Quick and simple: quality control of microarray data

MOTIVATION: Microarrays are high-throughput tools for parallel miniaturized detection of biomolecules. In contrast to experiments using ratios of signals in two channels, experiments with only one fluorescent dye cause special problems for data analysis. The present work compares algorithms for quality filtering on spot level as well as array/slide level. RESULTS: Methods for quantitative spot filtering are discussed and new sets of quality scores for data preprocessing are designed. As measures of spot quality also reflect the quality of protocols, they were employed to find the optimal print buffer in an optimization experiment. In order to determine problematic arrays within a set of replicates we tested methods of outlier detection which can suitably replace the visual inspection of slides. CONTACT: Ursula.Sauer@arcs.ac.at.     

Effects of relative humidity and buffer additives on the contact printing of microarrays by quill pins

DNA microarrays printed with quill pins exhibit significant variation in probe DNA spots. Interspot variations and nonuniform distribution of probe within spots are major sources of experimental uncertainty in microarray analysis. To gain better insight into the sources of variation, we analyzed 450 consecutive depositions printed at relative humidities between 40 and 80% using three print buffers. Increasing relative humidity improved printing performance by delaying pin failure but did not reduce the variability in spot characteristics. Adding either betaine or dimethyl sulfoxide (DMSO) to the print buffer also improved quill pin performance. Least interspot variation was observed with the DMSO additive printed at 80% relative humidity, but this additive also resulted in the greatest intraspot variation. Least intraspot variation was observed with 1.5M betaine printed at 60% relative humidity, but these conditions produced microarrays with high interspot variability. Evaporation of printing solution from the quill reservoir appeared to be the primary cause of interspot and intraspot variations. Our studies indicate that relative humidity and printing solution additives reduce evaporation. Based on the spot variability requirements for a particular application, humidity and additives may be chosen to optimize either inter- or intraspot variability.     

基于夹心免疫分析的抗体微阵列的构建

目的：建立一种基于夹心免疫分析的抗体微阵列构建的优化方法。方法：将MCP-1的捕获抗体点样于修饰后的玻片,标准抗原加样覆盖所点阵列,生物素标记抗体和链酶亲和素-cy3依次加样孵育, 激光共聚焦扫描仪获取图象并进行数据分析。对捕获抗体浓度、封闭液种类、系统可重复性和定量检测能力、两种因子平行性检测对信号分析的影响及点样后玻片稳定性进行分析和评价。结果：随着捕获抗体浓度的升高,信号强度逐渐增加;2℅ BSA/PBS和5℅ 酪蛋白可作为本系统的封闭液;所构建系统具有较好的可重复性（组内变异 1.3%,组间变异8.7%）和定量分析能力（所建立的抗原浓度-相对信号强度标准曲线相关系数达0.9995）;并实现了两因子的平行性分析和点样后玻片的稳定性。结论：确立了基于夹心免疫分析的抗体微阵列构建的优化方法,为进一步构建多因子定量检测抗体微阵列奠定了基础。     

Improving protein array performance: focus on washing and storage conditions

For protein microarrays, maintaining protein stability during the slide processing steps of washing, drying, and storage is of major concern. Although several studies have focused on the stability of immobilized antibodies in antibody microarrays, studies on protein-protein interaction arrays and enzyme arrays are lacking. In this paper we used five bait-prey protein interaction pairs and three enzymes to optimize the washing, drying, and storage conditions for protein arrays. The protein arrays for the study were fabricated by combining HaloTag technology and cell-free protein expression. The HaloTag technology, in combination with cell-free expression, allowed rapid expression and immobilization of fusion proteins on hydrogel-coated glass slides directly from cell extracts without any prior purification. Experimental results indicate enzyme captured on glass slides undergoes significant loss of activity when washed and spin-dried using only phosphate buffer, as is typically done with antibody arrays. The impact of washing and spin-drying in phosphate buffer on protein-protein interaction arrays was minimal. However, addition of 5% glycerol to the wash buffer helps retain enzyme activity during washing and drying. We observed significant loss of enzyme activity when slides were stored dry at 4 degrees C, however immobilized enzymes remained active for 30 days when stored at -20 degrees C in 50% glycerol. We also found that cell-free extract containing HaloTag-fused enzymes could undergo multiple freeze/thaw cycles without any adverse impact on enzyme activity. The findings indicate that for large ongoing studies, proteins of interest expressed in cell-free extract can be stored at -70 degrees C and repeatedly used to print small batches of protein array slides to be used over a few weeks.     

