Hunting for pigments in bacterial settlers of the Great Pacific Garbage Patch.

The Great Pacific Garbage Patch, a significant collection of plastic introduced by human activities, provides an ideal environment to study bacterial lifestyles on plastic substrates. We proposed that bacteria colonizing the floating plastic debris would develop strategies to deal with the ultraviolet-exposed substrate, such as the production of antioxidant pigments. We observed a variety of pigmentation in 67 strains that were directly cultivated from plastic pieces sampled from the Garbage Patch. The genomic analysis of four representative strains, each distinct in taxonomy, revealed multiple pathways for carotenoid production. These pathways include those that produce less common carotenoids and a cluster of photosynthetic genes. This cluster appears to originate from a potentially new species of the Rhodobacteraceae family. This represents the first report of an aerobic anoxygenic photoheterotrophic bacterium from plastic biofilms. Spectral analysis showed that the bacteria actively produce carotenoids, such as beta-carotene and beta-cryptoxanthin, and bacteriochlorophyll a. Furthermore, we discovered that the genetic ability to synthesize carotenoids is more common in plastic biofilms than in the surrounding water communities. Our findings suggest that plastic biofilms could be an overlooked source of bacteria-produced carotenoids, including rare forms. It also suggests that photoreactive molecules might play a crucial role in bacterial biofilm communities in surface water.

Hotspots of Floating Plastic Particles across the North Pacific Ocean.

The pollution of the marine environment with plastic debris is expected to increase, where ocean currents and winds cause their accumulation in convergence zones like the North Pacific Subtropical Gyre (NPSG). Surface-floating plastic (>330 µm) was collected in the North Pacific Ocean between Vancouver (Canada) and Singapore using a neuston catamaran and identified by Fourier-transform infrared spectroscopy (FT-IR). Baseline concentrations of 41,600-102,700 items km-2 were found, dominated by polyethylene and polypropylene. Higher concentrations (factors 4-10) of plastic items occurred not only in the NPSG (452,800 items km-2) but also in a second area, the Papaha̅naumokua̅kea Marine National Monument (PMNM, 285,200 items km-2). This second maximum was neither reported previously nor predicted by the applied ocean current model. Visual observations of floating debris (>5 cm; 8-2565 items km-2 and 34-4941 items km-2 including smaller "white bits") yielded similar patterns of baseline pollution (34-3265 items km-2) and elevated concentrations of plastic debris in the NPSG (67-4941 items km-2) and the PMNM (295-3748 items km-2). These findings suggest that ocean currents are not the only factor provoking plastic debris accumulation in the ocean. Visual observations may be useful to increase our knowledge of large-scale (micro)plastic pollution in the global oceans.

Spectrum of ANCA-specificities in eosinophilic granulomatosis with polyangiitis. A retrospective multicentre study.

OBJECTIVES: To determine the spectrum of anti-neutrophil cytoplasmic antibody (ANCA) antigen-specificities in eosinophilic granulomatosis with polyangiitis (EGPA), an ANCA-associated vasculitis (AAV) entity. METHODS: We conducted a retrospective analysis of 73 EGPA patients from three German tertiary referral centres for vasculitis. In addition to in-house ANCA testing, pentraxin 3 (PTX3)- and olfactomedin 4 (OLM4)-ANCA were determined using a prototype cell-based assay for research (EUROIMMUN, Lübeck, Germany). Patient characteristics and clinical manifestations were evaluated and compared based on ANCA status. RESULTS: Myeloperoxidase (MPO)-ANCA positive patients (n=8; 11%) significantly more frequently displayed peripheral nervous system (PNS) and pulmonary involvement and less frequently heart involvement compared to MPO-ANCA negative patients. PTX3-ANCA positive patients (n=5; 6.8%) had a significantly higher prevalence of ear, nose and throat, pulmonary, gastrointestinal and PNS involvement, and a lower prevalence of renal and central nervous system involvement compared to PTX3-ANCA negative patients. Proteinase 3 (PR3)-ANCA and OLM4-ANCA were detected in 2 patients (2.7%) each with multiorgan involvement. One PR3-ANCA positive patient was also positive for bactericidal permeability increasing protein (BPI)-ANCA. CONCLUSIONS: In addition to MPO, the spectrum of ANCA antigen specificities includes various other target antigens such as PR3, BPI, PTX3, and OLM4, potentially segregating further EGPA subgroups. A lower prevalence of MPO-ANCA was detected in this study compared with other studies. OLM4 is reported as novel ANCA antigen-specificity in EGPA, and thus AAV.

