Microarray-based kinetic colorimetric detection for quantitative multiplex protein phosphorylation analysis

Commonly used colorimetric detection applied to protein microarrays with enzymatic signal amplification leads to non‐linear signal production upon increase in analyte concentration, thereby considerably limiting the range and accuracy of quantitative readout interpretation. To extend the detection range, we developed a kinetic colorimetric detection protocol for the analysis of ELISA microarrays designed to measure multiple phosphorylated proteins using the platforms ArrayTube? and ArrayStrip?. With our novel quantification approach, microarrays were calibrated over a broad concentration range spanning four orders of magnitude of analyte concentration with picomolar threshold. We used this design for the simultaneous quantitative measurement of 15 phosphorylated proteins on a single chip.     

Systematic comparison of surface coatings for protein microarrays

To process large numbers of samples in parallel is one potential of protein microarrays for research and diagnostics. However, the application of protein arrays is currently hampered by the lack of comprehensive technological knowledge about the suitability of 2-D and 3-D slide surface coatings. We have performed a systematic study to analyze how both surface types perform in combination with different fluorescent dyes to generate significant and reproducible data. In total, we analyzed more than 100 slides containing 1152 spots each. Slides were probed against different monoclonal antibodies (mAbs) and recombinant fusion proteins. We found two surface coatings to be most suitable for protein and antibody (Ab) immobilization. These were further subjected to quantitative analyses by evaluating intraslide and slide-to-slide reproducibilities, and the linear range of target detection. In summary, we demonstrate that only suitable combinations of surface and fluorescent dyes allow the generation of highly reproducible data.     

1253例孕妇和新生儿TORCH检测结果分析

目的 了解TORCH感染情况,为妇幼保健提供参考依据.方法 采用捕获ELISA方法对1 253例孕妇和新生儿的血清进行TORCH-IgM抗体检测.结果 689例孕妇CMV、TOX、RV及HSVⅡ特异性IgM抗体阳性率分别为1.89％、0.87％、0.44％和0.73％;564例新生儿CMV-IgM、TOX-IgM、RV-IgM和HSVⅡ-IgM阳性率分别为6.91％、0.18％、0和0.35％.结论 检测TORCH特异性抗体对优生优育有重要关系.     

Quality control for a large-scale study using protein arrays and protein beads to measure immune response in serum and plasma

We report on quality control performed in the context of a large-scale, multi-institutional study of the immune response in blood samples from prostate cancer patients. The measurements were performed by two commercially available techniques/services: protein arrays and an automated bead-based ELISA-like technique on 871 patient samples. The project started with a wide screen using standard arrays with 8302 protein sequences for 113 patients, followed by three studies using custom arrays with 215 selected protein sequences. These studies were followed up by three studies using the bead-based approach on 57 protein sequences chosen from the 215 selected before. We find similar responses in plasma and serum samples. Samples from the two European projects from which the samples originated also appeared comparable. In the data from the high-density standard arrays, we see for ～12% of the protein sequences high cross-correlation (R(2) > 0.8) with signals from unrelated protein sequences that are physically nearby on the array, suggesting production issues. The custom array and bead-based techniques both have good reproducibility, but the techniques do not agree with each other for ～50% of the protein sequences measured. We discuss the consequences of the observed data quality for the design and interpretation of the study.     

Development of a "reverse capture" autoantibody microarray for studies of antigen-autoantibody profiling

Diagnosing cancers based on serum profiling is a particularly attractive concept. However, the technical challenges to analysis of the serum proteome arise from the dynamic range of protein amounts. Cancer sera contain antibodies that react with a unique group of autologous cellular antigens, which affords a dramatic amplification of signal in the form of antibodies relative to the amount of the corresponding antigens. The serum autoantibody repertoire from cancer patients might, therefore, be exploited for antigen-antibody profiling. To date, studies of antigen-antibody reactivity using microarrays have relied on recombinant proteins or synthetic peptides as arrayed features. However, recombinant proteins and/or synthetic peptides may fail to accurately detect autoantibody binding due to the lack of proper PTMs. Here we describe the development and use of a "reverse capture" autoantibody microarray. Our "reverse capture" autoantibody microarray is based on the dual-antibody sandwich immunoassay platform of ELISA, which allows the antigens to be immobilized in their native configuration. As "proof-of-principle", we demonstrate its use for antigen-autoantibody profiling with sera from patients with prostate cancer and benign prostate hyperplasia.     

