Application Note 1 Comparison of Manual vs. Automated Protein Microarray Processing Tecan HS Hybridization Station and ISAC ® protein microarrays for the determination of specific IgE Abstract In the recent years, the originally DNA fo- cused biochip technology has been adapted for the monitoring of multiple binding events on a protein level. The benefits of this inno- vative approach are the minute sample vol- umes, the high sensitivity and the enormous numbers of measurements conceivable in a single drop of sample liquid, e.g. human blood. Due to the heterogeneous nature of the proteins themselves, the monitoring of interactions involving complex mixtures of proteins is by far more challenging and more difficult to standardize than for DNA-based hybridizations. As a consequence, several technological hurdles still have to be over- come to make protein microarrays a reliable tool for basic research and routine laboratory in-vitro diagnostic (IVD) tools. Here, we show that the automated process- ing of a protein microarray based diagnostic test (ISAC ® , VBC-GENOMICS, Austria) in combination with a Tecan HS Hybridization Station greatly improves the assays perform- ance parameters, especially the reproducibil- ity and signal-to-noise ratio. By means of the automated procedure, the total assay dura- tion is reduced more than twofold due to an increased ligand binding rate. We suggest that assay automation will be an essential prerequisite for the standardization of protein microarray experiments in the field of basic research and for in vitro diagnostic applica- tions in particular. By improving the assay reproducibility we predict that it will be possi- ble to overcome current technological limita- tions, e.g. the inability to precisely quantify analytes in a multiplex microarray based measurement. Introduction For the analysis of gene-expression states on a whole-genome level DNA microarrays have become a widespread and reliable analytic tool [1]. The challenge of producing protein microarrays by employing a DNA biochip- based technological approach holds great promise for basic research as well as for IVD applications. In the last few years, reports of protein microarrays have been published by academic and industry based research groups, showing a broad range of potential applications of this new technology [reviewed in 2]. But aside from large scale proteomic
Transcript
Application Note
1
Comparison of Manual vs. Automated Protein Microarray Processing
Tecan HS Hybridization Station and ISAC® protein microarrays for the determination of specific IgE
Abstract In the recent years, the originally DNA fo-cused biochip technology has been adapted for the monitoring of multiple binding events on a protein level. The benefits of this inno-vative approach are the minute sample vol-umes, the high sensitivity and the enormous numbers of measurements conceivable in a single drop of sample liquid, e.g. human blood. Due to the heterogeneous nature of the proteins themselves, the monitoring of interactions involving complex mixtures of proteins is by far more challenging and more difficult to standardize than for DNA-based hybridizations. As a consequence, several technological hurdles still have to be over-come to make protein microarrays a reliable tool for basic research and routine laboratory in-vitro diagnostic (IVD) tools. Here, we show that the automated process-ing of a protein microarray based diagnostic test (ISAC®, VBC-GENOMICS, Austria) in combination with a Tecan HS Hybridization Station greatly improves the assays perform-ance parameters, especially the reproducibil-ity and signal-to-noise ratio. By means of the automated procedure, the total assay dura-
tion is reduced more than twofold due to an increased ligand binding rate. We suggest that assay automation will be an essential prerequisite for the standardization of protein microarray experiments in the field of basic research and for in vitro diagnostic applica-tions in particular. By improving the assay reproducibility we predict that it will be possi-ble to overcome current technological limita-tions, e.g. the inability to precisely quantify analytes in a multiplex microarray based measurement. Introduction For the analysis of gene-expression states on a whole-genome level DNA microarrays have become a widespread and reliable analytic tool [1]. The challenge of producing protein microarrays by employing a DNA biochip-based technological approach holds great promise for basic research as well as for IVD applications. In the last few years, reports of protein microarrays have been published by academic and industry based research groups, showing a broad range of potential applications of this new technology [reviewed in 2]. But aside from large scale proteomic
Application Note
2
