Optimization and clinical validation of a pathogen detection microarray

DNA microarrays used as 'genomic sensors' have great potential in clinical diagnostics. Biases inherent in random PCR-amplification, cross-hybridization effects, and inadequate microarray analysis, however, limit detection sensitivity and specificity. Here, we have studied the relationships between viral amplification efficiency, hybridization signal, and target-probe annealing specificity using a customized microarray platform. Novel features of this platform include the development of a robust algorithm that accurately predicts PCR bias during DNA amplification and can be used to improve PCR primer design, as well as a powerful statistical concept for inferring pathogen identity from probe recognition signatures. Compared to real-time PCR, the microarray platform identified pathogens with 94% accuracy (76% sensitivity and 100% specificity) in a panel of 36 patient specimens. Our findings show that microarrays can be used for the robust and accurate diagnosis of pathogens, and further substantiate the use of microarray technology in clinical diagnostics.     

Methods and applications of antibody microarrays in cancer research

Antibody microarrays have great potential for significant value in biological research. Cancer research in particular could benefit from the unique experimental capabilities of this technology. This article examines the current state of antibody microarray technological developments and assay formats, along with a review of the demonstrated applications to cancer research. Work is ongoing in the refinement of various aspects of the protocols and the development of robust methods for routine use. Antibody microarray experimental formats can be broadly categorized into two classes: (1) direct labeling experiments, and (2) dual antibody sandwich assays. In the direct labeling method, the covalent labeling of all proteins in a complex mixture provides a means for detecting bound proteins after incubation on an antibody microarray. If proteins are labeled with a tag, such as biotin, the signal from bound proteins can be amplified. In the sandwich assay, proteins captured on an antibody microarray are detected by a cocktail of detection antibodies, each antibody matched to one of the spotted antibodies. Each format has distinct advantages and disadvantages. Several applications of antibody arrays to cancer research have been reported, including the analysis of proteins in blood serum, resected frozen tumors, cell lines, and on membranes of blood cells. These demonstrations clearly show the utility of antibody microarrays for cancer research and signal the imminent expansion of this platform to many areas of biological research.     

Evaluation of microarray surfaces and arraying parameters for autoantibody profiling

Autoantigen microarrays are being used increasingly to study autoimmunity. Significant variation has been observed when comparing microarray surfaces, printing methods, and probing conditions. In the present study, 24 surfaces and several arraying parameters were analyzed using >500 feature autoantigen microarrays printed with quill pins. A small subset of slides, including FAST, PATH, and SuperEpoxy2, performed well while maintaining the sensitivity and specificity of autoantigen microarrays previously demonstrated by our laboratory. By optimizing the major variables in our autoantigen microarray platform, subtle differences in serum samples can be identified that will shed light on disease pathogenesis.     

Characterization of primate antibody responses to administered murine monoclonal immunoglobulin

Murine monoclonal antibodies (MAb) are currently being assessed for their utility as tools in cancer management. Anti-murine immunoglobulin responses have been observed in many patients receiving monoclonal antibody treatment. In this study, we evaluated the response of primates to the administration of a monoclonal antibody. MAb B6.2, an antibody generated against a human breast tumor metastasis, was used as a prototype MAb. Baboons were inoculated with MAb B6.2 whole IgG, Fab', or F(ab')2 fragments. Blood samples were drawn at periodic intervals post-inoculation and the sera collected. Anti-murine immunoglobulin responses were detected using a solid-phase radioimmunoassay. The specificity of the antibody response was analyzed to determine if the response was directed against the species of origin of the MAb (species specificity), against the class of the MAb (isotype specificity), or against the hypervariable region of the MAb (idiotype specificity). We found that primates develop a humoral immune response against all three forms of the monoclonal antibody [IgG, Fab', and F(ab')2]. Furthermore, this antibody response demonstrated a high degree of specificity for the antigen binding site suggesting an idiotypic specificity. Using a competitive radioimmunoassay, the antibody response was found to interfere with antigen binding of MAb B6.2. These studies suggest that monoclonal antibody treatment can generate an anti-idiotypic response which may alter the efficacy of this mode of treatment.     

