Michael G. Weller

Nitration enhances the allergenic potential of proteins

Background: Recent investigations have shown that proteins, including Bet v 1a, are nitrated by exposure to polluted urban air. We have investigated immunogenic and allergenic properties of in vitro nitrated allergens in in vivo models. Methods: Untreated and nitrated samples of ovalbumin or Bet v 1a were compared for their ability to stimulate proliferation and cytokine secretion in splenocytes from DO11.10 or from sensitized BALB/c mice, and for their ability to induce specific immunoglobulin (Ig)G1, IgG2a and IgE in sensitized mice. Additionally, sera from birch pollen-allergic individuals were analysed for IgE and IgG specific for nitrated Bet v 1a. Results: Upon splenocyte stimulation with nitrated as compared with unmodified allergens, proliferation as well as interleukin 5 and interferon-γ production were enhanced. Sera of mice sensitized with nitrated allergens showed elevated levels of specific IgE, IgG1 and IgG2a, compared with sera from mice sensitized with unmodified allergens. Moreover, cross-reactivity of antibodies against unrelated, nitrated allergens was observed in mice. We also found higher amounts of functional, specific IgE against nitrated than against untreated Bet v 1a in sera from birch pollen-allergic patients. Conclusions: Our findings suggest that nitration enhances allergic responses, which may contribute to an increased prevalence of allergic diseases in polluted urban environments.

Generic microcystin immunoassay based on monoclonal antibodies against Adda

A monoclonal antibody (clone AD4G2) was generated against a common part of microcystins and nodularins, the unusual amino acid Adda [(2S,3S,8S,9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4E,6E-dienoic acid]. A direct competitive ELISA based on this antibody was developed and the cross-reactivity pattern was measured. Different toxins showed a very similar response. The assay provides therefore a sum parameter of microcystins, nodularins and peptide fragments containing Adda. The IC50 for microcystin-LR was 0.33 microg L(-1) which leads to a detection limit of 0.07 microg L(-1). This is well below the concentration of 1 microg L(-1) proposed by the World Health Organisation (WHO) as the limit for drinking water. Microcystin-LR spiked water samples in the concentration range between 0.1 and 1 microg L(-1) were measured and a mean recovery of 113+/-23% was found. The antibody is well suited for the determination of microcystins in drinking as well as surface water.

A Unifying Review of Bioassay-Guided Fractionation, Effect-Directed Analysis and Related Techniques

The success of modern methods in analytical chemistry sometimes obscures the problem that the ever increasing amount of analytical data does not necessarily give more insight of practical relevance. As alternative approaches, toxicity- and bioactivity-based assays can deliver valuable information about biological effects of complex materials in humans, other species or even ecosystems. However, the observed effects often cannot be clearly assigned to specific chemical compounds. In these cases, the establishment of an unambiguous cause-effect relationship is not possible. Effect-directed analysis tries to interconnect instrumental analytical techniques with a biological/biochemical entity, which identifies or isolates substances of biological relevance. Successful application has been demonstrated in many fields, either as proof-of-principle studies or even for complex samples. This review discusses the different approaches, advantages and limitations and finally shows some practical examples. The broad emergence of effect-directed analytical concepts might lead to a true paradigm shift in analytical chemistry, away from ever growing lists of chemical compounds. The connection of biological effects with the identification and quantification of molecular entities leads to relevant answers to many real life questions.

Highly parallel affinity sensor for the detection of environmental contaminants in water

A parallel affinity sensor array (PASA) was implemented based on chemiluminescence labels (peroxidase/luminol) and CCD detection. Reagents like antibodies or haptens have been immobilized on a glass slide, which forms a biochip with an active area of about 1.8 cm 2 . The spot density on the biochip was up to 9 spots per mm 2 , which corresponds to an array of 1600 spots. Direct and indirect immunoassay formats have been tested successfully. Detection limits down to 20 ng l ˇ1 terbutylazine could be reached. The regeneration of the indirect assay chips could be performed more than 100 times. To improve the regenerability of direct assays an enzyme tracer with a cleavable spacer was introduced. The potential of the PASA for analyte identification by pattern recognition and subsequent analyte quantification could be shown. The application of the system is discussed for environmental, pharmaceutical, and other analytical fields. # 1999 Elsevier Science B.V. All rights reserved.

Immunoassays as high-throughput tools: Monitoring spatial and temporal variations of carbamazepine, caffeine and cetirizine in surface and wastewaters

Carbamazepine (CBZ), caffeine and cetirizine were monitored by enzyme-linked immunosorbent assays (ELISAs) in surface and wastewaters from Berlin, Germany. This fast and cost-efficient method enabled to assess the spatial and temporal variation of these anthropogenic markers in a high-throughput screening. CBZ and cetirizine were detected by the same antibody, which selectively discriminates between both compounds depending on the pH value used in the incubation step. To our best knowledge, this is the first dual-analyte immunoassay working with a single antibody. The frequent sampling with 487 samples being processed allowed for the repeated detection of unusually high concentrations of CBZ and caffeine. ELISA results correlate well with the ones obtained by liquid chromatography tandem mass spectrometry (LC-MS/MS). Caffeine concentrations found in surface waters were elevated by combined sewer overflows after stormwater events. During the hay fever season, the concentrations of the antihistamine drug cetirizine increased in both surface and wastewaters. Caffeine was almost completely removed during wastewater treatment, while CBZ and cetirizine were found to be more persistent. The maximum concentrations of caffeine, CBZ and cetirizine found in influent wastewater by LC-MS/MS were 470, 5.0 and 0.49 μg L(-1), while in effluent wastewater the concentrations were 0.22, 4.5 and 0.51 μg L(-1), respectively. For surface waters, concentrations up to 3.3, 4.5 and 0.72 μg L(-1) were found, respectively.