approaches which hold many technological pitfalls, protein microarrays are of particular interest for innovative in-vitro diagnostic applications, such as the monitoring of spe-cific antibodies directly from human serum. Because of their distinct advantages over conventional analytical devices - most im-portantly the small sample and reagent vol-umes, the sensitive detection methods and the possibility for massive parallel molecular analysis - protein microarrays are likely to revolutionize typical laboratory routines in the nearby future [3]. The ISAC® test system Recently, VBC Genomics have reported the development of a miniaturized immunoassay in a microarray format for the diagnosis of IgE mediated type I allergic diseases – the ISAC® test (Immuno Solid-phase Allergen Chip). In a number of publications the research group of VBC Genomics has demonstrated that ISAC® holds up against established immuno assay test systems regarding essential assay per-formance characteristics such as sensitivity and specificity [4-6]. A schematic overview of the ISAC® test sys-tem is shown in Figure 1. A panel of selected recombinant and purified allergens is immo-bilized onto a modified glass surface in an arrayed fashion (see Figure 1A). The dimen-sions (75x25mm) of the glass biochip are compatible with the standard laboratory in-strumentation for slide handling and microar-ray scanning. For the ISAC® test, the Proteo-Bind®TM chip surface coating has been de-veloped and optimized to efficiently and co-valently bind allergens (proteins or peptides) in a biologically active manner. Each allergen is spotted as a triplicate to assure maximum assay reliability. Additionally, increasing con-centrations of purified human IgE are immo-bilized in concert with the allergens for the purpose of assay calibration and quality con-trol (see Figure 1 A and E). Each ISAC® chip contains several arrays of allergens that allow the testing of more than one serum or several serum dilutions on a single chip (see Figure 1B). Each of the allergen arrays is sur-rounded by a thin layer of Teflon® in order to create individual reaction wells and to pre-vent sample overflow during the assay (Fig-ure 1B). 20 µl of serum per reaction well are sufficient to perform an ISAC® assay. Basi-cally, each chip well may contain up to 400 individual spots (e.g., allergens, auto-anti-gens, peptide epitopes). Following the incu-bation with patient’s serum and a short washing step, the allergen microarrays are
stained with a fluorescence-labeled anti-hu-man IgE antibody (Figure 1C). The chip is scanned using a confocal fluorescence laser-based biochip reader (Figure 1D). Figure 1E shows a false color image resulting of an ISAC® assay. The fluorescence image is analyzed using a specific software package. The fluorescence intensity values of the individual spots are quantified (Figure 1F) and bio-informatics-based quality control procedures are applied as described in details in [7]. ISAC® positive results are then assigned to three classes (low, medium and high) that indicate the allergen-specific IgE titer contained in the patient’s serum. For further details and the list of available purified and recombinant allergens see the manufacturer’s web page (www.vbc-genomics.com). The total assay duration is less than 300 minutes for the standard ISAC® assay procedure, including slide and reagent preparation, serum incuba-tion, washing steps, staining as well as image scanning and data acquisition. The total hands-on-time is approximately 10 minutes per ISAC® slide, depending, however, on the speed or automation of the biochip reader. Basically, using ISAC®, several thousand specific IgE determinations can be performed per day without further automation. For the user’s convenience, ISAC® is delivered as a kit that contains all necessary assay reagents (buffer, detection antibody and calibration serum). ISAC® takes advantage of the benefits of protein biochip technology, but also has to deal with the shortcomings of this new meth-odology which is still in its infancy. In par-ticular, reproducibility is always a critical factor for experiments conducted on a micro-array platform. Consequently, a step towards assay automation is likely to improve current limitations caused by insufficient assay re-producibility, and will pave the way for a reliable quantification of analytes on an ab-solute scale. The Tecan HS Hybridization Station Tecan has designed the HS Series of Hybridization Stations for fully automated hybridization of microarrays on slides, with the focus to offer improved reproducibility, sensitivity and reliability with reduced labor compared to traditional procedures. All steps of the hybridization procedure are performed without user intervention, from pre-hybridiza-tion and on-board denaturation up to auto-matic slide drying. This provides standardized
Application Note
3