Effects of leachates from UV-weathered microplastic on the microalgae Scenedesmus vacuolatus.

Plastics undergo successive fragmentation and chemical leaching steps in the environment due to weathering processes such as photo-oxidation. Here, we report the effects of leachates from UV-irradiated microplastics towards the chlorophyte Scenedesmus vacuolatus. The microplastics tested were derived from an additive-containing electronic waste (EW) and a computer keyboard (KB) as well as commercial virgin polymers with low additive content, including polyethylene (PE), polyethylene terephthalate (PET), polypropylene (PP), and polystyrene (PS). Whereas leachates from additive-containing EW and KB induced severe effects, the leachates from virgin PET, PP, and PS did not show substantial adverse effects in our autotrophic test system. Leachates from PE reduced algae biomass, cell growth, and photosynthetic activity. Experimental data were consistent with predicted effect concentrations based on the ionization-corrected liposome/water distribution ratios (Dlip/w) of polymer degradation products of PE (mono- and dicarboxylic acids), indicating that leachates from weathering PE were mainly baseline toxic. This study provides insight into algae toxicity elicited by leachates from UV-weathered microplastics of different origin, complementing the current particle- vs. chemical-focused research towards the toxicity of plastics and their leachates.

Equilibrium sampling of suspended particulate matter as a universal proxy for fish and mussel monitoring.

Bioaccumulation of persistent and hydrophobic organic compounds in the aquatic environment puts secondary consumers, such as fish, at risk. To assess their exposure, monitoring programs with high numbers of individuals have been conducted worldwide over decades that require major efforts and raise ethical issues. This study aimed at testing suspended particulate matter (SPM) as an alternative and accessible abiotic matrix to estimate the internal exposure concentrations of such chemicals in fish and mussels. Muscle tissues of bream (Abramis brama), tissues of zebra mussels (Dreissena polymorpha) and SPM were collected from four major German rivers, Elbe, Danube, Saar and Saale, in 2018 within the national monitoring program of the German Environmental Specimen Bank. We used (i) total solvent extraction for biota samples to quantify the lipid-normalized concentrations of polychlorinated biphenyls, polycyclic aromatic hydrocarbons and hexachlorobenzene and (ii) passive equilibrium sampling of SPM to derive equilibrium partitioning concentrations in lipids and (iii) set these independent data sets into context. Since the ratio of lipid-normalized concentration / equilibrium partitioning concentration in lipids was in most cases < 1.0, SPM may serve as a conservative proxy for the internal concentration of bream and mussels, although bream of high age (i.e., older than 10 years) showed a tendency for this ratio to exceed 1.0. This observation indicates that age-dependent biomagnification can exceed the predictions based on thermodynamic equilibrium relative to SPM.

Conditioning Film and Early Biofilm Succession on Plastic Surfaces.

In the context of environmental plastic pollution, it is still under debate if and how the "plastisphere", a plastic-specific microbial community, emerges. In this study, we tested the hypothesis that the first conditioning film of dissolved organic matter (DOM) sorbs selectively to polymer substrates and that microbial attachment is governed in a substrate-dependent manner. We investigated the adsorption of stream water-derived DOM to polyethylene terephthalate (PET), polystyrene (PS), and glass (as control) including UV-weathered surfaces by Fourier-transform ion cyclotron mass spectrometry. Generally, the saturated, high-molecular mass and thus more hydrophobic fraction of the original stream water DOM preferentially adsorbed to the substrates. The UV-weathered polymers adsorbed more polar, hydrophilic OM as compared to the dark controls. The amplicon sequencing data of the initial microbial colonization process revealed a tendency of substrate specificity for biofilm attachment after 24 h and a clear convergence of the communities after 72 h of incubation. Conclusively, the adsorbed OM layer developed depending on the materials' surface properties and increased the water contact angles, indicating higher surface hydrophobicity as compared to pristine surfaces. This study improves our understanding of molecular and biological interactions at the polymer/water interface that are relevant to understand the ecological impact of plastic pollution on a community level.

Weathering Plastics as a Planetary Boundary Threat: Exposure, Fate, and Hazards.