Sensitive single-molecule protein quantification and protein complex detection in a microarray format

Single-molecule protein analysis provides sensitive protein quantitation with a digital read-out and is promising for studying biological systems and detecting biomarkers clinically. However, current single-molecule platforms rely on the quantification of one protein at a time. Conventional antibody microarrays are scalable to detect many proteins simultaneously, but they rely on less sensitive and less quantitative quantification by the ensemble averaging of fluorescent molecules. Here, we demonstrate a single-molecule protein assay in a microarray format enabled by an ultra-low background surface and single-molecule imaging. The digital read-out provides a highly sensitive, low femtomolar limit of detection and four orders of magnitude of dynamic range through the use of hybrid digital-analog quantification. From crude cell lysate, we measured levels of p53 and MDM2 in parallel, proving the concept of a digital antibody microarray for use in proteomic profiling. We also applied the single-molecule microarray to detect the p53-MDM2 protein complex in cell lysate. Our study is promising for development and application of single-molecule protein methods because it represents a technological bridge between single-plex and highly multiplex studies.     

Prediction of recurrence of non muscle‐invasive bladder cancer by means of a protein signature identified by antibody microarray analyses

About 70% of newly diagnosed cases of bladder cancer are low‐stage, low‐grade, non muscle‐invasive. Standard treatment is transurethral resection. About 60% of the tumors will recur, however, and in part progress to become invasive. Therefore, surveillance cystoscopy is performed after resection. However, in the USA and Europe alone, about 54 000 new patients per year undergo repeated cystoscopies over several years, who do not experience recurrence. Analysing in a pilot study resected tumors from patients with (n = 19) and without local recurrence (n = 6) after a period of 5 years by means of an antibody microarray that targeted 724 cancer‐related proteins, we identified 255 proteins with significantly differential abundance. Most are involved in the regulation and execution of apoptosis and cell proliferation. A multivariate classifier was constructed based on 20 proteins. It facilitates the prediction of recurrence with a sensitivity of 80% and a specificity of 100%. As a measure of overall accuracy, the area under the curve value was found to be 91%. After validation in additional sample cohorts with a similarly long follow‐up, such a signature could support decision making about the stringency of surveillance or even different treatment options.     

Antibody microarray profiling of human prostate cancer sera: antibody screening and identification of potential biomarkers

We developed a practical strategy for serum protein profiling using antibody microarrays and applied the method to the identification of potential biomarkers in prostate cancer serum. Protein abundances from 33 prostate cancer and 20 control serum samples were compared to abundances from a common reference pool using a two-color fluorescence assay. Robotically spotted microarrays containing 184 unique antibodies were prepared on two different substrates: polyacrylamide based hydrogels on glass and poly-1-lysine coated glass with a photoreactive cross-linking layer. The hydrogel substrate yielded an average six-fold higher signal-to-noise ratio than the other substrate, and detection of protein binding was possible from a greater number of antibodies using the hydrogels. A statistical filter based on the correlation of data from "reverse-labeled" experiment sets accurately predicted the agreement between the microarray measurements and enzyme-linked immunosorbent assay measurements, showing that this parameter can serve to screen for antibodies that are functional on microarrays. Having defined a set of reliable microarray measurements, we identified five proteins (von Willebrand Factor, immunoglobulinM, Alpha1-antichymotrypsin, Villin and immunoglobulinG) that had significantly different levels between the prostate cancer samples and the controls. These developments enable the immediate use of high-density antibody and protein microarrays in biomarker discovery studies.     

Cerato-platanin protein is located in the cell walls of ascospores, conidia and hyphae of Ceratocystis fimbriata f. sp. platani

Cerato-platanin (CP), a protein of about 12.4 kDa from Ceratocystis fimbriata f. sp. platani (Cfp), accumulated in the mycelium and was located in the cell walls of Cfp ascospores, hyphae and conidia suggesting that this protein had a role in forming the fungal cell wall apart from the already known fact that it is secreted early in culture and elicits phytoalexin synthesis and/or plant cell death. The finding was obtained with three immunological techniques: a quantitative ELISA which determines the amount of CP in the mycelium, an immunofluorescence assay, and immunogold labelling to define the exact localization of CP in the Cfp cells.     

Development of a colorimetric colony‐screening assay for detection of defluorination by micro‐organisms

Aims: To develop a colorimetric colony‐screening assay to facilitate the isolation of micro‐organisms capable of defluorination. Methods and Results: A metal‐dye chelate, zirconium‐xylenol orange was used to detect fluoride ions released from a fluorinated substrate through microbial metabolism. Depolymerised zirconium reagent gave the greatest visual contrast for the presence of fluoride compared to more polymerised forms of zirconium reagent. The sensitivity of the assay was greatest when the molar ratio of depolymerised zirconium to xylenol orange was 1 : 2. Using depolymerised zirconium and xylenol orange (150 and 300 nmol l^?1 respectively), the assay could detect a fluoride application spot (5 mmol l^?1) containing 50 nmoles of fluoride ions. Most media constituents were well tolerated by the assay, although phosphate ions needed to be restricted to 0·1 g l^?1 and some proteins digest to between 1 and 5 g l^?1. A microbial enrichment culture growing on solidified medium containing 20 mmol l^?1 fluoroacetate was screened using the assay, and defluorinating bacteria belonging to the genus Burkholderia isolated. Conclusions: A method was developed that is sensitive, rapid and reliable for detecting defluorination by micro‐organisms growing on solidified medium. Significance and Impact of the Study: This method can be used to facilitate the isolation of micro‐organisms capable of defluorination.     