procedures and eliminates the risk of manual errors during microarray hybridization. Low chamber volumes with no dead volume help to save precious probe. A wider dynamic range of signals compared to manual meth-ods yields additional information from sensi-tive microarray experiments. Highly uniform signal-to-background ratios within and between slides provide consistent and reli-able results. With a choice of Hybridization Stations – the HS 400 and HS 4800 – Tecan meets the throughput needs for all research laboratories, irrespective of their size or ca-pacity requirements. Both instruments offer identical process performance and full proto-col compatibility. The superior performance of automated DNA microarray hybridization has been demon-strated recently. With the HS Series Hybridi-zation Stations the value of the coefficient of variation (CV) for signal intensities is typically much lower compared to the hybridization performed in the manual method. The same
applies to the CV of the background values and overall the signal-to-background ratios are significantly higher in the automated method. Therefore a wider dynamic range can be achieved in the HS Hybridization Station. Constant and high signal-to-back-ground ratios with very low inter- and intra-assay variation lead to improved reproduci-bility and reliability on the Tecan HS Hybridi-zation Station [9]. In this study the manual process flow of ISAC®-based IgE testing was compared to automated assay conduction with the Tecan HS 400 Hybridization Station concerning reproducibility, kinetic behavior, linearity and detection limit as well as signal-to-noise ratio. Therefore, the standard ISAC® procedure was adapted for the HS 400 with minor modi-fications of the standard protocol, mainly affecting the washing steps and incubation times.
Figure 1. ISAC® design and assay procedure. (A) Schematic representation of the allergen microarray layout. Individual allergens of a single biological source are depicted as circles bearing the same colour. (B) Incubation of a 4-well ISAC® chip with 4 individual serum samples (20 µl per serum). (C) Schematic representation of the ISAC® assay. Allergen molecules (blue) are covalently immobilized onto the chemically modified biochip surface to bind efficiently and in a biologically active manner. Allergens are recognized by serum IgE antibodies (red), which themselves are detected by a fluorescent labelled anti-human IgE antibody (yellow). (D) Schematic repre-sentation of fluorescence laser scanning. (E) ISAC® assay false colour sample image. In the upper part of the image, specific signals of fluorescence-labelled anti-human IgE antibodies bound to increasing concentrations of immobilised human IgE are shown. Fluorescence signals that correlate to the specific binding of labelled anti-human IgE to allergen triplicates are shown at the bottom of the image. (F) Schematic representation of an allergen triplicate quantification and the corresponding ISAC® classification range (low – intermediate – high).
Application Note
4
Methods ISAC protein microarrays A set of 8 well characterized recombinant allergens of birch (Bet v 1, Bet v 2), timothy grass (Phl p 1, Phl p 2, Phl p 5, Phl p 7) and Alternaria alternata (Alt a 1, Alt a 2) were spotted onto ProteoBind slides (VBC-Genomics, Vienna, Austria). All allergens were obtained from Biomay (Biomay, Vienna, Austria). Experiments described below were performed with two different sera of patients allergic to at least five of the 8 recombinant allergens and covering a wide range of spe-cific serum IgE concentrations, referred to in the text as serum A and serum B. Manually operated ISAC assay procedure A detailed ISAC® assay protocol can be downloaded from the manufacturers web page (www.vbc-genomics.com). In brief, ISAC® slides were washed with TBS-T for 60 minutes, rinsed with QD and then dried. In the next step, 20 µl of the sample serum were applied to the reaction well and the slides were incubated in a humid chamber at ambient temperature. The standard serum incubation time was 120 minutes, but varied for kinetic analysis between 5 and 120 min-utes. Following the incubation with serum and a short washing step (10 minutes TBS-T,
QD, drying), the allergen microarrays were stained with a fluorescence-labeled anti human IgE antibody for 60 minutes. Finally, the arrays were washed again (10 minutes TBS-T, QD, drying) and scanned using a confocal fluorescence laser-based biochip reader (GMS 428, Affymetrix, Santa Clara, CA). All steps were carried out at ambient temperature Automated ISAC assay procedure Automated protein microarray processing was performed in the Tecan HS 400 Hybridi-zation Station and different programs were conceived and optimized during a set-up phase. The standard protocol used for the determination of reproducibility, signal-to-noise ratios and dilution parallelism is shown in Figure 3. For the determination of the as-say kinetics, only the serum incubation time was varied. In contrast to manual ISAC® testing, on the used configuration of the HS 400 only one serum specimen per slide was applied. Consequently, for the HS 400 slide incubation, 70 µl of serum were necessary to cover the whole spotted area. All incubation and washing steps were run at a temperature of 37°C and agitated with high frequency (see Figure 3). Slides were dried automati-cally afterwards and scanned immediately.
