Analysis of multiple protein-protein interactions using microarray technology remains challenging, and site-specific immobilization of functional proteins is a key step in these approaches. Here we establish the efficient synthesis of protein-DNA conjugates for several members of a small family of GTPases. The family of Rab/Ypt GTPases is intimately involved in vesicular trafficking in yeast and serves as a model for the much larger group of analogous human proteins, the Rab protein family, with more than 60 members. The Ypt-DNA hybrid molecules described here are used for DNA-directed immobilization on glass-and silica-based microarrays. Methods for the detection of protein-DNA conjugates, as well as approaches for nucleotide exchange and distinguishing between GDP- and GTP-bound Ypts on microarrays, are reported. The high specificity of different Rab/Ypt-effector interactions, which also depends on the bound nucleotide, is shown by fluorescence readout of microarrays. Furthermore, initial experiments demonstrate that direct readout by mass spectrometry can be achieved with commercially available instruments. These developments will significantly contribute to the elucidation of complex transport networks in eukaryotic cells.

A mild, fast and flexible method for photoimmobilization of biomolecules based on the light‐initiated thiol–ene reaction has been developed. After investigation and optimization of various surface materials, surface chemistries and reaction parameters, microstructures and microarrays of biotin, oligonucleotides, peptides, and MUC1 tandem repeat glycopeptides were prepared with this photoimmobilization method. Furthermore, MUC1 tandem repeat glycopeptide microarrays were successfully used to probe antibodies in mouse serum obtained from vaccinated mice. Dimensions of biomolecule microstructures were shown to be freely controllable through photolithographic techniques, and features down to 5 μm in size covering an area of up to 75×25 mm were created. Use of a confocal laser microscope with a UV laser as UV‐light source enabled further reduction of biotin feature size opening access to nanostructured biochips.

AD is characterized as a protein misfolding disease due to the accumulation of abnormally folded amyloid beta protein and also considered a tauopathy. However, several proteins are involved in the pathogenesis and diagnosis of neurodegenerative disorders like AD. Some of these biomarkers are only available in lowest concentrations in CSF and/or blood samples during the early-stage of the disorders; other could only be detected in biopsies samples. Furthermore the sample size for testing is a critical issue. We addressed these challenges by improving immunoassays for different biomarkers. Immuno-PCR first described by Sano et al. is a highly promising technique for the ultrasensitive analysis of proteins, like biomarkers, biopharmaceutical compounds, therapeutic antibodies, cytokines and many more. Immuno-PCR combines the well-established ELISA methodology with the signal amplification power of the PCR. As a consequence Immuno-PCR does not lead only to an about 100 to 10,000-fold gain in sensitivity compared to conventional ELISA. It also reveals a set of additional advantages. We show the high sensitive detection of different Alzheimer's disease (AD) related biomarkers, e.g. phosphorylated TAU. Additionally, we could decrease the needed sample volume to only some microliter still showing an improved sensitivity compared to standard immunoassays. These results show ways for improved detection and prognosis of CNS biomarkers in AD and related dementias, including parkinsonian movement disorders, and ways for future developments.

The versatility of immunoassays for the detection of antigens can be combined with the signal amplification power of nucleic acid amplification techniques in a broad range of innovative detection strategies. This review summarizes the spectrum of both, DNA-modification techniques used for assay enhancement and the resulting key applications. In particular, it focuses on the highly sensitive immuno-PCR (IPCR) method. This technique is based on chimeric conjugates of specific antibodies and nucleic acid molecules, the latter of which are used as markers to be amplified by PCR or related techniques for signal generation and read-out. Various strategies for the combination of antigen detection and nucleic acid amplification are discussed with regard to their laboratory analytic performance, including novel approaches to the conjugation of antibodies with DNA, and alternative pathways for signal amplification and detection. A critical assessment of advantages and drawbacks of these methods for a number of applications in clinical diagnostics and research is conducted. The examples include the detection of viral and bacterial antigens, tumor markers, toxins, pathogens, cytokines and other targets in different biological sample materials.

The photochemical coupling of olefin-capped (bio)molecules to surface-bound thiols can be used to control protein immobilization on length scales of centimeters to sub-micrometers (see the fluorescence microscopy image of a nanopattern after treatment with labeled streptavidin). Two enzymes immobilized on the resulting patterns retained their enzymatic activity and underwent protein–protein interactions similar to those in the solution phase. Supporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2002/2008/z800101_s.pdf or from the author. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Welches Substrat darf's sein? Phosphotyrosin-Peptid-Mikroarrays ermöglichten die Zuordnung der Substratspezifitäten von zwei prototypischen Protein-Tyrosin-Phosphatasen (PTPs): PTP1B und PTPμ. Die erhaltenen Informationen wurden für molekulare Dockingstudien (Bild: Docking eines Peptids in die katalytische Tasche von PTPμ) und zum Design eines Inhibitors für PTPμ genutzt.

