Protein microarrays for research and diagnostic

Protein Microarray Applications
With the advent of proteome studies, i. e. the global analysis of overall protein expression, protein microarrays have moved into the focus of interest. Instrumentation originally developed for the generation of DNA microarrays is now successfully being used for the creation of protein microarrays with thousands of spots on a microscope slide, the current standard format. An increasing number of experimental methods have been published describing novel approaches for protein research. Table 1 summarizes a selection of very promising publications documenting the broad application range of protein microarrays in research.

Table 1: Applications of Protein Microarrays

Microarray-type	Description
Reverse phase	Cell lysates from different tissues 1, 2 or IEF protein fractions 3 were spotted; antigens were detected using various standard detection methods.
Antibody	Antibodies were spotted and incubated with biotinylated cell lysates4 , leucocytes (microscopic analysis)5 or GFP/RFP6
Antigen	Bacterial surface antigens were spotted and specific binding of IgM and IgG antibodies from human sera was detected7
Protein protein interactions	The yeast proteome was spotted, printed calmodulin binds specifically to calmodulin kinase and vice versa 8
Cytokine detection	Parallel quantification of human cytokines in one sample in a "protein microarray ELISA" 9
Protein-DNA interactions	Bacterial replication initiator DnaA recognizes specifically it’s Cy5-labelled cognate DNA sequence10
Kinase target identification	Arabidopsis proteins were spotted, novel targets for mitogen-activated protein kinases were identified, almost all targets were confirmed by other in-vitro methods11

Surface Chemistries for Protein Microarrays
Microscopic slides are available with different surface chemistries. S. Hanash’s group investigated aldehyde-, amino- and nitrocellulose-coated slides³. According to this study the sensitivity of detection was highest on nitrocellulose-coated FAST® Slides. The work published by Wu6 offers an explanation for the higher sensitivity of detection observed on FAST® Slides. Planar slides with aldehyde or aldehyde-type coating lost 95 % of spotted protein after incubation, washing, and detection while FAST® Slides kept ≥ 95 % of protein. The 3-dimensional sponge-like microporous nitrocellulose provides a much larger surface and higher binding capacity compared to all planar surface coatings. The group of Liotta defined the limit of detection on FAST® Slides in quantitative terms. They developed a reverse phase microarray to study cancerogenesis in different cell groups collected by laser capture microdissection¹. In this assay the sensitivity of detection was found to be in the zeptomolar range (10^-21 moles). Recombinant PSA (prostate specific antigen) was detected with linear and quantifiable signals from 1.25 to 20 fg. Table 2 gives an overview on different surface chemistries.

Table 2: Comparison of Protein Microarray Surfaces

	Detection Sensitivity 3, 6	Reverse phase arrays	Sample retention after spotting 6
Nitrocellulose-coated FAST ® Slides	++++	10-21moles PSA 1.25 fg PSA	≥ 95 %
Aldehyde-coated slides	++	n.a.	≤ 5 %
Amino-coated slides	+	n.a.	n.a.

n.a. - data not available

Stability and Reproducibility of Protein Microarrays
Since decades nitrocellulose membranes have proven to be a reproducible substrate for protein detection. Applications range from Western Blotting to immunochromatographic pregnancy tests (lateral flow immunoassays) with highly specific and sensitive detection. It is well known that antibodies and antigens immobilised on nitrocellulose retain their molecular binding and recognition specificities for much more than a year. This property is of crucial importance e.g. for commercially available immunodiagnostic Western blot test-strips and lateral flow immunoassay tests. Nitrocellulose-coated FAST® Slides show the same long term stability of immobilised proteins which can even be enhanced by using a specifically optimized arraying buffer (Fig. 1).

Human Cytokine Detection for Research
Recently, standard ELISAs for human cytokines have been transferred from microtiter plates to a protein microarray format with 16 identical microarrays on a single slide. This novel method allows for parallel detection of multiple different human cytokines in a single biological sample. Fig. 2. shows the general design of this assay system. It can be processed with multichannel pipettes thus bridging the gap between single protein microarray slides and convenient liquid handling in a microplate-format. For many cytokines the limit of detection and the linear range was determined in a protein microarray format and in a commercially available ELISA. Both the limit of detection and the linear range are better for most cytokines in the protein microarray as compared to the ELISA. In addition cytokines can be quantified in parallel in the same sample. This work has led to the development of a commercially available protein microarray for human cytokine quantification (FAST Quant9). In Fig. 3. the FAST Quant system has been used to measure the effect of an experimental drug on cytokine levels. The new mulitplex assay system offers significant savings in workload, reagent and sample consumption for cytokine quantification.

