BaP/kg body weight. In exposed trout, the liver, head kidney, and spleen contained similar levels of BaP, whereas EROD induction differed significantly between the organs, with liver showing the highest induction factor (132.8×), followed by head kidney (38.4×) and spleen (1.4×). Likewise, rates of microsomal metabolite formation experienced the highest induction in the liver of BaP-exposed trout, followed by the head kidney and spleen. Microsomes from control fish displayed tissue-specific differences in metabolite production. In contrast, in BaP-exposed trout, microsomes of all organs produced the potentially immunotoxic BaP-7,8-dihydrodiol as the main metabolite. The findings from this study show that PAHs, like BaP, are distributed into immune organs of fish and provide the first evidence that immune organs possess inducible PAH metabolism leading to in situ production of potentially immunotoxic PAH metabolites.

The relevance of monitoring of antibodies against the polycyclic aromatic hydrocarbon (PAH) and PAH-DNA adducts in serum in relation to lung cancer and chronic obstructive pulmonary disease (COPD).

Certain substances from the polycyclic aromatic hydrocarbons (PAHs) group are major inducers of respiratory tract carcinogenesis. The presented are the results of a serological epidemiological study aimed at monitoring the levels of anti-PAH antibodies and antibodies to PAH-DNA adducts in serum. The patients studied belonged both to the group of those with known lung disease (COPD and lung cancer), as well as to the healthy population of people who due to the work conditions or those at the place of residence can expect increased exposure to PAHs. In addition to the results proper that confirm increase of the genotoxic exposure risk to PAH in smoke-polluted places of residence and other PAH polluted environments. There has also been proved the relevance of still commonly used markers (DNA adducts), as well as the suitability of new markers, more favourable from the economic and practical viewpoints (anti-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide-DNA [anti-BPDE-DNA], anti-Benzo(a)pyrene antibodies of the IgA class).

Influence of immunization with non-genotoxic PAH-KLH conjugates on the resistance of organisms exposed to benzo(a)pyrene.

Polycyclic aromatic hydrocarbons (PAH) are recognized as common environmental pollutants released into the environment from many natural as well as man-made sources, and some have been classified as potent carcinogens. The main representative of the carcinogenic PAH is benzo(a)pyrene (B(a)P) which is known to induce genotoxic effects in vitro and in vivo, detected as PAH-DNA adducts. Long-term PAH exposure may be accompanied by an immunological response with the formation of antibodies against PAH as well as against PAH-DNA adducts. This paper describes the use of four PAH-keyhole-limpet haemocyanin (KLH) conjugates for the induction of specific and cross-reactive anti-PAH antibodies and focuses on the potential protective effects of anti-PAH antibodies produced after immunization of mice. In the in vitro experiments with HepG-2 cells, the genotoxicity of the PAH-KLH conjugates and the neutralizing effect of induced anti-PAH antibodies were evaluated. The titer of specific anti-PAH antibodies in sera and the amounts of DNA adducts in liver homogenates from immunized mice were investigated in vivo. The results show that anti-PAH antibodies of class IgG were induced during immunization. All the PAH-KLH conjugates tested were non-genotoxic and did not induce detectable DNA adducts in HepG2 cells or in the liver of immunized mice. The results show that only B(a)P-specific and B(a)P cross-reactive antibodies are able to neutralize B(a)P or its activated metabolites, which was revealed by a sudden decrease in the titer of anti-B(a)P antibodies in mouse sera after exposure to B(a)P. Furthermore, the anti-B(a)P antibodies produced by immunization were effective in reducing the amount of DNA adducts in mouse livers after intraperitoneal (i.p.) exposure to B(a)P. The results suggest that immunization with PAH-KLH conjugates can protect organisms against the adverse effects of carcinogenic PAH.

Polycyclic aromatic hydrocarbons regulate the pigmentation pathway and induce DNA damage responses in keratinocytes, a process driven by systemic immunity.

