evolution of tag antibodies: in-depth specificity and affinity analysis of Myc1-9E10 and Hyper-Myc.

One of the most widely used epitope tags is the myc-tag, recognized by the anti-c-Myc hybridoma antibody Myc1-9E10. Combining error-prone PCR, DNA shuffling and phage display, we generated an anti-c-Myc antibody variant (Hyper-Myc) with monovalent affinity improved to 18 nM and thermal stability increased by 37%. Quantification of capillary immunoblots and by flow cytometry demonstrated improved antigen detection by Hyper-Myc.

Expression of different L1 isoforms of papillomavirus as mechanism to circumvent adaptive immunity.

Although many high-risk mucosal and cutaneous human papillomaviruses (HPVs) theoretically have the potential to synthesize L1 isoforms differing in length, previous seroepidemiological studies only focused on the short L1 variants, co-assembling with L2 to infectious virions. Using the multimammate mouse as preclinical model, this is the first study demonstrating seroconversion against different L1 isoforms during the natural course of papillomavirus infection. Intriguingly, positivity with the cutaneous MnPV was accompanied by a strong seroresponse against a longer L1 isoform, but to our surprise, the raised antibodies were non-neutralizing.

High-density Peptide Arrays Help to Identify Linear Immunogenic B-cell Epitopes in Individuals Naturally Exposed to Malaria Infection.

High-density peptide arrays are an excellent means to profile anti-plasmodial antibody responses. Different protein intrinsic epitopes can be distinguished, and additional insights are gained, when compared with assays involving the full-length protein. Distinct reactivities to specific epitopes within one protein may explain differences in published results, regarding immunity or susceptibility to malaria.

High-flexibility combinatorial peptide synthesis with laser-based transfer of monomers in solid matrix material.

Laser writing is used to structure surfaces in many different ways in materials and life sciences. However, combinatorial patterning applications are still limited. Here we present a method for cost-efficient combinatorial synthesis of very-high-density peptide arrays with natural and synthetic monomers.

Printed peptide arrays identify prognostic TNC serumantibodies in glioblastoma patients.

Liquid biopsies come of age offering unexploited potential to monitor and react to tumor evolution. We developed a cost-effective assay to non-invasively determine the immune status of glioblastoma (GBM) patients. Employing newly developed printed peptide microarrays we assessed the B-cell response against tumor-associated antigens (TAAs) in 214 patients.

Particle-Based Microarrays of Oligonucleotides and Oligopeptides.

In this review, we describe different methods of microarray fabrication based on the use of micro-particles/-beads and point out future tendencies in the development of particle-based arrays. First, we consider oligonucleotide bead arrays, where each bead is a carrier of one specific sequence of oligonucleotides. This bead-based array approach, appearing in the late 1990s, enabled high-throughput oligonucleotide analysis and had a large impact on genome research.

Printing Peptide arrays with a complementary metal oxide semiconductor chip.

: In this chapter, we discuss the state-of-the-art peptide array technologies, comparing the spot technique, lithographical methods, and microelectronic chip-based approaches. Based on this analysis, we describe a novel peptide array synthesis method with a microelectronic chip printer. By means of a complementary metal oxide semiconductor chip, charged bioparticles can be patterned on its surface.

Purification of high-complexity peptide microarrays by spatially resolved array transfer to gold-coated membranes.

A method for the one-step purification of high-complexity peptide microarrays is presented. The entire peptide library is transferred from the synthesis support to a gold coated polyvinylidenfluoride (PVDF) membrane, whereby only full-length peptides covalently couple to the receptor membrane via an N-terminally added cysteine. Highly resolved peptide transfer and purification of up to 10 000 features per cm(2) is demonstrated.

Sensing immune responses with customized peptide microarrays.

The intent to solve biological and biomedical questions in high-throughput led to an immense interest in microarray technologies. Nowadays, DNA microarrays are routinely used to screen for oligonucleotide interactions within a large variety of potential interaction partners. To study interactions on the protein level with the same efficiency, protein and peptide microarrays offer similar advantages, but their production is more demanding.

Peptide arrays with a chip.

Today, lithographic methods enable combinatorial synthesis of >50,000 oligonucleotides per cm(2), an advance that has revolutionized the whole field of genomics. A similar development is expected for the field of proteomics, provided that affordable, very high-density peptide arrays are available. However, peptide arrays lag behind oligonucleotide arrays.

Combinatorial peptide synthesis on a microchip.

Microchips are used in the combinatorial synthesis of peptide arrays by means of amino acid microparticle deposition. The surface of custom-built microchips can be equipped with an amino-modified poly(ethylene glycol)methacrylate (PEGMA) graft polymer coating, which permits high loading of functional groups and resists nonspecific protein adsorption. Specific microparticles that are addressed to the polymer-coated microchip surface in a well defined pattern release preactivated amino acids upon melting, and thus allow combinatorial synthesis of high-complexity peptide arrays directly on the chip surface.

A novel combinatorial approach to high-density peptide arrays.

Combinatorial synthesis of peptides on solid supports (1), either as spots on cellulose membranes (2) or with split-pool-libraries on polymer beads (3), substantially forwarded research in the field of peptide-protein interactions. Admittedly, these concepts have specific limitations, on one hand the number of synthesizable peptide sequences per area, on the other hand elaborate decoding/encoding strategies, false-positive results and sequence limitations. We recently established a method to produce high-density peptide arrays on microelectronic chips (4).