蛋白质微阵列检测抗原-抗体相互作用

为了制备蛋白质微阵列和研究芯片表面抗原-抗体的相互作用,研究了如何在玻片表面固化蛋白质和用荧光染料（Cy3,Cy5）对蛋白质进行标记.结果表明,在醛基修饰的玻璃表面,通过共价偶联的方法将抗原或抗体固定到芯片表面,能使二者保持其特异性结合能力.同时,荧光标记后的抗原或抗体仍然具有特异性结合能力.蛋白质微阵列是通过机械手在玻片表面排阵制作的.芯片上的荧光信号获取采用了激光共焦荧光扫描系统.用不同浓度的抗原探针阵列,对其相应的抗体靶分子的特异性结合进行了分析和研究.此外,还通过在玻片表面固定兔IgG和固定鼠IgG,对羊抗兔和羊抗鼠抗体与其相应抗原的特异性相互作用进行了检测.     

Site-directed immobilisation of antibody fragments for detection of C-reactive protein

C-reactive protein, CRP antibody Fab'-fragments have been attached on pre-cleaned gold slides and protein repellent polymers have been used to block the remaining free space in between the antibody fragments. At optimal conditions the antibody fragments are site-directly immobilised on the surface and non-specific binding is reduced. The amount of Fab'-fragments in the polymer host monolayer has been optimised for various buffers. Binding of CRP to Fab'-fragment/polymer layers produced in phosphate buffered saline decreased with NaCl salt concentration. In a 1M NaCl phosphate buffer, the antibodies seem to be randomly oriented on the surface with a similar response to CRP as that of an antibody F(ab)(2)-fragment layer. In a 150 mM NaCl phosphate buffer, on the other hand, the fragments seem to be site-directly oriented and the response to CRP was fivefold. The highest response to CRP was obtained to a layer with a Fab'-fragment concentration of 60 microg/ml. CRP could be detected in a concentration range of 1 ng/ml to 50 microg/ml from a standard solution in phosphate buffer and in a range of 4 ng/ml to 50 microg/ml from serum/PBS. CRP was, moreover, successfully detected in patient samples with good reproducibility. The layer would thus be sensitive enough to analyse the CRP concentration in human serum for predicting cardiovascular disease.     

Antibody purification-independent microarrays (PIM) by direct bacteria spotting on TiO2-treated slides

The preparation of effective conventional antibody microarrays depends on the availability of high quality material and on the correct accessibility of the antibody active moieties following their immobilization on the support slide. We show that spotting bacteria that expose recombinant antibodies on their external surface directly on nanostructured-TiO(2) or epoxy slides (purification-independent microarray - PIM) is a simple and reliable alternative for preparing sensitive and specific microarrays for antigen detection. Variable domains of single heavy-chain antibodies (VHHs) against fibroblast growth factor receptor 1 (FGFR1) were used to capture the antigen diluted in serum or BSA solution. The FGFR1 detection was performed by either direct antigen labeling or using a sandwich system in which FGFR1 was first bound to its antibody and successively identified using a labeled FGF. In both cases the signal distribution within each spot was uniform and spot morphology regular. The signal-to-noise ratio of the signal was extremely elevated and the specificity of the system was proved statistically. The LOD of the system for the antigen was calculated being 0.4ng/mL and the dynamic range between 0.4ng/mL and 10μg/mL. The microarrays prepared with bacteria exposing antibodies remain fully functional for at least 31 days after spotting. We finally demonstrated that the method is suitable for other antigen-antibody pairs and expect that it could be easily adapted to further applications such as the display of scFv and IgG antibodies or the autoantibody detection using protein PIMs.     