Figure 3. The Tecan HS 400 standard protocol for ISAC® assays. All steps were carried out at 37°C. For the incubation steps the agitation frequency was set to “high”. Wash and soak times are shown for each step.
Application Note
5
Results and discussion The reproducibility of automated HS 400-based ISAC processing is increased when compared to the manually operated assay Both, the standard – manually operated - assay as well as the HS 400 standard assay were performed independently, two times per day on five consecutive workdays, by one operator, using the same serum specimen (serum A) for both procedures. Using these ten data sets, the coefficient of variation (CV) was calculated for each allergen individually, as well as the mean for all allergens collec-tively, resulting in a mean signal CV of 30.7 % for the manual operation, and 16.2 % for the HS 400 procedure. The fluorescence intensity mean values are illustrated in Figure 2A in concert with their standard deviation error bars. Additionally, the dataset was han-dled according to the NCCLS Guideline EP5-T for the evaluation of precision performance of clinical chemistry devices [10], to calculate the precision estimates for total, between-run, between-day and within-run precision. The precision estimates are separately listed in Table 1A, expressed as percent of the total signal mean of all allergen specific measure-ments. Summarizing, the mean signal CV value calculated for all allergens was almost two-fold higher in case of the manual handling (34 %) than for the automated procedure (19 %), as shown in Table 1. With exception of the allergen Phl p 7, all allergens displayed a superior reproducibility when the assay was
performed with the HS 400. This demon-strates clearly that the automation of the assay handling is a crucial factor for the re-producibility, even if all assays are conducted by only one single operator. A study including more than one operator results in even higher CV values for manual handling [un-published data]. Interestingly, we found a significant and re-producible difference in the signal intensity means of Phl p 1 and Phl p 2 when com-pared between manual and automated han-dling, although the same batch of slides was used for both methods. Supposedly, the affinity of the allergen-specific IgE antibodies towards the immobilized allergen was ad-versely influenced by the agitation of the sample fluid in the HS 400 device, whereas during the manual handling the sample is incubated without agitation. Since the reper-toire of specific IgEs against a certain aller-gen is probably heterogeneous concerning the individual IgE’s affinity towards the aller-gen epitopes, low affinity IgE antibodies might bind to their allergens more efficiently without extensive fluid agitation, whereas high affinity antibodies might result in more or less uninfluenced signal intensities if incu-bated with or without agitation. Consequently, the allergen specific signal intensities ob-tained might not only depend on the total amount of antibody present in the serum, but also on the ratio of high and low affinity anti-bodies. However, this will be subject to fur-ther investigations using a broader collection of serum samples.
Table 1. Comparison of precision estimates for manually and automatically processed protein microarrays. Within-run, between-run and between-day precision for both methods are listed for each allergen separately, as well as the mean value for all allergens. For Alt a 2 and Bet v 2 the precision estimates were not determined (n.d.) since the serum did not react with these allergens.
