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Ron Wacker

Publications and source records attributed to Ron Wacker.

13 recordsLinked to original sources
PubMed2006

A generic building block for C- and N-terminal protein-labeling and protein-immobilization.

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.

Azides↗
PubMed2005

Immuno-PCR: high sensitivity detection of proteins by nucleic acid amplification.

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.

Immunosorbent Techniques↗
PubMed2004

Performance of antibody microarrays fabricated by either DNA-directed immobilization, direct spotting, or streptavidin-biotin attachment: a comparative study.

Antibody microarrays have the potential to revolutionize protein diagnostics. The major problems in the fabrication of antibody arrays, however, concern the reproducibility and homogeneity of the attachment of the proteins on the solid substrate. We here compare the DNA-directed immobilization (DDI) method with two conventional strategies for immobilization of antibodies on glass substrates. DDI is based on the self-assembly of semisynthetic DNA-streptavidin conjugates which converts an array of DNA oligomers into an antibody microarray. DDI was compared with direct spotting of antibodies on chemically activated glass slides and with immobilization of biotinylated antibodies on streptavidin-coated slides. The immobilized antibodies were used as capture reagents in a two-sided (sandwich) immunoassay for the quantification of rabbit IgG as a model antigen. Detection limits down to 0.001nM (150 pg/mL) were attained with all three array formats; however, DDI and direct spotting of the antibodies led to the highest fluorescence intensities. DDI led to the best spot homogeneity and intra- and interexperimental reproducibility. Moreover, DDI allowed highly economical use of antibody materials; that is, at least 100-fold less antibody is needed for preparing an array by DDI instead of by direct spotting. Taking into account the greater versatility and convenience of handling of the self-assembly approach, this study demonstrates that DDI is an advantageous alternative for generating versatile and robust protein arrays.

Animals↗
PubMed2004

DDI-microFIA--A readily configurable microarray-fluorescence immunoassay based on DNA-directed immobilization of proteins.

We describe a chip-based immunoassay for multiplex antigen detection, based on the self-assembly of semi-synthetic DNA-protein conjugates to generate an easily configurable protein microarray. The general principle of this microarray-fluorescence immunoassay (microFIA) is similar to that of a two-sided (sandwich) immunoassay. However, covalent single-stranded DNA-streptavidin conjugates are employed for the efficient immobilization of biotinylated capture antibodies through hybridization to complementary surface-bound DNA oligomers. In a model system, we use the DNA-directed immobilization (DDI) of antibodies to generate an antibody microarray for the parallel detection of the tumor marker human carcinoembryonic antigen (CEA), recombinant mistletoe lectin rViscumin (rVis), ceruloplasmin (CEP), and complement-1-inactivator (C1A) in human blood serum samples. Detection limits down to 400 pg mL(-1) are reached. In addition, we describe a method for the internal standardization of protein microarray analyses, based on the simultaneous measurement of constant amounts of the blood proteins CEP and C1 A, intrinsically present in human serum, to compensate for interexperimental variations usually occurring in microarray analyses. The standardization leads to a significantly higher data reliability and reproducibility in intra- and interassay measurements. We further demonstrate that the DDI-microFIA can also be carried out in a single step by tagging of the analyte simultaneously with both capture and detection antibody and subsequent immobilization of the immunocomplex formed, on the DNA microarray capture matrix. This protocol significantly reduces handling time and costs of analysis.

Antibodies↗
PubMed2004

Microarray-based in vitro evaluation of DNA oligomer libraries designed in silico.

We report on the microarray-based in vitro evaluation of two libraries of DNA oligonucleotide sequences, designed in silico for applications in supramolecular self-assembly, such as DNA computing and DNA-based nanosciences. In this first study which is devoted to the comparison of sequence motif properties theoretically predicted with their performance in real-life, the DNA-directed immobilization (DDI) of proteins was used as an example of DNA-based self-assembly. Since DDI technologies, DNA computing, and DNA nanoconstruction essentially depend on similar prereguisites, in particular, large and uniform hybridization efficiencies combined with low nonspecific cross-reactivity between individual sequences, we anticipate that the microarray approach demonstrated here will enable rapid evaluation of other DNA sequence libraries.

Base Sequence↗
PubMed2004

Covalent coupling of DNA oligonucleotides and streptavidin.

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.

Cross-Linking Reagents↗
PubMed2003

A real-time immuno-PCR assay for routine ultrasensitive quantification of proteins.

A fast and robust assay, based on the combination of the highly sensitive immuno-PCR (IPCR), employing standardized self-assembled DNA-protein conjugates as reagents, and the well-established, reliable, and fast real-time PCR detection by means of the TaqMan principle is introduced in this work. The use of anti-species immunoglobulin reagents allows one for easy adaptation of this assay to basically any existing ELISA application. The use of an internal competitor in the real-time IPCR (rtIPCR) further increases the sensitivity and significance of this assay; 0.1-0.01 amol (500-50 fg/mL) IgG from several species (mouse, rabbit, goat, and human) were detectable using direct, indirect, and sandwich model rtIPCR assays, thereby increasing the detection limit of the analogous ELISA tests about 100- to 1000-fold. The robustness of this method was demonstrated in two typical applications by detecting 40 pg/mL of the novel anti-cancer drug rViscumin in human plasma samples as well as 100 pg/mL of a research antibody in cell culture media. In both cases, a comparable ELISA was 1000-fold less sensitive.

Animals↗
PubMed2003

Combination of DNA-directed immobilization and immuno-PCR: very sensitive antigen detection by means of self-assembled DNA-protein conjugates.

An assay for very sensitive antigen detection is described which takes advantage of the self- assembly capabilities of semi-synthetic conjugates of DNA and proteins. The general scheme of this assay is similar to a two-sided (sandwich) enzyme-linked immunoassay (ELISA); however, covalent single-stranded DNA-streptavidin (STV) conjugates, capable of hybridizing to complementary surface-bound DNA oligomers, are utilized for the effective immobilization of either capture antibodies or antigens, rather than the chemi- or physisorption usually applied in ELISA. Immuno-PCR (IPCR) is employed as a method for signal generation, utilizing oligomeric reagents obtained by self-assembly of STV, biotinylated DNA and antibodies. In three different model systems, detecting human IgG, rabbit IgG or carcinoembryonic antigen, this combination allowed one to increase the sensitivity of the analogous ELISA approximately 1000-fold. For example, <0.1 amol/ micro l (15 pg/ml) of rabbit IgG was detectable. The immunoassay can be carried out in a single step by tagging the analyte with both reagents for capture and read-out simultaneously, thereby significantly reducing handling time and costs of analysis. Moreover, as the spatial selectivity of target immobilization is determined by the specificity of DNA base pairing, the assay is particularly suited for miniaturized microfluidics and lab-on-a-chip devices.

Animals↗