We described in 2017 how weathering plastic litter in the marine environment fulfils two of three criteria to impose a planetary boundary threat related to "chemical pollution and the release of novel entities": (1) planetary-scale exposure, which (2) is not readily reversible. Whether marine plastics meet the third criterion, (3) eliciting a disruptive impact on vital earth system processes, was uncertain. Since then, several important discoveries have been made to motivate a re-evaluation. A key issue is if weathering macroplastics, microplastics, nanoplastics, and their leachates have an inherently higher potential to elicit adverse effects than natural particles of the same size. We summarize novel findings related to weathering plastic in the context of the planetary boundary threat criteria that demonstrate (1) increasing exposure, (2) fate processes leading to poorly reversible pollution, and (3) (eco)toxicological hazards and their thresholds. We provide evidence that the third criterion could be fulfilled for weathering plastics in sensitive environments and therefore conclude that weathering plastics pose a planetary boundary threat. We suggest future research priorities to better understand (eco)toxicological hazards modulated by increasing exposure and continuous weathering processes, to better parametrize the planetary boundary threshold for plastic pollution.

Bioavailable Environmental Pollutant Patterns in Sediments from Passive Equilibrium Sampling.

Sediment-associated risks depend on the bioavailable fraction of organic chemicals and cannot be comprehended by their total concentrations. The present study investigated contamination patterns of bioavailable chemicals in sediments from various sites around the globe by using passive equilibrium sampling. The extracts had been characterized previously for mixture effects by in vitro reporter gene assays and were in this study analyzed using gas chromatography-high resolution mass spectrometry for 121 chemicals including both legacy and emerging contaminants. The spatial distribution of the detected chemicals revealed distinct contamination patterns among sampling sites. We identified compounds in common at the different sites but most contaminant mixtures were site-specific. The mixture effects of the detected chemicals were predicted with a mixture toxicity model from effect concentrations of bioactive single chemicals and detected concentrations, applying a joint model for concentration addition and independent action. The predicted mixture effects were dominated by polycyclic aromatic hydrocarbons, and among the chemicals with available effect data, 17% elicited oxidative stress response and 18% activated the arylhydrocarbon receptor. Except for two sites in Sweden, where 11 and 38% of the observed oxidative stress response were explained by the detected chemicals, less than 10% of effects in both biological end points were explained. These results provide a comprehensive investigation of bioavailable contamination patterns of sediments and may serve as an example of employing passive equilibrium sampling as a monitoring technique to integrate the risk of bioavailable sediment-associated chemicals in aquatic environments.

Influence of Co-Dosed Lipids from Biota Extracts on the Availability of Chemicals in In Vitro Cell-Based Bioassays.

Extraction of chemicals from biota leads to co-extraction of lipids. When dosing such extracts into in vitro bioassays, co-dosed lipids act as an additional phase that can reduce the bioavailability of the chemicals and the apparent sensitivity of the assay. Equilibrium partitioning between medium, cells, and co-dosed lipids was described with an existing equilibrium partitioning model for cell-based bioassays extended by an additional lipid phase. We experimentally investigated the influence of co-dosed lipids on the effects elicited by four test chemicals of different hydrophobicity in two bioassays, indicative of the aryl hydrocarbon receptor and oxidative stress response (AREc32). The partitioning model explained the effect of the test chemicals in the presence of spiked triolein within a factor of 0.33-5.83 between the measured and predicted effect concentration (EC). We applied the model to marine mammal blubber extracted with silicone. Extracts dosed in the AREc32 bioassay showed a linear increase of apparent EC with increasing lipid fraction. The partitioning model was used to interpret the role of the co-extracted lipid. A quantitative lipid correction of bioassay results in the presence of co-dosed lipids was possible for known compounds and defined mixtures, while we could only estimate a range for mixtures of unknown chemicals.

Sorptive Capacities of Nonpolymeric Plant Lipids for Hydrophobic Chemicals Determined by Passive Dosing.

Vegetation plays an important role in the partitioning, transport, and fate of semivolatile hydrophobic organic chemicals (HOCs) in the environment. Leaf/air partition ratios ( Kleaf/air) of HOCs are highly variable for different plant species. The differences cannot be fully explained by the fraction of lipids in the leaves or the thickness of the cuticle. Our goal was to elucidate the importance of nonpolymeric lipids in determining Kleaf/air. To do this, we extracted organic matter from 7 plant species using solvents that do not extract the polymeric lipids cutin and cutan, to yield extractable organic matter (EOM). We used passive dosing to determine the partition ratios of selected HOCs between the EOM of the leaves and our reference lipid, olive oil ( KEOM/olive oil). In addition, we measured analogous partition ratios for three lipid standards. Proton nuclear magnetic resonance (NMR) spectroscopy was used to characterize the composition of lipids. Differences in KEOM/olive oil of two polychlorinated biphenyls and four chlorinated benzenes were below a factor of 2 in the plant species studied, indicating that the reported differences in Kleaf/air are not caused by differences in the sorptive capacities of nonpolymeric lipids or that our EOM is not representative of all nonpolymeric leaf lipids.

