Seeing Better through a MIST:  Evaluation of Monoclonal Recombinant Antibody Fragments on Microarrays

Angenendt, Philipp; Wilde, Jeannine; Kijanka, Gregor; Baars, Theodor; Cahill, Dolores J.; Kreutzberger, Jürgen; Lehrach, Hans; Konthur, Zoltán; Glökler, Jörn

doi:10.1021/ac035357a

Cited by 46 publications

(28 citation statements)

References 27 publications

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“…The principle of the assays is based on the multiple spotting technology (MIST) [11,12] and comprises two spotting steps in which the fluorogenic substrates are spotted in a first round of spotting, followed by the transfer of enzyme solution in a second round of spotting. Since the enzyme solution contains hygroscopic additives, such as glycerol, the total evaporation of the transferred solution, which is initially about 0.6 nL [13], is prevented.…”

Section: Resultsmentioning

confidence: 99%

Subnanoliter enzymatic assays on microarrays

et al. 2005

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Many areas of research today are based on enzymatic assays most of which are still performed as enzyme-linked immunosorbent assays in microtiter plates. The demand for highly parallel screening of thousands of samples eventually led to a miniaturization and automation of these assays. However, the final transfer of enzymatic assays from a microtiter-based technology to microarrays has proven to be difficult for various reasons, such as the inability to maintain unbound reaction products on the spot of reaction or the missing capability of multiplexing. Here, we have conducted multiplex enzymatic assays in subnanoliter volumes on a single microarray using the multiple spotting technology. We were able to measure enzymatic activity with a sensitivity down to 35 enzyme molecules, applying only conventional flat microarray surfaces and standard microarray hardware. We have performed assays of inhibition and applied this format for the detection of prognostic markers, such as cathepsin D. The new approach allows the rapid and multiplex screening of thousands of samples on a single microarray with applications in drug screening, metagenomics, and high-throughput enzyme assays.

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Section: Resultsmentioning

confidence: 99%

Subnanoliter enzymatic assays on microarrays

et al. 2005

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“…In summary, we present an in situ expression of proteins on a chip as a new application of the multiple spotting technique that complements its application in the detection of proteins and antibodies (27), in the selection of single chain Fv fragments from phage display selections (37), and the characterization of enzymatic activities and inhibitors thereof (38). The in situ expression involves the spotting of DNA templates in a first spotting step followed by the spotting of a cell-free transcription and translation mixture in a second spotting step on top of the DNA templates.…”

Section: Figmentioning

confidence: 99%

Generation of High Density Protein Microarrays by Cell-free in Situ Expression of Unpurified PCR Products

Angenendt

Kreutzberger

Glökler

et al. 2006

Molecular & Cellular Proteomics

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Due to the success of DNA microarrays and the growing numbers of available protein expression clones, protein microarrays have become more and more popular for the high throughput screening of protein interactions. However, the widespread applicability of protein microarrays is currently hampered by the large effort associated with their production. Apart from the requirement for a protein expression library, expression and purification of the proteins themselves and the lacking stability of many proteins remain the bottleneck. Here we present an approach that allows the generation of high density protein microarrays from unbound DNA template molecules on the chip. It is based on the multiple spotting technique and comprises the deposition of a DNA template in a first spotting step and the transfer of a cell-free transcription and translation mixture on top of the same spot in a second spotting step. Using wild-type green fluorescent protein as a model protein, we demonstrated the time and template dependence of this coupled transcription and translation and showed that enough protein was produced to yield signals that were comparable to 300 g/ml spotted protein. Plasmids as well as unpurified PCR products can be used as templates, and as little as 35 fg of PCR product (ϳ22,500 molecules) were sufficient for the detectable expression of full-length wild-type green fluorescent protein in subnanoliter volumes. We showed that both aminopropyltrimethoxysilane and nickel chelate surfaces can be used for capture of the newly synthesized proteins. Surprisingly we observed that nickel chelate-coated slides were binding the newly synthesized proteins in an unspecific manner. Finally we adapted the system to the high throughput expression of libraries by designing a single primer pair for the introduction of the required T7 promoter and demonstrated the in situ expression using 384 randomly chosen clones. Molecular & Cellular Proteomics 5:1658 -1666, 2006.The understanding of complex cellular networks necessitates tools that are amenable to the analysis of different parameters in a highly parallel manner (1). Although in the last years DNA microarrays were the technology of choice to monitor the abundance of several thousands of mRNA transcripts at a time, such studies provide us with little information on the proteins that are encoded by these transcripts (2, 3). However, because proteins rather than DNA carry out cellular functions, there is large interest to analyze proteins and their entirety, the proteome, in a manner comparable to DNA microarrays. One technology that is envisaged to meet the demands of high throughput protein interaction and modification screening is protein microarray technology (4 -8).Protein microarrays have been applied in different areas of application, such as the analysis of protein-protein interactions (9 -11), the identification of substrates for protein kinases (12-14), or the elucidation of potential diagnostic markers in bacterial or autoimmune diseases (15-18). All of them share the bas...

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“…In a first study, enzymatic activity of enzymes such as alkaline phosphatase, ß-galactosidase or cathepsin D was detected [189]. Moreover, the applicability of MIST was shown for multiplex immunosorbent assays [190,191] and for the screening of large populations of recombinant antibody fragments on a microarray [192].…”

Section: Applicationsmentioning

confidence: 99%

Microarray Technology as a Universal Tool for High-Throughput Analysis of Biological Systems

Sobek¹,

Bartscherer²,

Jacob³

et al. 2006

CCHTS

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Over the last years microarray technology has become one of the principal platform technologies for the highthroughput analysis of biological systems. Starting with the construction of first DNA microarrays in the 1990s, microarray technology has flourished in the last years and many different new formats have been developed. Peptide and protein microarrays are now applied for the elucidation of interaction partners, modification sites and enzyme substrates. Antibody microarrays are envisaged to be of high importance for the high-throughput determination of protein abundances in translational profiling approaches. First cell microarrays have been constructed to transform microarray technology from an in vitro technology to an in vivo functional analysis tool. All of these approaches share a common prerequisite: the solid support on which they are generated. The demands on this solid support are thereby as manifold as the applications themselves. This review is aimed to display the recent developments in surface chemistry and derivatization, and to summarize the latest developments in the different application areas of microarray technology.

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Seeing Better through a MIST: Evaluation of Monoclonal Recombinant Antibody Fragments on Microarrays

Cited by 46 publications

References 27 publications

Subnanoliter enzymatic assays on microarrays

Subnanoliter enzymatic assays on microarrays

Generation of High Density Protein Microarrays by Cell-free in Situ Expression of Unpurified PCR Products

Microarray Technology as a Universal Tool for High-Throughput Analysis of Biological Systems

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