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.     

Protein microarrays for highly parallel detection and quantitation of specific proteins and antibodies in complex solutions

Background  

We have developed and tested a method for printing protein microarrays and using these microarrays in a comparative fluorescence assay to measure the abundance of many specific proteins in complex solutions. A robotic device was used to print hundreds of specific antibody or antigen solutions in an array on the surface of derivatized microscope slides. Two complex protein samples, one serving as a standard for comparative quantitation, the other representing an experimental sample in which the protein quantities were to be measured, were labeled by covalent attachment of spectrally resolvable fluorescent dyes.     

Toward optimized antibody microarrays: a comparison of current microarray support materials

With the advent of protein and antibody microarray technology several different coatings and protocols have been published, which may be broadly divided into two types: gel-coated surfaces and plain non-gel-coated glass or plastic surfaces, some with chemical groups attached. We have screened 11 different array surfaces of both types and compared them with respect to their detection limit, inter- and intrachip variation, and storage characteristics. Five different antibodies were immobilized onto each type of microarray support, with total protein concentrations ranging from 40 fmol to 25 amol per spot. From these results, it was seen that some antibodies were more suited for use on antibody arrays. All measurements were performed in quadruplicate, and the results revealed high signal uniformity and reproducibility of most plain glass and plastic slides. Lower detection limits were obtained with polyacrylamide-coated slides, making them more suitable for the detection of very low concentrations of antigen. All microarray coatings could be stored for a period of 8 weeks; however, improved results were seen after 2 weeks of storage. In conclusion, the results indicate the need to test each antibody to be used on an antibody array and to select the microarray coating based on experimental requirements.     

Protein microarrays on hybrid polymeric thin films prepared by self-assembly of polyelectrolytes for multiple-protein immunoassays

We report here the development and characterization of protein microarrays fabricated on nanoengineered 3-D polyelectrolyte thin films (PET) deposited on glass slide by consecutive adsorption of polyelectrolytes via self-assembly technique. Antibodies or antigens were immobilized in the PET-coated glass slides by electrostatic adsorption and entrapment of porous structure of the 3-D polymer film and thus establishing a platform for parallel analysis. Both antigen and antibody microarrays were fabricated on the PET-coated slides, and direct and indirect immunoassays on protein microarrays for multiple-analyte detection were demonstrated. Microarrays produced on these PET-coated slides have consistent spot morphology and provide performance features needed for proteomic analysis. The protein microarrays on the PET films provide LOD as low as 6 pg/mL and dynamic ranges up to three orders of magnitude, which are wider than the protein microarrays fabricated on aldehyde and poly-L-lysine functionalized slides. The PET films constructed by self-assembly technique in aqueous solution is green chemistry based, cost-effective method to generate 3-D thin film coatings on glass surface, and the coated slide is well suited for immobilizing many types of biological molecules so that a wide variety of microarray formats can be developed on this type of slide.     

Creating highly dense and uniform protein and DNA microarrays through photolithography and plasma modification of glass substrates