BACKGROUND: Urban pollution is correlated with an increased prevalence of skin pigmentation disorders, however the physiological processes underlying this association are unclear. OBJECTIVES: To delineate the relationship between polycyclic aromatic hydrocarbons (PAHs), a key constituent of atmospheric pollution, and immunity/skin pigmentation pathways. METHODS: We exposed peripheral blood mononuclear cells (PBMC) to PAHs and performed cytokines/chemokine profiling. We then examined the effect of immune activation on pigmentation by co-culturing PBMC and Benzo(a)pyrene (BaP) with reconstructed human pigmented epidermis (RHPE). To study the mechanism, we treated keratinocytes with conditioned medium from BaP-exposed PBMC and studied DNA damage responses, aryl hydrocarbon receptor (AhR) activation and pro-pigmentation factor, proopiomelanocortin (POMC) secretion. RESULTS: PAHs induced up-regulation of inflammatory cytokines/chemokine in PBMC. Co-culturing of RHPE with PBMC+BaP resulted in increased melanin content and localization. BaP-conditioned medium significantly increased DNA damage, p53 stabilization, AhR activation and POMC secretion in keratinocytes. We found that IFNγ induced DNA damage, while TNFα and IL-8 potentiated POMC secretion in keratinocytes. Importantly, BaP-conditioned medium-induced DNA damage and POMC secretion is prevented by antioxidants vitamin E, vitamin C and sulforaphane, as well as the prototypical corticosteroid dexamethasone. Finally, vitamin C and sulforaphane enhanced the genome protective and depigmentation effects of dexamethasone, providing proof-of-concept for a combinatorial approach for the prevention and/or correction of PAH-induced pigment spots formation. CONCLUSION: Our study reveals the importance of systemic immunity in regulating PAH-induced skin pigmentation, and provide a new keratinocyte DNA damage response mechanistic target for the prevention or reversal of pollution-associated skin pigmentation.

Disruption of human plasma cell differentiation by an environmental polycyclic aromatic hydrocarbon: a mechanistic immunotoxicological study.

BACKGROUND: The AhR is a ligand-activated transcription factor that mediates immunosuppression induced by environmental PAH and HAH. Recently, a critical role for the AhR in development of T cells involved in autoimmunity (Th17 and Treg) has been demonstrated, supporting the hypothesis that the AhR plays a key role in immune regulation both in the presence and absence of environmental ligands. Despite these results with T cells systems, little is known of the role that the AhR plays in B cell development. We have demonstrated that B cell activation with CD40 ligand, a stimulus that models adaptive immunity, induces AhR expression in primary human B cells, suggesting that activation may increase human B cell sensitivity to AhR ligands and that the AhR may play a role in B cell development. METHODS: To test these possibilities, we developed an in vitro system in which activated human B cells expressing high AhR levels are induced to differentiate into plasma cells. Consequently, the effects of benzo [a]pyrene, a prototypic environmental AhR ligand, on plasma cell differentiation could be investigated and this chemical could be exploited essentially as drug probe to implicate the role of the AhR in plasma cell development. RESULTS: A previously unattainable level of B cell differentiation into plasma cells (up to 45% conversion) was observed. Benzo [a]pyrene significantly suppressed that differentiation. gamma-Irradiation after an initial proliferation phase induced by CD40 ligand and immediately prior to initiation of the differentiation phase blocked cell growth but did not affect cell viability or plasma cell differentiation. B [a]P suppressed differentiation whether or not cell growth was inhibited by gamma-irradiation. CONCLUSIONS: 1) Extensive proliferation is not required during the differentiation phase per se for CD40L-activated human B cells to undergo plasma cell differentiation, and 2) an environmental PAH blocks both proliferation and differentiation of AhR expressing B cells. The results uncover a new mechanism by which environmentally ubiquitous PAHs may negatively impact human B cell-mediated immunity.

The development and evaluation of monoclonal antibodies for the detection of polycyclic aromatic hydrocarbons.

