Optical microarray biosensors.
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Biosensing Techniques↗
Biomedical subjects
Michael G Weller
Publications and source records attributed to Michael G Weller.
Comparison of nitrotyrosine antibodies and development of immunoassays for the detection of nitrated proteins.
Three monoclonal antibodies (mAb) and three polyclonal antibodies (pAb) have been characterized and compared with respect to their cross-reactivities and affinities for 3-nitrotyrosine, eight aromatic compounds with similar chemical structures, a peptide containing a single nitrotyrosine residue, and fourteen nitrated protein standards (bovine serum albumin, BSA) containing different numbers of nitrotyrosine residues per protein molecule (0.2 to 16.8). In indirect competitive immunoassays, mAb Alexis 39B6 exhibited the highest affinity for free 3-nitrotyrosine (10(6) L mol(-1)), while the pAb Oxis 24312 from sheep exhibited the highest affinities for nitrated proteins (up to 10(8) L mol(-1)). The apparent affinities determined in the indirect competitive assays were inversely correlated with the limits of detection (LOD) determined in one-sided immunoassays. With the sheep pAb minimum LOD on the order of 10 pmol L(-1) were achieved for highly nitrated proteins, corresponding to effective LOD on the order of 100 pmol L(-1) for nitrotyrosine residues. In the one-sided assays, however, the LOD for nitrated proteins increased proportionally with increasing background concentrations of native proteins in the investigated samples. Sandwich immunoassays combining pAb and mAb for selective enrichment and detection of nitrated proteins allowed to eliminate this native protein matrix effect and to achieve LOD on the order of 300 pmol L(-1) for highly nitrated proteins independent of native protein background concentrations.
Air Pollutants↗
Automated microarray system for the simultaneous detection of antibiotics in milk.
A parallel affinity sensor array (PASA) for the rapid automated analysis of 10 antibiotics in milk is presented, using multianalyte immunoassays with an indirect competitive ELISA format. Microscope glass slides modified with (3-glycidyloxypropyl)trimethoxysilane were used for the preparation of hapten microarrays. Protein conjugates of the haptens were immobilized as spots on disposable chips, which were processed in a flow cell. Monoclonal antibodies against penicillin G, cloxacillin, cephapirin, sulfadiazine, sulfamethazine, streptomycin, gentamicin, neomycin, erythromycin, and tylosin allowed the simultaneous detection of the respective analytes. Antibody binding was detected by a second antibody labeled with horseradish peroxidase generating enhanced chemiluminescence, which was recorded with a sensitive CCD camera. All liquid handling and sample processing was fully automated, and one analysis was carried out in milk within less than 5 min. The detection limits ranged from 0.12 (cephapirin) to 32 microg/L (neomycin). Penicillin G could be detected at the maximum residue limit (MRL); the detection limits for all other analytes were far below the respective MRLs. The PASA system proved to be the first immunochemical biosensor platform having the potential to test for numerous antibiotics in parallel, such being of considerable interest for the control of milk in the dairy industry.
Animals↗
Development of antibodies for the detection of N-acetyl-glufosinate.
Glufosinate is a widely used herbicide, which is difficult to detect by conventional analytical techniques. For many other herbicides, suitable antibodies have been raised for immunoassay development. Unfortunately, glufosinate is a very small molecule and difficult to immunize with. Thus, a derivatization-assisted immunoassay (DAIA) using the target analyte N-acetyl-glufosinate (NAG) was constructed. The activated hapten was synthesized by a new approach, using a homobifunctional cross-linker suberic acid bis(N-hydroxysuccinimide ester). The preparation of a suitable conjugate, the immunization, and the characterization of polyclonal antibodies are shown. The determination of the conjugation density (hapten density) of the immunogens was performed by four different methods (high-performance liquid chromatography with a refractive index detector, total reflection X-ray fluorescence, inductively coupled plasma mass spectrometry, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry), which gave similar results. The limit of detection was 17 mug/L NAG in water for the direct competitive enzyme immunoassay. NAG is also a main metabolite of glufosinate in resistant transgenic plants. The antibodies might be useful for the selective detection of NAG in the presence of the parent compound glufosinate (cross-reactivity 0.13%) and other metabolites.
Acetylation↗
Microarrays for the screening of allergen-specific IgE in human serum.
The described in vitro test system for allergy diagnosis is based on microscope glass slides activated with (3-glycidyloxypropyl)trimethoxysilane. Allergen solutions are immobilized as small droplets (approximately 10 nL) on the activated glass slides with a piezoelectric arrayer. In contrast to other tests for specific IgE, such as Pharmacia CAP FEIA, AlaSTAT, or FAST, only a 25-microL serum sample is needed for the screening of allergen-specific IgE against a multitude of allergens and the test can be performed in less than 1 h. Compared with multiallergen dipstick screening tests (e.g., IgEquick, CMG Immunodot) based on multiallergen-coated nitrocellulose strips, the measurement of the microarray-based system can be performed automatically. The chemiluminescence intensities are detected with a sensitive CCD camera. Allergen extracts and recombinant/purified allergens (24 preparations) have been used on the same modified surface for the screening of allergen-specific IgE. With these disposable microarray slides, it is possible to distinguish between patients with and without elevated levels of allergen-specific IgE. Repeated measurements of serum samples demonstrated a sufficient reproducibility. Detection limits (microg/L) of 0.35 (r Betvl), 0.16 (PLA2), and 1.9 (Der p1) were achieved.
Allergens↗
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.
Air Pollutants↗
Classification of protein microarrays and related techniques.
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Immunoblotting↗
Liquid- and gas-phase nitration of bovine serum albumin studied by LC-MS and LC-MS/MS using monolithic columns.
Post-translational nitration of proteins was analyzed by capillary reversed-phase high-performance liquid chromatography (RP-HPLC) on-line interfaced to electrospray ionization mass spectrometry (ESI--MS) or tandem mass spectrometry (ESI--MS/MS). Both methods were compared using a tryptic digest of bovine serum albumin (BSA) and yielded sequence coverages of 95% and 33% with RP-HPLC--ESI--MS and RP-HPLC--ESI--MS/MS, respectively. At least 95% of the tyrosines were covered by the former method, whereas the latter method only detected less than 50% of the tyrosine-containing peptides. Upon liquid-phase nitration of BSA in aqueous solution using an excess of tetranitromethane, at least 16 of the 20 tyrosine residues were found to be nitrated. After exposure of solid BSA samples to gaseous nitrogen dioxide and ozone at atmospherically relevant concentration levels, only 3 nitrated peptides were detected. By use of such a model system, RP-HPLC--ESI--MS proved to be a rapid and highly efficient method for the comprehensive and quantitative detection of protein nitration.
Amino Acid Sequence↗