Autoantibody Detection for Disease Diagnostic
Protein micoroarray technology can also enhance disease diagnostics. In a most recent development multiple collagenosis and vasculitis related antigens are printed together with an internal calibrator on 16 pad FAST Slides (Fig 4.). Autoantibodies against these antigens are accepted as clinical markers for various autoimmune diseases thus creating a novel multiparameter in-vitro diagnostic tool. The assay is developed in cooperation with H. Appelhans and H.-P. Seelig from the Privates Institut für Immunologie und Molekulargenetik in Karlsruhe, Germany. The clinical validation has shown that the assay performance for autoantibody detection against the analytes listed in Table 3 matches that of current standard methods. Patients will benefit from an enhanced autoimmune disease profile while reference labs can reduce their workload through multiplexing. An additional benefit is that a single platform is sufficient whereas currently different methods have to be used. For some of the analytes a semi-quantitave determination will be possible for the first time. This novel system is expected to be introduced in the beginning of 2006 as a CE-marked in-vitro diagnostic test system.

Table 3: Antigens for autoimmune disease diagnostic protein microarray

U1-70k (snRNP)	ds-DNA
Sm	Scl-70
SS-A/Ro-52	Jo-1
SS-A/Ro-60	Pr3-ANCA
SS-B/La	MPO-ANCA
Pm/Scl-75	Mi-2
Pm/Scl-100

Outlook
Protein microarrays are becoming a crucial tool in applications ranging from basic proteome research to drug discovery (e.g. identification of novel kinase targets). Formats enabling higher throughput with more samples to be processed on a single slide are likely to increase in importance. The trend is towards a smaller number of analytes (< 50) and quantitative multiplex determinations. Nitrocellulose-coated FAST® Slides in different formats are becoming a worldwide standard due to their high sensitivity of detection, reproducible manufacturing and long term stability of arrayed proteins.

Figures:

Fig 1. Stability of Protein Microarrays on FAST® Slides

Caption Fig. 1:
Monoclonal antibody against creatine kinase (0.1 mg/ml) diluted in PBS (phosphate buffered saline) or in proprietary FAST PAK array buffer was spotted on FAST® Slides. Slides were incubated with Cy5-labelled creatine kinase at the indicated times and detected with a confocal laser imager. A.) Absolute signal intensities after different storage times B.) Relative signal intensities of FAST PAK array buffer vs. PBS.

 

Fig 2. Design FAST Quant Assay System

Caption Fig. 2:
Four 16 pad FAST slides in a FAST Frame showing a typical array result. Inserts show the array map for FAST Quant, D represents spots containing donkey anti-goat Ig to act as landing light controls.

 

Fig 3: Cytokine Expression in PBMC cells

Caption Fig 3:
Cytokine expression levels in cell culture supernatants from control and drug treated PBMC cells. The data shown is derived from triplicate array spots on FAST Quant. Cytokine levels are determined via the use of a standard curve in ArrayVision - FAST software.

 

Fig 4: Autoimmune Disease Diagnostic Microarray

Caption Fig 4:
Layout of antigens (see also Table 3) printed on 16 pad FAST Slides for autoantibody profiling of collagenosis and vasculits related autoimmune diseases. The grey spots on the top left are varying amounts of human IgG serving as an internal calibrator for semi-quantitative determinations.

 

Literature:
[1] Paweletz et al. 2001. Reverse phase protein microarrays which capture disease progression show activation of pro-survival pathways at the cancer invasion front. Oncogene 20, 1981 - 1989
[2] Nishizuka et al. 2003. Proteomic profiling of the NCI-60 cancer cell lines using new high-density reverse-phase lysate microarrays, Proc. Natl. Acad. Sci. USA 100:14229-14234.
[3] Madoz-Gurpide et al. 2001. Protein based microarrays: A tool for probing the proteome of cancer cells and tissues. Proteomics 1, 1279-1287
[4] Knezevic et al. 2002. Proteomic profiling of the cancer microenvironment by antibody arrays. Proteomics 1:1271-1278
[5] Belov et al. 2001. Immunphenotyping of leukemias using a cluster of differentiation antibody microarray. Cancer Research 61, 4483 – 4489
[6] Kukar et al. 2002. Protein microarrays to detect protein-protein interactions using red and green fluorescent proteins. Analytical Biochemistry 306, 50 – 54
[7] Wang et al. 2002. Carbohydrate microarrays for the recognition of cross-reactive molecular markers of microbes and host cells. Nature Biotechnology 20, 275 – 281
[8] Schweitzer et al. 2003. Microarrays to characterize protein interactions on a whole-proteome scale. Proteomics 3:2190-2199.
[9] Stillman et al. 2004. Applying multiplexed microspot immunoassays. Genetic Engineering News. March 15, Vol.24
[10] Kersten et al. 2004. Protein microarray technology and ultraviolet crosslinking combined with mass spectrometry for the analysis of protein–DNA interactions. Analytical Biochemistry 331: 303–313.
[11] Feilner et al. 2005, High-throughput identification of potential Arabidopsis MAP kinases substrates. Mol Cell Proteomics. 4:1558–1568, 2005