Alt a 1 Alt a 2 Bet v 1 Bet v 2 Phl p 1 Phl p 2 Phl p 5 Phl p 7 MeanWithin Run 43,5 n.d. 29,4 n.d. 28,5 30,8 50,0 36,4 36,4
Between Run 26,7 n.d. 18,1 n.d. 16,8 18,3 31,8 18,7 21,7Between Day 22,3 n.d. 16,0 n.d. 16,8 11,7 27,2 7,9 17,0
Total 41,6 n.d. 27,8 n.d. 28,7 27,5 47,1 31,8 34,1
Alt a 1 Alt a 2 Bet v 1 Bet v 2 Phl p 1 Phl p 2 Phl p 5 Phl p 7 MeanWithin Run 20,2 n.d. 16,8 n.d. 11,5 15,0 18,6 32,0 19,0
Between Run 9,0 n.d. 9,8 n.d. 5,3 9,1 10,6 13,5 9,6Between Day 11,4 n.d. 0,6 n.d. 6,7 8,3 8,8 5,6 6,9
Total 21,5 n.d. 13,6 n.d. 11,8 14,7 21,8 33,6 19,5
Manual operation
HS 400
Application Note
6
The ISAC® assay kinetics are positively influenced by the HS 400 automated procedure We then sought to determine differences between the assay kinetics of manually oper-ated ISAC assays - without temperature control or sample agitation - and the auto-mated assay processing at 37 °C and with rapid sample agitation. For the experiments summarized in Figure 2B, all assay parame-ters except the serum incubation time are identical. The serum incubation times were: 5, 10, 30, 45, 60 and 120 minutes. Again, serum A was used for all experiments, and the fluorescence intensity values obtained for the allergens Phl p 1 and Phl p 2 differed reproducibly between the two methods (see Table 2). Each time course assay was performed two times independently and the mean value of the two individual data sets was calculated.
The mean fluorescence intensities of all allergens with positive results were employed for the graphical data visualization in Figure 2B. The kinetic measurements were sub-jected to linear regression analysis. As the figure shows, signal increase as a function of the incubation time is almost 40 % higher for the HS 400 than for the manual handling. This implies a beneficial influence of the agitation and temperature control on the assay kinetics. Moreover, the obtained signal intensities were significantly higher with the HS 400 than with manual operation. The difference in the curve offsets indicates a particular boost of the kinetic in the first few minutes of the incubation, which we did not monitor (< 5 min.) The kinetic curves for both methods were still linear after 120 minutes of serum incubation and did not display a sign of achieving satu-ration at this time point.
Table 2. Comparison of assay kinetics for manually and automatically processed protein microarrays. Assay signals were monitored after 5, 10, 30, 45, 60 and 120 minutes for both methods. The total assay signal was calculated by summarizing all allergen specific fluorescence intensities.