We describe an innovative modification of the Immuno-PCR technology for automatable high sensitive antigen detection. The Magneto Immuno-PCR (M-IPCR) is based on antibody-functionalized biogenic magnetosome nanoparticles revealing major advantages over synthetic magnetic particles. The general principle of the M-IPCR is similar to that of a two-sided (sandwich) immunoassay. However, antibody-functionalized magnetosome conjugates were employed for the immobilization and magnetic enrichment of the signal generating detection complex enabling the establishment of a surface independent immunoassay. To this end, the M-IPCR was carried out by simultaneously tagging the antigen with the reagent for read-out, i.e., a conjugate comprising the specific antibody and DNA fragments, in the presence of the antibody-functionalized magnetosomes. To demonstrate the general functionality of the M-IPCR, the detection of recombinant Hepatitis B surface Antigen (HBsAg) in human serum was established. We observed a detection limit of 320 pg/ml of HBsAg using the M-IPCR, which was about 100-fold more sensitive than the analogous Magneto-ELISA, established in parallel for comparison purposes.

The quantitative immuno-PCR (qIPCR) technology combines the advantages of flexible and robust immunoassays with the exponential signal amplification power of PCR. The qIPCR allows one to detect antigens using specific antibodies labeled with double-stranded DNA. The label is used for signal generation by quantitative PCR. Because of the efficiency of nucleic acid amplification, qIPCR typically leads to a 10- to 1,000-fold increase in sensitivity compared to an analogous enzyme-amplified immunoassay. A standard protocol of a qIPCR assay to detect human interleukin 6 (IL-6) using a sandwich immunoassay combined with real-time PCR readout is described here. The protocol includes initial immobilization of the antigen, and coupling of this antigen with antibody–DNA conjugates is then carried out by (a) the stepwise assembly of biotinylated antibody, streptavidin and biotinylated DNA, (b) the use of a biotinylated antibody and an anti-biotin–DNA conjugate or (c) the employment of an anti-IL-6 antibody–DNA conjugate. Following the assembly of signal-generating immunocomplexes, real-time PCR is used to amplify and record the signal. Depending on the coupling strategy, the qIPCR assays require 4–7 h with only about 3 h hands-on-time. The use of qIPCR assays enables the detection of rare biomarkers in complex biological samples that are poorly accessible by conventional immunoassays. Therefore, qIPCR offers novel opportunities for the biomedical analysis of, for instance, neurodegenerative diseases and viral infections as well as new tools for the development of novel pharmaceuticals.

Which substrate will it be? Phosphotyrosine peptide microarrays have allowed the substrate specificity to be mapped for two prototypical protein-tyrosine phosphatases (PTPs): PTP1B and PTPμ. The knowledge gained was used for molecular docking studies (see picture: the docking of a peptide in the binding pocket of PTPμ) and the design of an inhibitor for PTPμ.

Protein an Glas: Die ortsspezifische Immobilisierung von Proteinen auf einer Glasoberfläche könnte sich zum Aufbau von Protein-Mikroarrays eignen. Der Ansatz beruht auf einer Staudinger-Ligation zwischen den Azidgruppen modifizierter Proteine und Phosphanfunktionen auf der Oberfläche.

Expressed protein ligation (EPL) and bioconjugation based on the maleimide group (MIC-conjugation) provide powerful tools for protein modification. In the light of the importance of site-selectively modified proteins for the study of protein function, a flexible method for the introduction of tags and reporter groups into the C-terminus of proteins employing EPL and MIC-conjugation was developed. We describe the solid-phase synthesis of a generic building block, equipped with fluorescence markers or different functional groups. This generic building block allows for a flexible incorporation of different tags into proteins and was used for the introduction of fluorescence markers into the C-terminus of Rab and Ras GTPases by EPL or MIC-conjugation techniques. In addition, a building block appropriately modified for the incorporation of an azide into proteins was synthesized. Azide-functionalized Ras protein was immobilized on a phosphane-modified surface by means of Staudinger ligation providing a highly chemoselective ligation method for the immobilization of proteins.

Auf Proteinfang: In Protein-DNA-Mikroarrays gebundene Proteine können durch MALDI-Massenspektrometrie direkt ausgelesen werden. Hierzu wurden Fängerproteine auf der Oberfläche von Siliciumchips hoch selektiv und reversibel durch DNA-gerichtete Immobilisierung verankert (siehe Bild). Die Analytproteine, die an ihre Partner auf der Oberfläche binden, können aus Zelllysat nachgewiesen werden.