A highly sensitive enzyme-linked immunosorbent assay (ELISA) for the detection of 3- to 5-ring polycyclic aromatic hydrocarbons (PAHs) has been developed. A functional derivative of dibenzothiophene was synthesized and covalently linked to carrier proteins that were used to produce monoclonal antibodies (mAbs). During the conjugation step, the conjugation efficiency was improved by the presence of 25% N,N-dimethylformamide (DMF). Antibodies were selected based on a competitive inhibition assay to isolate those with the highest sensitivity for free PAHs. When using the mAb in an ELISA format, free PAHs were detected at a concentration as low as 0.1 microg/L (0.1 ppb) in aqueous samples.

A pilot study comparing T-regulatory cell function among healthy children in different areas of Gansu, China.

Immune system is critical to protecting human health from toxic substances. Our previously published research had found an important link between polycyclic aromatic hydrocarbons (PAHs) in ambient air and changes at the DNA level in immune cells that led to impaired function of regulatory T (Treg) cells in children living in California, USA. But molecular and cellular pathways of these changes remain unclear. The present study aims to explore whether exposure to PAHs leads to changes in Treg cells functions of children living in Gansu, China, where ambient air pollution levels are much higher than those in California, and to explore potential mechanisms of PAH-induced immunological dysfunctions. Air pollutions in Lanzhou and Lintao, Gansu Province, were measured from December 2015 to June 2016. Healthy children were recruited from both cities and enrolled in this pilot study. Demographic information was collected by questionnaires. Blood samples were collected. Peripheral blood Treg cells were analyzed for Treg cells percentage by flow cytometry. Gene expression of forkhead box transcription factor 3 (Foxp3), transforming growth factor-ß (TGF-ß), and interleukin 35 (IL35) were examined by reverse transcription-polymerase chain reaction (RT-PCR). The results indicated PAH concentration (as sum of 16 PAHs) in Lintao was over two times higher than that was in Lanzhou (707 vs. 326 ng/m3), whereas PM2.5 concentration was comparable in two cities (55.3 in Lintao vs. 65.7 µg/m3 in Lanzhou). Notably, we observed lower gene expressions for Foxp3 (P < 0.05), IL35 (P < 0.05), and TGF-ß, in children living in Lintao, suggesting an impairment of Treg cells function potentially associated with higher PAH exposure in Lintao. However, no significant difference was observed in Treg cells % among CD4+ T cells between Lanzhou and Lintao groups.

Immunization with soot from a non-combustion process provokes formation of antibodies against polycyclic aromatic hydrocarbons.

The emission of soot during combustion processes used in transportation, manufacturing, and power generation is of increasing concern because of its serious adverse health effects. In particular, the ability to modulate the immune system has recently been established. In the present investigation, an artificial soot sample that was prepared by fragmentation of acetylene in a laser-induced plasma was used as an antigen for the immunization of a rabbit. A highly sensitive competition curve in an indirect competitive ELISA using a benzo[a]pyrene-BSA conjugate as a coating antigen could be constructed for benzo[a]pyrene with an IC50 of 2.94 mug/l (11.65 nmol/l). In contrast to the high affinity, the soot antiserum dilution (antibody titer) of 1:750 was rather low. The cross-reactivity was tested with 16 parent polycyclic aromatic hydrocarbons, 7 nitrated polycyclic aromatics, and 3 methylated, hydroxylated or butyric acid derivatives. The results obtained suggest that the vertebrate immune system can respond to an immunization with soot by the generation of high affinity IgG class antibodies against polycyclic aromatics. It is likely that antibodies are raised against the molecular structures which form the framework of the soot particles and not against adsorbed and extractable polyaromatic compounds. The experiments suggest that if soot is considered a T-independent antigen, the isotype switch, essentially from IgM to IgG, could have been caused by co-inoculation with a T-dependent antigen, i.e., mycobacteria contained in Freunds complete adjuvant. However, at the cellular level the mechanism remains to be uncovered.

Real-time immuno-PCR for ultrasensitive detection of pyrene and other homologous PAHs.