HS 400 based ISAC® assays permit a higher S/N ratio than the manually operated procedure For the determination of the average signal-to-noise ratios of both methods, we per-formed standard assays on five slides (4 arrays per slide) by either manual operation or the HS 400, using serum A for all experi-ments. For each allergen and IgE standard spot in the individual microarrays (see Figure 1), the signal-to-noise ratio was calculated individually by the following formula: S/N = (FI – BG) / SD BG, where FI is the fluores-cence signal intensity of the according spot,
BG represents the surrounding spot back-ground fluorescence intensity, and SD BG stands for the standard deviation of the background pixels. For all 5 experiments, the mean, median, maximum and standard de-viations of the signal-to-noise ratios of each spot were calculated and are provided in Table 3. Figure 2C summarizes the mean values for all five experiments. As shown there, the mean S/N ratio values were signifi-cantly higher for the HS 400 (1:43) than for the manual assay processing (1:30). We therefore conclude that the agitation of the sample, the omission of the drying steps
Time Alt a 1 Alt a 2 Bet v 1 Bet v 2 Phl p 1 Phl p 2 Phl p 5 Phl p 7 Total5 1217 0 4781 1356 2245 1053 4700 686 16038
during the assay and the fully automated handling have a beneficial effect on the as-say background noise, which in turn will in-crease the assay sensitivity by allowing to use a lower signal threshold. The HS 400-based and the manually operated assay behave similarly in dilution experiments Since the ISAC® test is used for a semi-quantitative analysis of allergen-specific IgE titers, a linear response behavior for diluted samples is essential. Consequently, we monitored assay linearity with a broad range of sample dilutions for both manual handling and the HS 400 assay processing. We therefore performed standard assays for both methods with serum B diluted in threefold steps (1:1, 1:3, 1:9, 1:57, 1:81, 1:243, 1:729, 1:2187). Again, each experiment was per-formed twice and the mean value of the two replicates was used for further data interpre-tation (Table 4A). ISAC®-based measurement of serum B resulted in positive results for the allergens Bet v 1, Bet v 2, Phl p 1 and Phl p 5. The absolute IgE concentrations of serum B for the according allergens as determined with UniCAP (Pharmacia, Sweden) analysis are shown in Table 4B. The fluorescence
intensities for these allergens in the serial dilution experiments are shown in Table 4A. Fluorescence values below the defined aller-gen threshold (500 FI) were set to 0 and considered as IgE negative. The resulting data are plotted against the calculated abso-lute IgE concentrations for the according dilutions in Figure 2D. Both methods displayed a linear behavior over at least 4 orders of magnitude, with a slight advantage with respect to the detection limit for the manual operation. This might be due to washing conditions, the high agitation level during the incubation or an effect of the significantly shorter incubation time in the HS 400 (60 minutes versus 120 minutes for manual incubation). Previously, we demon-strated that the signal-to-noise ration is better for the HS 400, and accordingly the use of a lower signal threshold for HS 400 experi-ments would be valid and likely to result in a similar or even better sensitivity for automatic array handling than plotted in Figure 2D. However, for both methods the detection limit was below 0.1 kUA/L, which corresponds to 0.24 ng IgE per ml serum. For routine diag-nostic testing, the generally defined clinically relevant specific IgE level is >= 0.35 kUA/L.
Table 3. Comparison of signal-to-noise ratios of manually and automatically processed protein microarrays. For each method, five experiments (1-5) were applied to calculate the mean, median, maximum and standard deviation of the signal-to-noise ratios from each individual spot’s signal-to-noise ratios in the arrays. For details see text.
Table 4A. Comparison of dilution parallelism and detection limits for manually and automatically processed protein microarrays. A serum was diluted in threefold steps, and for each dilution the according allergen specific fluorescence intensities are listed for both methods. Only those allergens that dis-played positive signals are listed. Values > 0 were ISAC positive.
Manual operation HS 400Dilution Bet v 1 Bet v 2 Phl p 1 Phl p 5 Bet v 1 Bet v 2 Phl p 1 Phl p 5
Specific IgE conc. (kUA/L) Bet v 1 Bet v 2 Phl p 1 Phl p 5 184.40 14.50 42.70 40.00
* 1 kUA/L = 2.4 ng / ml
Table 4B. Specific IgE concentrations for serum B as determined with UniCAP.
Figure 2. (A) Determination of differences in the reproducibility of the ISAC® assay when performed manually or with the HS 400. Mean fluorescence intensity values of 10 experiments performed on 5 days for the allergens Alt a 1, Bet v 1, Phl p 1, Phl p 2, Phl p 5 and Phl p 7 are shown together with the corresponding standard deviations error bars. (B) Differences in the kinetic behaviour of the ISAC® assay when performed manually or with the HS 400. Total fluorescence intensity values of 2 experiments for the allergens Alt a 1, Bet v 1, Phl p 1, Phl p 2, Phl p 5 and Phl p 7 are plotted against the according incubation times. Linear regression equations of the two curves are also provided. (C) Signal-to-noise ratios of the ISAC® assay when performed manually or with the HS 400. Mean, median, maximum as well as the standard deviations of all S/N ratios are drawn for both methods. For details see text. (D) Determination of differences in the dilution parallelism and detection limit of the ISAC® assay when performed manually or with the HS 400. The fluorescence intensities of the allergens Bet v 1, Bet v 2, Phl p 1 and Phl p 5 are plotted against the specific IgE values calculate for the according dilution from the original value of the undiluted serum (see Table 2B).