Semisynthetic DNA-streptavidin conjugates are synthesized by covalent coupling of thiol-modified DNA oligonucleotides and streptavidin (STV). The resulting conjugates have binding capacities for four equivalents of biotin and a complementary nucleic acid sequence. The conjugates are purified to homogeneity by ultrafiltration and chromatography, and are characterized by spectrophotometry and gel electrophoresis. Subsequently, the conjugates are applied as molecular connectors in the DNA-directed immobilization (DDI) of biotinylated antibodies using DNA microarrays as immobilization matrices. The results are protein microarrays that can be used for the multiplexed detection of various antigens.

Chimera Biotec, founded in 2000 as a spin-off from academic research in Germany, has implemented its proprietary DNA-antibody conjugates within the highly innovative Imperacer product line, enabling the ultra-sensitive detection of proteins and other antigens. The ready-to-use, commercially available Imperacer kits offer a performance-enhancing tool to improve the limit of detection of conventional ELISA protocols by more than 1,000-fold. Hence, the detection and quantification of trace amounts of basically any antigen, ranging from large proteins to small molecules such as hormones or biogenic amines, can be accomplished with an Imperacer-boosted immunoassay.

Nucleic acid amplification techniques are used for signal generation in antibody-based immunoassays, thereby dramatically enhancing the sensitivity of conventional immunoassays. Methodological aspects, as well as applications of this novel approach, are summarized in this review, with an emphasis on immuno-polymerase chain reaction (IPCR). IPCR is based on chimeric conjugates of specific antibodies and nucleic acid molecules, the latter of which are used as markers to be amplified by PCR for signal generation. The enormous efficiency of nucleic acid amplification typically leads to a 100-10 000-fold increase in sensitivity, as compared with the analogous enzyme-amplified immunoassay. The evolution of IPCR included the development of efficient reagents, the design of assay formats and the maintenance of functionality, even within complex biological matrices. Eventually, IPCR crossed the border from being a research method to a routine laboratory technique, enabling a broad range of applications in immunological research and clinical diagnostics.

Nucleic acid amplification techniques are used for signal generation in antibody-based immunoassays, thereby dramatically enhancing the sensitivity of conventional immunoassays. Methodological aspects, as well as applications of this novel approach, are summarized in this review, with an emphasis on immuno-polymerase chain reaction (IPCR). IPCR is based on chimeric conjugates of specific antibodies and nucleic acid molecules, the latter of which are used as markers to be amplified by PCR for signal generation. The enormous efficiency of nucleic acid amplification typically leads to a 100-10 000-fold increase in sensitivity, as compared with the analogous enzyme-amplified immunoassay. The evolution of IPCR included the development of efficient reagents, the design of assay formats and the maintenance of functionality, even within complex biological matrices. Eventually, IPCR crossed the border from being a research method to a routine laboratory technique, enabling a broad range of applications in immunological research and clinical diagnostics.

Catching proteins: Proteins from a protein–DNA microarray can be directly detected by using MALDI mass spectrometry. Capture proteins are attached to silicon chips in a highly selective and reversible way by DNA-directed immobilization (see picture). The analyte proteins bound specifically to their partners on the microarray can be detected from cell lysates.

Mild attachment: Proteins can be ligated chemoselectively and immobilized on functionalized glass surfaces by means of Diels–Alder cycloadditions (see scheme).

The cover picture shows the self‐assembly of antibody molecules tagged with short DNA oligomers at surfaces containing arrays of complementary oligonucleotides. In their full paper on p. 453 ff, Wacker and Niemeyer report that this DNA‐directed immobilization (DDI) of antibodies forms the basis of a variety of readily configurable immunoassays that allow for the simultaneous and highly reproducible detection of various antigens from biological samples. DDI‐based assays are particularly suited for analyses in microfuluidic systems, such as the plastic, capillary‐driven microfluidic microtiterplate shown at the bottom. Graphical materials are courtesy of CHIMERA BIOTEC and STEAG microParts, Dortmund.

Semisynthetic DNA-protein conjugates are synthesized by covalent coupling of thiol-modified DNA oligonucleotides and streptavidin. The resulting conjugates have a binding capacity for four equivalents of biotin and one nucleic acid of complementary sequence. The conjugates are purified to homogeneity by ultrafiltration and chromatography and characterized by photometry and gel electrophoresis. Subsequently, the conjugates are applied as molecular linkers in the DNA-directed immobilization of a biotinylated enzyme on a microplate, containing complementary capture oligonucleotides.