Polycyclic aromatic hydrocarbons (PAHs) are significant environmental pollutant that can lead to cancer and endocrine system disrupting. Here we developed a real-time immuno-PCR (RT-IPCR) assay based on a biotinylated reporter DNA system for ultrasensitive detection of pyrene (PYR) and homologous PAHs in water. The PAHs in sample compete with PYR-OVA coated on PCR plate to bind with monoclonal antibody (McAb). The biotinylated goat anti-mouse IgG (Bio-IgG) can be captured by the McAb bound with PYR-OVA. Then streptavidin is bound with biotin on Bio-IgG. Finally biotinylated reporter DNA is captured by the streptavidin and quantified by real-time PCR using FastStart universal SYBR Green Master (ROX) kit. The linear range of the assay was from 500 fmol L(-1) to 5 nmol L(-)) with a detection limit of 450 fmol L(-1). The average recoveries of PYR and homologous PAHs from lake water, tap water and commercial mineral water were 96.8%, 101.4% and 99.6% respectively, indicating that water samples had little interfere with the assay. The results demonstrated that the developed RT-IPCR might be a potential method for ultrasensitive detection of PYR and homologous PAHs in drinking and environment water sample.

Polyaromatic hydrocarbons administered in humans by dermal route increase total IgE.

Inhalation of polyaromatic hydrocarbons (PAHs) extracted from diesel exhaust particles (DEP) enhances local (nasal) production of IgE in humans. The aim of the present research is to investigate whether in humans dermal exposure to PAHs which are not extracted from DEPs increases serum IgE, and whether host factors modify the immunologic effect. In thirty-two patients with acute psoriatic lesions, a cream containing 3% of coal tar (which holds a variety of PAHs) was applied to the skin for 24 hours. Serum IgE were measured before (IgE0) and four (IgE4) and eight (IgE8) days after application. Replicated means were compared by analysis of variance for repeated measures and by the Newman-Keuls' test. IgE0, IgE4 and IgE8 were 151.19, 159.69 (a 6% excess) and 170.90 kU/L (a 13% excess) respectively; pairwise comparison showed IgE8 was significantly higher than IgE0 (p<0.05). At multiple linear regression analysis, the percentage increase in serum IgE across observation days was the dependent variable against age, sex, cigarettes/day, urinary 1-pyrenol, atopy, skin area treated, and grams of cream. Of the independent variables, only age had a significant (p<0.028) influence: the younger the age, the higher the IgE response to PAHs. We conclude that whatever the source and the route of entry (skin or respiratory tract), PAHs increase total serum IgE, mainly in younger age groups.

Evaluation of two direct immunoassays for rapid detection of petroleum products on marine birds.

To reduce rehabilitation time and improve survival, a practical, objective test is needed to determine if marine birds are contaminated with oil before they enter captivity. The RaPID Assays and EnviroGard immunoassays for detection of polycyclic aromatic hydrocarbons (PAHs) in soil were evaluated for their ability to detect petroleum on feather samples from 30 intermediate fuel oil contaminated and 30 uncontaminated common murres (Uria aalge). Sensitivity, specificity, positive and negative predictive value, precision, and time required to run each assay were determined. The RaPID Assays was 96.7% sensitive and specific, while the EnviroGard assay was 93.3% sensitive, and 90.0% specific. Sensitivity decreased at higher dilutions for both assays. Intra-replicate variation was less than 20%. Our evaluation showed that these immunoassays are rapid and cost-effective methods for detecting oil-contamination on the plumage of seabirds, with the EnviroGard assay being more practical in most oil spill response situations due to ease of use and rapidity of results.

[New approaches in evaluating the antitumor immune response in breast cancer]. / Novye podkhody k otsenke protivoopukholevoi immunoreaktivnosti pri rake molochnoi zhelezy.