Application Note
9
Conclusions In this study, we compared the assay per-formance of ISAC® allergen microarrays by employing two conceptually different meth-ods: A manual assay operation and an auto-mated assay procedure adapted for the Tecan HS 400. As has been described above, the benefits of assay automation are manifold. Without influencing the assay per-formance negatively, we were able to reduce the total assay time from approximately 300 minutes for the manual procedure to 120 minutes with the HS 400. This was due to the reduced washing and incubation times, and the omission of drying steps during the assay steps. At the same time, the hands-on time was reduced significantly, since the only manual interventions necessary for the HS 400 protocol were the insertion of the slides into the device and the sample injection following later. As we have shown in detail, the assay per-formance of the Tecan HS 400 compared to the manual assay processing was superior in most belongings, in particular for the assay reproducibility, assay kinetics and the signal-to-noise ratio. The reproducibility increased almost twofold during the automated assay handling. The resulting fluorescence signal intensities were higher when using the HS 400 despite the shorter incubation time. With respect to the better signal-to-noise ratios, the automated array processing would permit the use of a lower signal threshold for the scoring of IgE positive signals than the man-ual handling, which is likely to further in-
crease the sensitivity of automated assay processing and outperform the manual handling. Moreover, the automation of the assay bears additional advantages, such as the exclusion of errors inflicted by the operator’s handling (e.g. by sample confusion), the potentiality of data tracking and the conformity with quality control guidelines. We therefore conclude that the automation of protein microarray experiments with the Tecan HS 400 or a similar device will be an important prerequisite to ensure data con-sistency and reliability in all experimental fields dealing with protein microarray experi-ments, and for in vitro diagnostic microarray applications such as the ISAC® allergen chips in particular. Acknowledgements The experiments performed were possible due to the support and expert advice from the following institutes and persons: Sabine Hutter1, Gerald Probst2, Claudia Kirisits1, Reinhard Hiller1 and Christian Harwanegg1
Literature 1. Gershon D. 2002. Microarray technology: an array of opportunities. Nature 416:885-91. 2. MacBeath G. 2002. Protein microarrays and proteomics. Nat Genet 32 Suppl:526-32. 3. Templin MF, Stoll D, Schrenk M, Traub PC, Vohringer CF, Joos TO. 2002. Protein microarray technology. Trends Biotechnol 20:160-6. 4. Hiller R, Laffer S, Harwanegg C, Huber M, Schmidt WM, Twardosz A, Barletta B, Becker WM, Blaser K, Breiteneder H, Chapman M, Crameri R, Duchene M, Ferreira F, Fiebig H, Hoffmann-Sommergruber K, King TP, Kleber-Janke T, Kurup VP, Lehrer SB, Lidholm J, Muller U, Pini C, Reese G, Scheiner O, Scheynius A, Shen HD, Spitzauer S, Suck R,
Swoboda I, Thomas W, Tinghino R, Van Hage-Hamsten M, Virtanen T, Kraft D, Muller MW, Valenta R. 2002. Microarrayed allergen molecules: diagnostic gatekeepers for allergy treatment. FASEB J 16:414-6. 5. Harwanegg C, Laffer S, Hiller R, Mueller MW, Kraft D, Spitzauer S, Valenta R. 2003. Microarrayed recombinant allergens for diagnosis of allergy. Clin Exp Allergy 33:7-13. 6. Jahn-Schmid B, Harwanegg C, Hiller R, Bohle B, Ebner C, Scheiner O, Mueller MW. 2003. Allergen microarray: comparison of microarray using recombinant allergens with conventional diagnostic methods to detect allergen-specific serum immunoglobulin E. Clin Exp Allergy 33:1443-9.