Effects of Leachates from UV-Weathered Microplastic in Cell-Based Bioassays.

Standard ecotoxicological testing of microplastic does not provide insight into the influence that environmental weathering by, e.g., UV light has on related effects. In this study, we leached chemicals from plastic into artificial seawater during simulated UV-induced weathering. We tested largely additive-free preproduction polyethylene, polyethylene terephthalate, polypropylene, and polystyrene and two types of plastic obtained from electronic equipment as positive controls. Leachates were concentrated by solid-phase extraction and dosed into cell-based bioassays that cover (i) cytotoxicity; (ii) activation of metabolic enzymes via binding to the arylhydrocarbon receptor (AhR) and the peroxisome proliferator-activated receptor (PPARγ); (iii) specific, receptor-mediated effects (estrogenicity, ERα); and (iv) adaptive response to oxidative stress (AREc32). LC-HRMS analysis was used to identify possible chain-scission products of polymer degradation, which were then tested in AREc32 and PPARγ. Explicit activation of all assays by the positive controls provided proof-of-concept of the experimental setup to demonstrate effects of chemicals liberated during weathering. All plastic leachates activated the oxidative stress response, in most cases with increased induction by UV-treated samples compared to dark controls. For PPARγ, polyethylene-specific effects were partially explained by the detected dicarboxylic acids. Since the preproduction plastic showed low effects often in the range of the blanks future studies should investigate implications of weathering on end consumer products containing additives.

Strategies for Transferring Mixtures of Organic Contaminants from Aquatic Environments into Bioassays.

Mixtures of organic contaminants are ubiquitous in the environment. Depending on their persistence and physicochemical properties, individual chemicals that make up the mixture partition and distribute within the environment and might then jointly elicit toxicological effects. For the assessment and monitoring of such mixtures, a variety of cell-based in vitro and low-complexity in vivo bioassays based on algae, daphnids or fish embryos are available. A very important and sometimes unrecognized challenge is how to combine sampling, extraction and dosing to transfer the mixtures from the environment into bioassays, while conserving (or re-establishing) their chemical composition at adjustable levels for concentration-effect assessment. This article outlines various strategies for quantifiable transfer from environmental samples including water, sediment, and biota into bioassays using total extraction or polymer-based passive sampling combined with either solvent spiking or passive dosing.

Organic Carbon/Water and Dissolved Organic Carbon/Water Partitioning of Cyclic Volatile Methylsiloxanes: Measurements and Polyparameter Linear Free Energy Relationships.

The sorption of cyclic volatile methyl siloxanes (cVMS) to organic matter has a strong influence on their fate in the aquatic environment. We report new measurements of the partition ratios between freshwater sediment organic carbon and water (KOC) and between Aldrich humic acid dissolved organic carbon and water (KDOC) for three cVMS, and for three polychlorinated biphenyls (PCBs) that were used as reference chemicals. Our measurements were made using a purge-and-trap method that employs benchmark chemicals to calibrate mass transfer at the air/water interface in a fugacity-based multimedia model. The measured log KOC of octamethylcyclotetrasiloxane (D4), decamethylcyclopentasiloxane (D5), and dodecamethylcyclohexasiloxane (D6) were 5.06, 6.12, and 7.07, and log KDOC were 5.05, 6.13, and 6.79. To our knowledge, our measurements for KOC of D6 and KDOC of D4 and D6 are the first reported. Polyparameter linear free energy relationships (PP-LFERs) derived from training sets of empirical data that did not include cVMS generally did not predict our measured partition ratios of cVMS accurately (root-mean-squared-error (RMSE) for logKOC 0.76 and for logKDOC 0.73). We constructed new PP-LFERs that accurately describe partition ratios for the cVMS as well as for other chemicals by including our new measurements in the existing training sets (logKOC RMSEcVMS: 0.09, logKDOC RMSEcVMS: 0.12). The PP-LFERs we have developed here should be further evaluated and perhaps recalibrated when experimental data for other siloxanes become available.

Equilibrium sampling to determine the thermodynamic potential for bioaccumulation of persistent organic pollutants from sediment.