Enzyme immunoassays for the investigation of protein nitration by air pollutants

Two enzyme immunoassays have been developed, characterised, and applied to investigate protein nitration in birch pollen extract (BPE) and bovine serum albumin (BSA) samples exposed to air pollutants. The monoclonal antibody CAY-189542 against nitrotyrosine (raised against peroxynitrite-treated keyhole limpet hemocyanine) was characterised in an indirect competitive assay (affinity and cross-reactivities) and applied in a new one-sided enzyme immunoassay for nitrated proteins. The one-sided assay was calibrated against a nitrated BSA standard with an average of 14 nitrotyrosine residues per molecule (nitro-(14)-BSA; detection limit 8.3 pmol L(-1)), and the sensitivity of the test was found to be significantly enhanced by a multivalent binding mode of the monoclonal antibody (bonus effect of multivalency). The same antibody and a polyclonal antibody against Bet v 1, the most prominent birch pollen allergen, were used in a new sandwich immunoassay for specific determination of nitrated Bet v 1. This assay was calibrated against a nitrated Bet v 1 standard with an average of 3 nitrotyrosine residues per molecule (nitro-(3)-Bet v 1; detection limit 0.2 nmol L(-1)). Bet v 1 and BSA exposed to polluted urban outdoor air and to synthetic gas mixtures containing NO2 and O3 at atmospherically relevant concentration levels were found to be efficiently nitrated within hours to days. Pronounced correlations of nitro-(14)-BSA equivalent concentrations with exposure time and with nitro-(3)-Bet v 1 equivalent concentrations in nitrated BPE samples were observed. Test experiments indicated that the efficiency of protein nitration was strongly enhanced by reactive species formed upon interaction of NO2 with O3 and H2O (e.g. NO3 and HNO3). Potential implications of protein nitration by air pollutants are outlined and discussed.

Increased sensitivity of an enzyme immunoassay (ELISA) for the determination of triazine herbicides by variation of tracer incubation time

Immunoassays for triazine herbicides were tested for their reaction to the variation of the tracer incubation time. By application of a sequential technique the measuring range of atrazine could be expanded to five decades and the total duration of the test could be reduced to about 30 min. In an optimized version a lower detection limit of 9 pmol/l (2 ng/l) was achieved. The detection limit of a sensitive immunoassay for terbuthylazine is also below the concentration limit demanded of the German drinking water regulation (100 ng/l) and reaches 130 pmol/l (30 ng/l). Short tracer incubation times did not lead to increased cross-reactivities in contrast to theoretical models [1, 2]. Different mechanisms, which could cause a shift of the center point of the calibration curve, are discussed, including kinetic considerations.

MeCAT—new iodoacetamide reagents for metal labeling of proteins and peptides

Besides protein identification via mass spectrometric methods, protein and peptide quantification has become more and more important in order to tackle biological questions. Methods like differential gel electrophoresis or enzyme-linked immunosorbent assays have been used to assess protein concentrations, while stable isotope labeling methods are also well established in quantitative proteomics. Recently, we developed metal-coded affinity tagging (MeCAT) as an alternative for accurate and sensitive quantification of peptides and proteins. In addition to absolute quantification via inductively coupled plasma mass spectrometry, MeCAT also enables sequence analysis via electrospray ionization tandem mass spectrometry. In the current study, we developed a new labeling approach utilizing an iodoacetamide MeCAT reagent (MeCAT-IA). The MeCAT-IA approach shows distinct advantages over the previously used MeCAT with maleinimide reactivity such as higher labeling efficiency and the lack of diastereomer formation during labeling. Here, we present a careful characterization of this new method focusing on the labeling process, which yields complete tagging with an excess of reagent of 1.6 to 1, less complex chromatographic behavior, and fragmentation characteristics of the tagged peptides using the iodoacetamide MeCAT reagent.

Novel Aflatoxin Derivatives and Protein Conjugates

Aflatoxins, a group of structurally related mycotoxins, are well known for their toxic and carcinogenic effects in humans and animals. Aflatoxin derivatives and protein conjugates are needed for diverse analytical applications. This work describes a reliable and fast synthesis of novel aflatoxin derivatives, purification by preparative HPLC and characterisation by ESI-MS and one- and two-dimensional NMR. Novel aflatoxin bovine serum albumin conjugates were prepared and characterised by UV absorption and MALDI-MS. These aflatoxin protein conjugates are potentially interesting as immunogens for the generation of aflatoxin selective antibodies with novel specificities.

Immunoassays and Biosensors for the Detection of Cyanobacterial Toxins in Water

Algal blooms are a frequent phenomenon in nearly all kinds of fresh water. Global warming and eutrophication by waste water, air pollution and fertilizers seem to lead to an increased frequency of occurrence. Many cyanobacteria produce hazardous and quite persistent toxins, which can contaminate the respective water bodies. This may limit the use of the raw water for many purposes. The purification of the contaminated water might be quite costly, which makes a continuous and large scale treatment economically unfeasible in many cases. Due to the obvious risks of algal toxins, an online or mobile detection method would be highly desirable. Several biosensor systems have been presented in the literature for this purpose. In this review, their mode of operation, performance and general suitability for the intended purpose will be described and critically discussed. Finally, an outlook on current developments and future prospects will be given.