Application Note
10
Tecan Asia (Pte) Ltd., 80, Marine Parade, #13-04, Singapore 449269, Singapore, T +65 644 41 886, F +65 644 41 836 Tecan Sales Austria GmbH, Untersbergstrasse 1a, A-5082 Grödig / Salzburg, Austria, T +43 62 46 89 33, F +43 62 46 72 770 Tecan Sales International GmbH, Untersbergstrasse 1a, A-5082 Grödig / Salzburg, Austria, T +43 62 46 89 33, F +43 62 46 72 770 Tecan Benelux B.V.B.A., Vaartdijk 55, B-2800 Mechelen, Belgium, T +32 15 42 13 19, F +32 15 42 16 12 Tecan Benelux B.V.B.A., Industrieweg 30, NL-4283 GZ Giessen, Netherlands, T +31 18 34 48 17 4, F +31 18 34 48 06 7 Tecan Deutschland GmbH, Theodor-Storm-Straße 17, D-74564 Crailsheim, Germany, T +49 79 51 94 170, F +49 79 51 50 38 Tecan France S.A., Parc d’Activités de Pissaloup, Bâtiment Hermes II, Rue Edouard Branly, F-78190 Trappes, France, T +33 1 30 68 81 50, F +33 1 30 68 98 13 Tecan Italia S.r.l., Via F.lli Cervi, Palazzo Bernini, Centro Direzionale Milano 2, I-20090 Segrate (Mi), Italy, T +39 02 215 21 28, F +39 02 215 97 441 Tecan Japan Co. Ltd, Meiji Seimei Fuchu Building 10F, 1-40 Miyamachi, Fuchu City, Tokyo, Japan, T +81 42 334 88 55, F +81 42 334 04 01 Tecan Nordic AB, Box 208, SE-431 23 Mölndal, Sweden, T +46 31 75 44 000, F +46 31 75 44 010 Tecan Portugal, Quinta da Fonte - Edificio Pedro I, 2780-730 Paço d'Arcos, Portugal, T +351 21 000 82 16, F +351 21 000 16 75 Tecan Sales Switzerland AG, Seestrasse 103, CH-8708 Männedorf, Switzerland, T +41 1 922 89 22, F +41 1 922 89 23 Tecan Spain, Sabino de Arana, 32, E-08028 Barcelona, Spain, T +34 93 490 01 74, F +34 93 411 24 07 Tecan UK, Theale Court, 11-13 High Street, Theale, UK-Reading RG7 5AH, United Kingdom, T +44 11 89 300 300, F +44 11 89 305 671 Tecan US, P.O. Box 13953, Research Triangle Park, NC 27709, USA, T +1 919 361 5200, F +1 919 361 5201 www.tecan.com
7. Harwanegg C, Spitzauer S, Valenta R, Mueller MW, Hiller R. 2004. Protein biochips for the profiling of allergen-specific antibodies. In M. Schena (Ed.), Protein Microarrays, Jones & Bartlett Publishers, Sudbury, MA. In press. 8. Valenta R, Lidholm J, Niederberger V, Hayek B, Kraft D, Gronlund H. 1999. The recombinant allergen-based concept of component-resolved diagnostics and immunotherapy (CRD and CRIT). Clin Exp Allergy 29:896-904.
9. Probst G, Posch J, Hagenbüchle O, Wyniger J, Porter G. 2003. A comparison of manual and automated hybridization methods with a focus on homogeneity and reproducibility. ABRF Conference 2003: P158-Th. 10. Wayne PA. 1984. NCCLS Tentative Guideline EP5-T. User evaluation of precision performance of clinical chemistry devices.: National Committee for Clinical Laboratory Standards.