An optimized predictor panel for colorectal cancer diagnosis based on the combination of tumor-associated antigens obtained from protein and phage microarrays

Humoral response in cancer patients appears early in cancer progression and can be used for diagnosis, including early detection. By using human recombinant protein and T7 phage microarrays displaying colorectal cancer (CRC)-specific peptides, we previously selected 6 phages and 6 human recombinant proteins as tumor-associated antigens (TAAs) with high diagnostic value. After completing validation in biological samples, TAAs were classified according to their correlation, redundancy in reactivity patterns and multiplex diagnostic capabilities. For predictor model optimization, TAAs were reanalyzed with a new set of samples. A combination of three phages displaying peptides homologous to GRN, NHSL1 and SREBF2 and four proteins PIM1, MAPKAPK3, FGFR4 and ACVR2B, achieved an area under the curve (AUC) of 94%, with a sensitivity of 89.1% and specificity of 90.0%, to correctly predict the presence of cancer. For early colorectal cancer stages, the AUC was 90%, with a sensitivity of 88.2% and specificity of 82.6%. In summary, we have defined an optimized predictor panel, combining TAAs from different sources, with highly improved accuracy and diagnostic value for colorectal cancer. This article is part of a Special Issue entitled: Translational Proteomics.     

Design of recombinant antibody microarrays for membrane protein profiling of cell lysates and tissue extracts

Generating global protein expression profiles, including also membrane proteins, will be crucial for our understanding of biological processes in health and disease. In this study, we have expanded our antibody microarray technology platform and designed the first human recombinant antibody microarray for membrane proteins targeting crude cell lysates and tissue extracts. We have optimized all key technological parameters and successfully developed a setup for extracting, labeling and analyzing non-fractionated membrane proteomes under non-denaturing conditions. Finally, the platform was also extended and shown to be compatible with simultaneous profiling of both membrane proteins and water-soluble proteins.     

Development of peptide aptamer microarrays for detection of HPV16 oncoproteins in cell extracts

Protein microarrays represent an emerging technology that promises to facilitate high-throughput proteomics. The major goal of this technology is to employ peptides, full-length proteins, antibodies, and small molecules to simultaneously screen thousands of targets for potential protein–protein interactions or modifications of the proteome. This article describes the performance of a set of peptide aptamers specific for the human papillomavirus (HPV) type 16 oncoproteins E6 and E7 in a microarray format. E6 and E7 peptide aptamer microarrays were probed with fluorescence-labeled lysates generated from HPV-infected cervical keratinocytes expressing both E6 and E7 oncoproteins. Peptide aptamer microarrays are shown to detect low levels of E6 and E7 proteins. Peptide aptamers specific for cellular proteins included on these microarrays suggested that expression of CDK2, CDK4, and BCL-6 may be affected by HPV infection and genome integration. We conclude that peptide aptamer microarrays represent a promising tool for proteomics and may be of value in biological and clinical investigations of cervical carcinogenesis.     

Quantum dot-based protein micro- and nanoarrays for detection of prostate cancer biomarkers

In this article, we demonstrate the fabrication and detection of cancer protein biochips consisting of micro- and nanoarrays whereby pegylated quantum dots (QDs) conjugated to antibodies (Abs) of prostate specific antigens (PSA) were used for the detection of clinical biomarkers such as PSA. BSA which acts as an efficient blocking layer in microarrays, tends to show an interaction with QDs. In view of this fact, we investigated two series of samples which were fabricated in the presence and absence of BSA blocking layer. Variation in the incubation time required for the antigen-antibody interaction to take place, different proteins as controls and the effect of bare QDs on these microarrays, were the three main parameters which were studied in these two series. Samples fabricated in the absence of BSA blocking layer exhibited an extremely high specificity in the detection of cancer proteins and were also marked by negligible nonspecific binding effects of QDs, in stark contrast to the samples fabricated using BSA as a blocking layer. Fabrication of nanoarrays of QD-conjugated PSA Abs having a spot size of nearly 900 nm has also been demonstrated. Thus, we show the potential offered by QDs in in vitro analysis of cancer biomarker imaging.     