We demonstrate a method to create high density protein microarrays with excellent spot uniformity using photolithography and plasma processing on low cost commercially available microscope glass slides. Protein deposition and fluorescence signal evaluation on these substrates are performed by standard arrayers and scanners. To this end, spots of commercial photoresists (AZ5214, SU8 and Ormocomp(?)) were defined through lithography on glass substrates followed by short SF(6) plasma treatment and selective protein adsorption on these spots with respect to glass (spot to background fluorescence signal ratios 30:1 to 40:1) was demonstrated using model protein binding assays. Among the photoresists tested, Ormocomp was selected since it provided the highest protein binding capacity. No ageing of Ormocomp/glass substrates in terms of protein binding capacity was observed for at least two months. Besides to protein microarrays, DNA microarrays were also developed by spotting streptavidin-biotinylated oligonucleotide conjugates corresponding to wild- and mutant-type sequences of four deleterious BRCA1 gene mutations. For all of the examined mutations, higher specific hybridization signals (1.5-4 times) and improved discrimination ratios between wild- and mutant-type sequences as well as higher spot uniformity and repeatability were demonstrated on Ormocomp/glass substrates with intra- and inter-spot CVs of 8.0% and 4.5%, respectively, compared to commercial polystyrene (intra- and inter-spot CVs 36% and 18%) and epoxy-coated glass (intra- and inter-spot CVs 26% and 20%) slides. Thus, the proposed substrates can be readily applied to protein and DNA microarrays fabrication and, moreover, the described method for selective protein adsorption can be advantageously implemented in various analytical microdevices for multi-analyte detection.     

An Activated GOPS‐poly‐L‐Lysine‐ Coated Glass Surface for the Immobilization of 60mer Oligonucleotides

To explore a method for enhancing the immobilization and hybridization efficiency of oligonucleotides on DNA microarrays, conventional protocols of poly‐L‐lysine coating were modified by means of surface chemistry, namely, the slides were prepared by the covalently coupling of poly‐L‐lysine to a glycidoxy‐modified glass surface. The modified slides were then used to print microarrays for the detection of the SARS coronavirus by means of 60mer oligonucleotide probes. The characteristics of the modified slides concerning immobilization efficiency, hybridization dynamics, and probe stripping cycles were determined. The improved surface exhibited high immobilization efficiency, a good quality uniformity, and satisfactory hybridization dynamics. The spotting concentration of 10 μmol/L can meet the requirements of detection; the spots were approximately 170 nm in diameter; the mean fluorescence intensity of the SARS spots were between 3.2 × 10⁴ and 5.0 × 10⁴ after hybridization. Furthermore, the microarrays prepared by this method demonstrated more resistance to consecutive probe stripping cycles. The activated GOPS‐PLL slide could undergo hybridization stripping cycles for at least three cycles, and the highest loss in fluorescence intensity was found to be only 11.9 % after the third hybridization. The modified slides using the above‐mentioned method were superior to those slides treated with conventional approaches, which theoretically agrees with the fact that modification by surface chemistry attaches the DNA covalently firmly to the slides. This protocol may have great promise in the future for application in large‐scale manufacture.     

A label-free visual immunoassay on solid support with silver nanoparticles as plasmon resonance scattering indicator

By taking silver nanoparticles (Ag-NPs) as plasmon resonance scattering (PRS) indicator considering that Ag-NPs have strong plasmon resonance light scattering signals corresponding to their plasmon resonance absorption (PRA), we propose a label-free visual immunoassay on the solid support of glass slides. Our investigations showed that Ag-NPs could be adsorbed on the surface of glass slides where immunoreactions between a previously immobilized antigen and its antibody have occurred if the glass slides were immersed in an Ag-NP suspension whose pH value has been carefully adjusted. The optimal pH of the Ag-NP suspension depends on the nature of previously immobilized antigen and its antibody. It was found that the adsorption of negative-charged Ag-NPs on the surface of glass slides depends only on the content of antibody under optimal conditions. With a common spectrofluorometer to measure the PRS signals of the Ag-NPs adsorbed on the surface, we could detect antibody in the range of 10 to 160 ng ml⁻¹. If a white light-emitting diode (LED) torch is employed to illuminate the glass slides, we can make visual detection of the antibody by the naked eye.     