Different classes of antibodies to polycyclic aromatic hydrocarbons (PAH) and immunogenicity of carcinoembryonic antigen (CEA) were investigated in breast cancer patients versus course. Prior to treatment, the levels of IgM, IgG and IgA antibodies to PAHs were 5.9, 7.7 and 32.5%, respectively. In cases of tumor regression, they were 13.8, 25.0 and 19.2%, while in those of progression-2.4, 2.8 and 12.8%, respectively. The immunogenicity of CEA, i.e. the power of forming circulating immune complexes, appeared to be higher in cases of tumor regression than in those of progression. CEA concentration in immune complexes exceeding 5ng/ml was observed in 24.0 and 15.1% of cases, respectively, while in whole serum, the concentration in excess of 10 ng/ml was in 8.0 and 42.4%, respectively.

On stabilization of a neutral aromatic ligand by π-cation interactions in monoclonal antibodies.

It has been shown that anti-PAH mAb can bind a particular cross-reactant by adopting two distinct "red" and "blue" conformations of its binding sites [N.M. Grubor et al. PNAS 102, 2005, 7453-7458]. In the case of red conformation of pyrene (Py)/anti-PAH mAb (with a broad fluorescence (0,0)-band with fwhm ~140 cm(-1)), the central role in complex formation was played by π-π interactions. The nature of the blue-shifted conformation with very narrow fluorescence (0,0)-band (fwhm ~75 cm(-1)) was left unclear due to the lack of suitable data for comparison. In this work, we suggest spectroscopic and modeling results obtained for the blue conformation of Py in several mAb (including 4D5 mAb) are consistent with π-cation interactions, underscoring the importance of π-cation interaction in ligand binding and stabilization in agreement with earlier modeling studies [J-L. Pellequer, et al. J. Mol. Biol. 302, 2000, 691-699]. We propose considerable narrowing of the fluorescence origin band of ligand in the protein environment could be regarded as a simple indicator of π-cation interactions. Since 4D5 mAb forms only the blue-shifted conformation, while anti-PAH and 8E11 mAbs form both blue- and red-shifted conformations, we suggest mAb interactions, with Py molecules lacking H-bonding functionality, may induce distinct conformations of mAb binding sites that allow binding by π-π and/or π-cation interactions.

Enhanced immunological and detoxification responses in Pacific oysters, Crassostrea gigas, exposed to chemically dispersed oil.

The aim of this study was to evaluate the effects of chemically dispersed oil on an economically and ecologically important species inhabiting coasts and estuaries, the Pacific oyster Crassostrea gigas. Studies were carried out with juveniles, known to generally be more sensitive to environmental stress than adults. A set of enzyme activities involved in immune defence mechanisms and detoxification processes, i.e. superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), catecholase-type phenoloxidase (PO), laccase-type PO and lysozyme were analysed in different oyster tissues, i.e. the gills, digestive gland and mantle, and in the plasma and the haemoycte lysate supernatant (HLS) of the haemolymph. Results indicated that total PAH body burdens were 2.7 times higher in the presence than in the absence of the chemical dispersant. After 2 days of exposure to chemically dispersed oil, alkylated naphthalenes accounted for 55% of the total PAH body burden, whereas alkylated fluorenes and alkylated dibenzothiophenes accounted for 80% when the chemical dispersant was absent. Importantly, a higher number of enzyme activities were modified when oil was chemically dispersed, especially in the plasma and gills. Moreover, independently of the presence or absence of chemical dispersant, oil exposure generally inhibited enzyme activities in the gills and plasma, while they were generally activated in the mantle and haemocytes. These results suggest that the gills and plasma constitute sensitive compartments in C. gigas, and that the mantle and haemocytes may play an important role in protection against xenobiotics. Among the six enzyme activities that were analysed in these body compartments, five were modulated in the chemical dispersion (CD) treatment while only half of the enzyme activities were modulated in the mechanical dispersion treatment. Furthermore, CD treatment effects were often observed following exposure, but also during depuration periods. These results suggest that immune and/or detoxification responses are likely to be affected when dispersants are used to treat oil spills in shallow waters.

A new danger in the air: how pulmonary innate immunity copes with man-made airborne xenobiotics.