High-density peptide arrays.

Arrays promise to advance biology by allowing parallel screening for many different binding partners. Meanwhile, lithographic methods enable combinatorial synthesis of > 50,000 oligonucleotides per cm(2), an advance that has revolutionized the whole field of genomics. A similar development is expected for the field of proteomics, provided that affordable, very high-density peptide arrays are available.

Particle-based synthesis of peptide arrays.

Lithographic methods allow for the combinatorial synthesis of >50,000 oligonucleotides per cm(2), and this has revolutionized the field of genomics. High-density peptide arrays promise to advance the field of proteomics in a similar way, but currently lag behind. This is mainly due to the monomer-by-monomer repeated consecutive coupling of 20 different amino acids associated with lithography, which adds up to an excessive number of coupling cycles.

PEGMA/MMA copolymer graftings: generation, protein resistance, and a hydrophobic domain.

We synthesized various graft copolymer films of poly(ethylene glycol) methacrylate (PEGMA) and methyl methacrylate (MMA) on silicon to examine the dependency of protein-surface interactions on grafting composition. We optimized atom transfer radical polymerizations to achieve film thicknesses from 25 to 100 nm depending on the monomer mole fractions, and analyzed the resulting surfaces by X-ray photoelectron spectroscopy (XPS), ellipsometry, contact angle measurements, and atomic force microscopy (AFM). As determined by XPS, the stoichiometric ratios of copolymer graftings correlated with the concentrations of provided monomer solutions.

Precise selective deposition of microparticles on electrodes of microelectronic chips.

We examined the high precision deposition of toner and polymer microparticles with a typical size of approximately 10 microm on electrode arrays with electrodes of 100 microm and below using custom-made microelectronic chips. Selective desorption of redundant particles was employed to obtain a given particle pattern from preadsorbed particle layers. Microparticle desorption was regulated by dielectrophoretic attracting forces generated by individual pixel electrodes, tangential detaching forces of an air flow, and adhesion forces on the microchip surface.

The effect of vaccination against porcine circovirus type 2 in pigs suffering from porcine respiratory disease complex.

A field study was conducted to investigate the effect of vaccination against porcine circovirus type 2 (PCV2) in pigs suffering from porcine respiratory disease complex (PRDC). A total of 1542 pigs were allocated randomly into two treatment groups at approximately 20 days of age. Groups received either a Baculovirus-expressed recombinant PCV2 Open Reading Frame (ORF) 2 vaccine or placebo by single intramuscular injection.

Combinatorial synthesis of peptide arrays onto a microchip.

Arrays promise to advance biology through parallel screening for binding partners. We show the combinatorial in situ synthesis of 40,000 peptide spots per square centimeter on a microchip. Our variant Merrifield synthesis immobilizes activated amino acids as monomers within particles, which are successively attracted by electric fields generated on each pixel electrode of the chip.

Noncontact charge measurement of moving microparticles contacting dielectric surfaces.

In this study examples for a noncontact procedure that allow the description of instant electric charging of moving microparticles that contact dielectric surfaces, for instance, of a flow hose are presented. The described principle is based on the measurement of induced currents in grounded metal wire probes, as moving particles pass close to the probe. The feasibility of the approach was tested with laser printer toner particles of a given size for different basic particle flow and charging conditions.

Multifunctional CMOS microchip coatings for protein and peptide arrays.

Complementary metal oxide semiconductor (CMOS) microelectronic chips fulfill important functions in the field of biomedical research, ranging from the generation of high complexity DNA and protein arrays to the detection of specific interactions thereupon. Nevertheless, the issue of merging pure CMOS technology with a chemically stable surface modification which further resists interfering nonspecific protein adsorption has not been addressed yet. We present a novel surface coating for CMOS microchips based on poly(ethylene glycol)methacrylate graft polymer films, which in addition provides high loadings of functional groups for the linkage of probe molecules.

A novel glass slide-based peptide array support with high functionality resisting non-specific protein adsorption.

Glass slides have been modified with a multifunctional poly(ethylene glycol) (PEG)-based polymer with respect to array applications in the growing field of proteome research. We systematically investigated the stepwise synthesis of the PEG films starting from self-assembled alkyl silane monolayers via monolayer peroxidation and subsequent graft polymerization of PEG methacrylate (PEGMA). Chemical composition was examined by X-ray photoelectron spectroscopy (XPS); infrared spectroscopy provided information about order and composition of the films as well; film thickness was determined by ellipsometry; using fluorescence microscopy and again XPS, the amount of proteins adsorbed on the slides was investigated.

Identification and characterization of a novel RanGTP-binding protein in the yeast Saccharomyces cerevisiae.

The small Ras-like GTPase Ran plays an essential role in the transport of macromolecules in and out of the nucleus and has been implicated in spindle (1,2 ) and nuclear envelope formation (3,4 ) during mitosis in higher eukaryotes. We identified Saccharomyces cerevisiae open reading frame YGL164c encoding a novel RanGTP-binding protein, termed Yrb30p. The protein competes with yeast RanBP1 (Yrb1p) for binding to the GTP-bound form of yeast Ran (Gsp1p) and is, like Yrb1p, able to form trimeric complexes with RanGTP and some of the karyopherins.