Effects of organic pH buffers on a cell growth and an antibody production of human-human hybridoma HB4C5 cells in a serum-free culture

Human-human hybridoma cells secreting a human monoclonal antibody were cultured in a serum-free medium containing various organic pH buffers in order to clarify their effects on cell growth and antibody production. Organic pH buffers having either one sulfonic acid and several acyclic amine moieties, or several cyclic amine moieties containing two amino nitrogen did not inhibit cell growth; while other organic buffers sulfonic acid moiety plus several cyclic amine moieties containing one amino nitrogen slightly decreased cell growth, but enhanced antibody production. Using Fujita's organic conceptual diagram, a relationship between the organicity and inorganicity of a pH buffer to cell growth and antibody production was found. pH buffers with large inorganicity and small organicity values were favorable for cell growth, and buffers with small inorganicity and large organicity values were preferred to enhance antibody production. Although the pH buffering range affects cell growth, its effect on antibody production is not clear. In conclusion, 2-morpholinoethanesulfonic acid (MES), 3-morpholino-propanesulfonic acid (MOPS) and 1, 2-N, N-bis[N, N-di(2-sulfonoethyl)piperazinyl]ethane (Bis-PIPES) are shown to be the most optimal of the buffers tested, because they enhanced antibody production without decreasing the cell growth among the pH buffers tested here.     

Design of recombinant antibody microarrays for complex proteome analysis: choice of sample labeling-tag and solid support

Antibody-based microarray is a novel technology with great potential within high-throughput proteomics. The process of designing high-performing antibody (protein) microarrays has, however, turned out to be a challenging process. In this study, we have developed further our human recombinant single-chain variable-fragment (scFv) antibody microarray methodology by addressing two crucial technological issues, choice of sample labeling-tag and solid support. We examined the performance of a range of dyes in a one- or two-color approach on a selection of solid supports providing different surface and coupling chemistries, and surface structures. The set-ups were evaluated in terms of sensitivity, specificity, and selectivity. The results showed that a one-color approach, based on NHS-biotin (or ULS-biotin) labeling, on black polymer Maxisorb slides (or Nexterion slide H) was the superior approach for targeting low-abundant (pg/mL) analytes in nonfractionated, complex proteomes, such as human serum or crude cell supernatants. Notably, microarrays displaying adequate spot morphologies, high S/Ns, minimized nonspecific binding, and most importantly a high selectivity, specificity, and sensitivity (>or=fM range) were obtained. Taken together, we have designed the first generation of a high-performing recombinant scFv antibody microarray technology platform on black polymer Maxisorb slides for sensitive profiling of low-abundant analytes in nonfractionated biotinylated complex proteomes.     

Signal stability of Cy3 and Cy5 on antibody microarrays

Background  

The antibody microarray technique is a newly emerging proteomics tool for differential protein expression analyses that uses fluorescent dyes Cy 3 and Cy 5. Environmental factors, such as light exposure, can affect the signal intensity of fluorescent dyes on microarray slides thus, it is logical to scan microarray slides immediately after the final wash and drying processes. However, no research data are available concerning time-dependent changes of fluorescent signals on antibody microarray slides to this date. In the present study, microarray slides were preserved at -20°C after regular microarray experiments and were rescanned at day 10, 20 and 30 to evaluate change in signal intensity.     

免疫荧光检测HIV—1抗体方法的建立和应用

免疫荧光法检测HIV抗体是确认HIV感染的方法之一［l］,与蛋白印迹法相比,它的特异性更高,易于鉴别非特异反应,更为经济、快速、容易操作。为了拥有更多的HIV抗体检测手段,我们用感染和未感染HIV－l的HeLaCD“细胞制备抗原片,建立了免疫荧光检测HIV抗体的方法,并进行了初步应用。材料和方法1细胞和病毒HeLaCD4”细胞,引自美国BruceChesebro博士的实验室,用含10％热灭活小牛血清的RPMI1640培养基培养和传代。HIV－l株引自美国。2血清艾滋诊断试剂国家参比品,批号9602Z16份经蛋白印迹确认的HIV抗体阳性血清;10份健康…