Preparation of biomolecule microstructures and microarrays by thiol-ene photoimmobilization.

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 microm in size covering an area of up to 75x25 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.

Sensitivity by combination: immuno-PCR and related technologies.

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.

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.

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.

Synthesis of protein-nucleic acid conjugates by expressed protein ligation.

The synthesis of covalent conjugates of proteins and polyamide nucleic acids (PNA) is accomplished by expressed protein ligation of intein-fusion proteins and a PNA-cysteine conjugate.

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.

Hapten-Functionalized DNA-Streptavidin Nanocircles as Supramolecular Reagents in a Competitive Immuno-PCR Assay.

Analysis with nanorings: The endogeneous proteins of supramolecular DNA nanocircles, obtained in high yields from oligomeric precursors containing bisbiotinylated DNA and streptavidin, are conveniently functionalized with biotinylated hapten moieties. These modular conjugates can be used as reagents in a novel competitive immunoassay for the high-sensitivity detection of low molecular weight analytes.

Matrix-assisted laser desorption and ionization-time-of-flight mass spectrometry, 16S rRNA gene sequencing, and API 32E for identification of Cronobacter spp.: a comparative study.

Twenty-two isolates of the family Enterobacteriaceae, with focus on Cronobacter isolated from infant formula and the environment of milk powder plants, were comparatively identified using API 32E (bioMérieux, Marcy l'Etoile, France), 16S rRNA gene sequencing (Accugenix, Newark, USA), and matrix-assisted laser desorption and ionization-time-of-flight mass spectrometry (MALDI-TOF MS; Mabritec, Riehen, Switzerland and AnagnosTec, Potsdam, Germany). With API 32E, 22% of the isolates were assigned to species, 64% were assigned to a genus, and 14% could not be discriminated at any taxonomic level. Both 16S rRNA gene sequencing and MALDI-TOF MS assigned 100% of the isolates to species, but the identifications based on MALDI-TOF MS results were more discriminating and unequivocal. Our data indicate that MALDI-TOF MS provides the most rapid and unambiguous identification of Cronobacter and closely related Enterobacteriaceae isolates.

Modeling the occurrence of Mycobacterium avium subsp. paratuberculosis in bulk raw milk and the impact of management options for exposure mitigation.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causal agent of paratuberculosis (Johne's disease) in cattle and other farm ruminants. The potential role of MAP in Crohn's disease in humans and the contribution of dairy products to human exposure to MAP continue to be the subject of scientific debate. The occurrence of MAP in bulk raw milk from dairy herds was assessed using a stochastic modeling approach. Raw milk samples were collected from bulk tanks in dairy plants and tested for the presence of MAP. Results from this analytical screening were used in a Bayesian network to update the model prediction. Of the 83 raw milk samples tested, 4 were positive for MAP by culture and PCR. We estimated that the level of MAP in bulk tanks ranged from 0 CFU/ml for the 2.5th percentile to 65 CFU/ml for the 97.5th percentile, with 95% credibility intervals of [0, 0] and [16, 326], respectively. The model was used to evaluate the effect of measures aimed at reducing the occurrence of MAP in raw milk. Reducing the prevalence of paratuberculosis has less of an effect on the occurrence of MAP in bulk raw milk than does managing clinically infected animals through good farming practices.

Detecting antigens by quantitative immuno-PCR.

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.

Magneto immuno-PCR: a novel immunoassay based on biogenic magnetosome nanoparticles.

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 320pg/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.

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.

Synthesis of covalent oligonucleotide-streptavidin conjugates and their application in DNA-directed immobilization (DDI) of proteins.

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.

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.