The pulmonary innate immune system has evolved over millions of years to provide swift detection of inhaled microbial agents and trigger well-balanced protective responses. Much more recent on the evolutionary scale is human activity, which has resulted in the release of a new class of potentially harmful, non-microbial compounds into the air. These xenobiotics include combustion by-products such as reactive oxygen species and polycyclic aromatic hydrocarbons. This review will summarize evidence showing how airborne xenobiotics can engage pulmonary innate immunity components at many levels. We will focus on potential effects of xenobiotics on airway dendritic cells, as these constitute key innate immune sensors in the lung, with the unique ability to initiate adaptive immunity. We propose that the aberrant processing of inhaled xenobiotics by an innate immune system that is now evolutionarily maladapted underlies the increase in chronic inflammatory lung diseases in modern times.

Color encoded microbeads-based flow cytometric immunoassay for polycyclic aromatic hydrocarbons in food.

Food contamination caused by chemical hazards such as persistent organic pollutants (POPs) is a worldwide public health concern and requires continuous monitoring. The chromatography-based analysis methods for POPs are accurate and quite sensitive but they are time-consuming, laborious and expensive. Thus, there is a need for validated simplified screening tools, which are inexpensive, rapid, have automation potential and can detect multiple POPs simultaneously. In this study we developed a flow cytometry-based immunoassay (FCIA) using a color-encoded microbeads technology to detect benzo[a]pyrene (BaP) and other polycyclic aromatic hydrocarbons (PAHs) in buffer and food extracts as a starting point for the future development of rapid multiplex assays including other POPs in food, such as polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs). A highly sensitive assay for BaP was obtained with an IC(50) of 0.3 microg L(-1) using a monoclonal antibody (Mab22F12) against BaP, similar to the IC(50) of a previously described enzyme-linked immunosorbent assay (ELISA) using the same Mab. Moreover, the FCIA was 8 times more sensitive for BaP compared to a surface plasmon resonance (SPR)-based biosensor immunoassay (BIA) using the same reagents. The selectivity of the FCIAs was tested, with two Mabs against BaP for 25 other PAHs, including two hydroxyl PAH metabolites. Apart from BaP, the FCIAs can detect PAHs such as indenol[1,2,3-cd]pyrene (IP), benz[a]anthracene (BaA), and chrysene (CHR) which are also appointed by the European Food Safety Authority (EFSA) as suitable indicators of PAH contamination in food. The FCIAs results were in agreement with those obtained with gas chromatography-mass spectrometry (GC-MS) for the detection of PAHs in real food samples of smoked carp and wheat flour and has great potential for the future routine application of this assay in a simplex or multiplex format in combination with simplified extraction procedure which are under development.

Development of redox-labeled electrochemical immunoassay for polycyclic aromatic hydrocarbons with controlled surface modification and catalytic voltammetric detection.

A redox-labeled direct competitive electrochemical immunoassay for polycyclic aromatic hydrocarbons (PAHs) was developed. A ruthenium tris(bipyridine)-pyrenebutyric acid conjugate was synthesized as the redox-labeled tracer. Its recognition by an anti-PAH monoclonal antibody was confirmed by surface plasmon resonance. In the immunoassay, the antibody was immobilized on (3-glycidoxypropyl)-trimethoxysilane (GPTMS)-modified indium tin oxide (ITO) electrodes. The assay was quantified by measuring the electro-catalytic current of the redox label in an oxalate-containing electrolyte which served as a sacrificial electron donor to amplify the current signal. Formation of GPTMS film on ITO and subsequent antibody immobilization were characterized by X-ray photoelectron spectroscopy (XPS) and electrochemistry. Using a ruthenium tris(bipyridine)-conjugated IgG (IgG-Ru) as the surface-bound redox probe, the highest electrochemical signal was obtained on GPTMS electrodes with 1 h modification. Under the optimized conditions for ITO modification, antibody immobilization and tracer concentration, competition curves for benzo[a]pyrene and pyrenebutyric acid were obtained with a detection limit of 2.4 and 10 ng mL(-1), respectively. The redox-labeled electrochemical immunoassay with signal amplification mechanism offers a potential analytical method for the simultaneous detection of multiple environmental organic pollutants on antibody biochips.