Equilibrium partitioning (EqP) theory is currently the most widely used approach for linking sediment pollution by persistent hydrophobic organic chemicals to bioaccumulation. Most applications of the EqP approach assume (I) a generic relationship between organic carbon-normalized chemical concentrations in sediments and lipid-normalized concentrations in biota and (II) that bioaccumulation does not induce levels exceeding those expected from equilibrium partitioning. Here, we demonstrate that assumption I can be obviated by equilibrating a silicone sampler with chemicals in sediment, measuring chemical concentrations in the silicone, and applying lipid/silicone partition ratios to yield concentrations in lipid at thermodynamic equilibrium with the sediment (CLip⇌Sed). Furthermore, we evaluated the validity of assumption II by comparing CLip⇌Sed of selected persistent, bioaccumulative and toxic pollutants (polychlorinated biphenyls (PCBs) and hexachlorobenzene (HCB)) to lipid-normalized concentrations for a range of biota from a Swedish background lake. PCBs in duck mussels, roach, eel, pikeperch, perch and pike were mostly below the equilibrium partitioning level relative to the sediment, i.e., lipid-normalized concentrations were ≤CLip⇌Sed, whereas HCB was near equilibrium between biota and sediment. Equilibrium sampling allows straightforward, sensitive and precise measurement of CLip⇌Sed. We propose CLip⇌Sed as a metric of the thermodynamic potential for bioaccumulation of persistent organic chemicals from sediment useful to prioritize management actions to remediate contaminated sites.

Assessment, distribution, and ecological risk of contaminants of emerging concern in a surface water-sediment-fish system impacted by wastewater.

The presence of contaminants of emerging concern in aquatic ecosystems represents an ever-increasing environmental problem. Aquatic biota is exposed to these contaminants, which can be absorbed and distributed to their organs. This study focused on the assessment, distribution, and ecological risk of 32 CECs in a Spanish river impacted by effluents from a wastewater treatment plant, analyzing the organs and plasma of common carp. Environmental concentrations in water and sediment were examined at sites upstream and downstream of the wastewater treatment plant. The two downstream sites showed 15 times higher total concentrations (12.4 µg L-1 and 30.1 µg L-1) than the two upstream sites (2.08 µg L-1 and 1.66 µg L-1). Half of the CECs were detected in fish organs, with amantadine having the highest concentrations in the kidney (158 ng g-1 w.w.) and liver (93 ng g-1 w.w.), followed by terbutryn, diazepam, and bisphenol F in the brain (50.2, 3.82 and 1.18 ng g-1 w.w.). The experimental bioaccumulation factors per organ were compared with the bioconcentration factors predicted by a physiologically based pharmacokinetic model, obtaining differences of one to two logarithmic units for most compounds. Risk quotients indicated a low risk for 38 % of the contaminants. However, caffeine and terbutryn showed an elevated risk for fish. The mixed risk quotient revealed a medium risk for most of the samples in the three environmental compartments: surface water, sediment, and fish.

Chemometers: an integrative tool for chemical assessment in multimedia environments.

We propose novel chemometers - passive equilibrium samplers of, e.g., silicone - as an integrative tool for the assessment of hydrophobic organic compounds in multimedia environments. The traditional way of assessing levels of organic pollutants across different environmental compartments is to compare the chemical concentration normalized to the major sorptive phase in two or more media. These sorptive phases for hydrophobic organic compounds differ between compartments, e.g., lipids in biota and organic carbon in sediments. Hence, comparability across media can suffer due to differences in sorptive capacities, but also extraction protocols and bioavailability. Chemometers overcome these drawbacks; they are a common, universal and well-defined polymer reference phase for sampling of a large range of nonpolar organic pollutants in different matrices like biota, sediment and water. When bringing the chemometer into direct contact with the sample, the chemicals partition between the sample and the polymer until thermodynamic equilibrium partitioning is established. At equilibrium, the chemical concentrations in the chemometers can be determined and directly compared between media, e.g., between organisms of different trophic levels or inhabiting different areas, between organs within an organism or between biotic and abiotic compartments, amongst others. Chemometers hence allow expressing the data on a common basis, as the equilibrium partitioning concentrations in the polymer, circumventing normalizations. The approach is based on chemical activity rather than total concentrations, and as such, gives a measure of the "effective concentration" of a compound or a mixture. Furthermore, chemical activity is the main driver for partitioning, biouptake and toxicity. As an additional benefit, the extracts of the chemometers only require limited cleanup efforts, avoiding introduction of a bias between chemicals of different persistence, and can be submitted to both chemical analysis and/or bioanalytical profiling.