An ELISA based on a truncated soluble ORF2 protein for the detection of PCV2 antibodies in domestic pigs

Postweaning multisystemie wasting syndrome (PMWS) is an important swine disease that is closely associated with porcine circovirus type 2 (PCV2). The capsid protein (Cap protein) is a major structural protein that has at least three immunoreactive regions, and it can be a suitable candidate antigen for detecting the specific antibodies of a PCV2 infection. In the present study, an indirect enzyme-linked immunosorbent assay (TcELISA)based on a truncated soluble Cap protein produced in Escherichia coli (E.coli) was established and validated for the diagnostic PCV2 antibodies in swine. The TcELISA was validated by comparison with an indirect immunofluorescence assay (IIFA). The diagnostic sensitivity (DSN), specificity (DSP), and accuracy of the TcELISA were 88.6%, 90.7% and 89.4%, respectively. The agreement rate was 89.38% between results obtained with TcELISA and IIFA on 113 field sera. A cross-reactivity assay showed that the method was PCV2-specific by comparison with other sera of viral disease. Therefore ,the TcELISA will be helpful for the development of a reliable serology diagnostic test for large scale detection of PCV2 antibodies and for the evaluation of vaccine against PCV2 in swine.     

Electrochemical biosensor based on base-stacking-dependent DNA hybridization assay for protein detection

An antibody-based electrochemical biosensing platform has been developed and used for the detection of protein. In the presence of the target, an antibody pair binds to the protein simultaneously, which causes two oligo-DNAs conjugated with the antibody pair to hybridize to each other and become a big “stem–loop” structure. Subsequently, the longer oligo-DNA of the stem, with a methylene blue (MB) label at the terminal, hybridizes stably with capture DNA owing to the enhancement of base stacking. The strong redox current signal of MB is used for protein quantification. Using α-fetoprotein (AFP) as a model, the proposed method could detect AFP at a concentration as low as 2 pg ml⁻¹ with a dynamic range of 4 orders of magnitude, which approaches traditional assays such as enzyme-linked immunosorbent assay.     

Evaluation of homo- and hetero-functionally activated glass surfaces for optimized antibody arrays

Antibody arrays hold great promise for biomedical applications, but they are typically manufactured using chemically functionalized surfaces that still require optimization. Here, we describe novel hetero-functionally activated glass surfaces favoring oriented antibody binding for improved performance in protein microarray applications. Antibody arrays manufactured in our facility using the functionalization chemistries described here proved to be reproducible and stable and also showed good signal intensities. As a proof-of-principle of the glass surface functionalization protocols described in this article, we built antibody-based arrays functionalized with different chemistries that enabled the simultaneous detection of 71 human leukocyte membrane differentiation antigens commonly found in peripheral blood mononuclear cells. Such detection is specific and semi-quantitative and can be performed in a single assay under native conditions. In summary, the protocol described here, based on the use of antibody array technology, enabled the concurrent detection of a set of membrane proteins under native conditions in a specific, selective, and semi-quantitative manner and in a single assay.     

Blocking and detection chemistries affect antibody performance on reverse phase protein arrays

Antibody specificity is critical for RP protein arrays (RPA). The effects of blocking and detection chemistries on antibody specificity were evaluated for Western blots and RPA. Blocking buffers significantly affected nonspecific banding on Western blots, with corresponding effects on arrays. Tyramide signal amplification (TSA) increased both specific and nonspecific signals on Westerns and arrays, masking the expected gradations in signal intensity. These results suggest that consistent blocking and detection conditions should be used for antibody validation and subsequent RPA experiments.     

Development of multiplexed protein profiling and detection using near infrared detection of reverse-phase protein microarrays

Protein microarrays have been recently employed for signal pathway profiling and high-throughput protein expression analysis. Reversephase arrays, where the array consists of immobilized analytes and lysates has especially shown promise in low abundance analyte detection and signal pathway profiling using phospho-specific antibodies. A limitation to current reverse phase array methodology is the inability to multiplex proteomic-based endpoints as each array can only report one analyte endpoint. In this study, we report on the use of a dual dye based approach that can effectively double the number of endpoints observed per array allowing, for example, both phosphospecific and total protein levels to be measured and analyzed at once. The method utilizes antibody bound dyes that emit in the infrared spectral region as a means of sensitive and specific detection.     

Identification of Mycoplasmas using a fluorophore-free microarray and infrared chemical imaging (IRCI)

A novel application of mid-infrared chemical imaging (IRCI) for the fluorophore-free detection and identification of mycoplasma species is reported for the first time. The PCR-amplified biotinylated targets hybridized to microarray probes were treated with streptavidin-gold nanoparticles followed by silver enhancement. This modification has the potential to expand the implementation of DNA microarray techniques in laboratories involved in the detection of cell substrates, other biological products, and clinical materials for the presence of mycoplasmas.