Characterizing the marine mammal exposome by iceberg modeling, linking chemical analysis and in vitro bioassays.

The present study complements work on mixture effects measured with in vitro bioassays of passive equilibrium sampling extracts using the silicone polydimethylsiloxane (PDMS) in organs from marine mammals with chemical profiling. Blubber, liver, kidney and brain tissues of harbor porpoise (Phocoena phocoena), harbor seal (Phoca vitulina), ringed seal (Phoca hispida) and orca (Orcinus orca) from the North and Baltic Seas were investigated. We analyzed 117 chemicals including legacy and emerging contaminants using gas chromatography-high resolution mass spectrometry and quantified 70 of those chemicals in at least one sample. No systematic differences between the organs were found. Only for single compounds a clear distribution pattern was observed. For example, 4,4'-dichlorodiphenyltrichloroethane, enzacamene and etofenprox were mainly detected in blubber, whereas tonalide and the hexachlorocyclohexanes were more often found in liver. Furthermore, we compared the chemical profiling with the bioanalytical results using an iceberg mixture model, evaluating how much of the biological effect could be explained by the analyzed chemicals. The mixture effect predicted from the quantified chemical concentrations explained 0.014-83% of the aryl hydrocarbon receptor activating effect (AhR-CALUX), but less than 0.13% for the activation of the oxidative stress response (AREc32) and peroxisome-proliferator activated receptor (PPARγ). The quantified chemicals also explained between 0.044-45% of the cytotoxic effect measured with the AhR-CALUX. The largest fraction of the observed effect was explained for the orca, which was the individuum with the highest chemical burden. This study underlines that chemical analysis and bioassays are complementary to comprehensively characterize the mixture exposome of marine mammals.

Sensitive equilibrium sampling to study polychlorinated biphenyl disposition in Baltic Sea sediment.

An equilibrium sampling approach using glass jars with µm thin coatings of the silicone polydimethylsiloxane (PDMS) was validated and applied to background sediment samples from a >50 km transect in the Stockholm Archipelago. Equilibrium between the sediment and the PDMS was demonstrated using different coating thicknesses. From the concentrations of polychlorinated biphenyls (PCBs) in the PDMS, we assessed (i) freely dissolved concentrations in the sediment interstitial porewater (C(Sediment_free)); (ii) the equilibrium status between sediment and water; (iii) the equilibrium status between sediment and biota; and (iv) site-specific sediment/water distribution ratios (K(D)). The results showed that (i) Stockholm was a source of PCBs to the Baltic Sea as evidenced by significantly higher C(Sediment_free) in Stockholm Harbor; (ii) the fugacity in sediment exceeded that in water (monitoring samples collected in February) by an average factor of 4.0; (iii) the fugacity in sediment exceeded that in herring by an average factor of 5.2; and (iv) K(D) near Stockholm Harbor was 0.3-1.7 log units greater than in the outer archipelago. The coated glass jar method with its high precision and built-in QA/QC opens new possibilities to study the disposition of hydrophobic chemicals at trace levels (C(Sediment_free) down to 1.06 fg/L) in background environments.

Clonality characterization of natural epitope-specific antibodies against the tumor-related antigen topoisomerase IIa by peptide chip and proteome analysis: a pilot study with colorectal carcinoma patient samples.

Patient-specific sequential epitopes were identified by peptide chip analysis using 15mer peptides immobilized on glass slides that covered the topoisomerase IIa protein with a frameshift of five amino acids. Binding specificities of serum antibodies against sequential epitopes were confirmed as being mono-specific by peptide chip re-analysis of epitope-affinity-purified antibody pools. These results demonstrate that serum samples from colon carcinoma patients contain antibodies against sequential epitopes from the topoisomerase IIa antigen. Interactions of patients' antibodies with sequential epitopes displayed by peptides on glass surfaces may thus mirror disease-specific immune situations. Consequently, these data suggest epitope-antibody reactivities on peptide chips as potential diagnostic readouts of individual immune response characteristics, especially because monospecific antibodies can be interrogated. Subsequently, the clonality of the antibodies present in the mono-specific antibody pools was characterized by 2D gel electrophoresis. This analysis suggested that the affinity-purified antibodies were oligoclonal. Similarly to large-scale screening approaches for specific antigen-antibody interactions in order to improve disease diagnostic, we suggest that "protein-wide" screening for specific epitope-paratope interactions may help to develop novel assays for monitoring of personalized therapies, since individual properties of antigen-antibody interactions remain distinguishable.