Evaluation of a high-affinity QCM immunosensor using antibody fragmentation and 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer

This study evaluated construction of a highly affinitive quartz crystal microbalance (QCM) immunosensor using anti-C-reactive protein (CRP) antibody and its fragments for CRP detection. Three types of antibody were immobilized on the surface of a QCM via covalent-bounding. Then affinity was evaluated through antigen-antibody binding between CRP and its antibody. Affinity between antigen-antibody was shown to be highest when anti-CRP F(ab')2-IgG antibody (70 microg/mL) was immobilized on the QCM. In case of anti-CRP F(ab')2-IgG antibody, affinity which was attributable to antigen-antibody binding was almost twice that of anti-CRP IgG antibody, which is used conventionally for QCM immunosensors. In addition, when it was treated with 2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate, so-called MPC polymer, highly affinitive and selective immunosensing for CRP was achieved without non-specific binding from plasma proteins in human serum. When anti-CRP F(ab')2-IgG antibody was immobilized on the QCM, the detection limit and the linearity of CRP calibration curve were achieved at concentrations from 0.001 to 100 microg/dL even during investigation in serum samples. Experimental results verified the successful construction of a highly affinitive and selective QCM-immunosensor which was modified with anti-CRP F(ab')2-IgG antibody and MPC polymer.     

Immunological properties of a radioiodinated monoclonal antibody and its F(ab')2mu fragments directed against the polymorphic epithelial mucin of human breast cancer.

The purification of the IgM monoclonal antibody 436 against a breast tumor antigen from mouse ascitic fluid is reported. The purified immunoglobulin was radioiodinated and the resulting product assessed for its binding capacity and binding specificity. Purified IgM-436 served for F(ab')2 mu preparation which was tested for its antigen binding capacity. Radioiodinated IgM-436 and its F(ab')2 mu retained their immunological activity which was never lower than those of the corresponding cold products.     

Development and validation of sandwich ELISA microarrays with minimal assay interference

Sandwich enzyme-linked immunosorbent assay (ELISA) microarrays are emerging as a strong candidate platform for multiplex biomarker analysis because of the ELISA's ability to quantitatively measure rare proteins in complex biological fluids. Advantages of this platform are high-throughput potential, assay sensitivity and stringency, and the similarity to the standard ELISA test, which facilitates assay transfer from a research setting to a clinical laboratory. However, a major concern with the multiplexing of ELISAs is maintaining high assay specificity. In this study, we systematically determine the amount of assay interference and noise contributed by individual components of a multiplexed 24-assay system. We find that nonspecific reagent cross-reactivity problems are relatively rare. We did identify the presence of contaminant antigens in a "purified antigen". We tested the validated ELISA microarray chip using paired serum samples that had been collected from four women at a 6-month interval. This analysis demonstrated that protein levels typically vary much more between individuals than within an individual over time, a result which suggests that longitudinal studies may be useful in controlling for biomarker variability across a population. Overall, this research demonstrates the importance of a stringent screening protocol and the value of optimizing the antibody and antigen concentrations when designing chips for ELISA microarrays.     

Antibody microarrays: current status and key technological advances

Antibody-based microarrays are among the novel classes of rapidly evolving proteomic technologies that holds great promise in biomedicine. Miniaturized microarrays (< 1 cm2) can be printed with thousands of individual antibodies carrying the desired specificities, and with biological sample (e.g., an entire proteome) added, virtually any specifically bound analytes can be detected. While consuming only minute amounts (< microL scale) of reagents, ultra- sensitive assays (zeptomol range) can readily be performed in a highly multiplexed manner. The microarray patterns generated can then be transformed into proteomic maps, or detailed molecular fingerprints, revealing the composition of the proteome. Thus, protein expression profiling and global proteome analysis using this tool will offer new opportunities for drug target and biomarker discovery, disease diagnostics, and insights into disease biology. Adopting the antibody microarray technology platform, several biomedical applications, ranging from focused assays to proteome-scale analysis will be rapidly emerging in the coming years. This review will discuss the current status of the antibody microarray technology focusing on recent technological advances and key issues in the process of evolving the methodology into a high-performing proteomic research tool.     

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.     

Nanosphere lithography-based platform for developing rapid and high sensitivity microarray systems

A novel gold nanoarray (NA)-based platform was developed for microarray applications. This novel approach is based upon the principle of nanosphere lithography and can be used for one-step antibody immobilization. The developed platform was checked by functionalizing with cysteine followed by capturing biotinylated antibody and detecting it with dye-conjugated steptravidin. An immunoassay was performed with spiked samples containing human fetuin A antigen. The minimum limits of detection (LOD) of human fetuin A for NA-based and conventional microarray platforms were 50 pg/mL and 50 ng/mL, respectively. The developed approach was highly reproducible and unlike conventional microarray approaches the use of a spotting system was omitted because immobilization was controlled and directed on the predefined arrays. This approach could be an ideal alternative for developing microarrays. And, the ease of the strategy also allows the high throughput production of the microarrays.     

Optimized normalization for antibody microarrays and application to serum-protein profiling

The measurements of coordinated patterns of protein abundance using antibody microarrays could be used to gain insight into disease biology and to probe the use of combinations of proteins for disease classification. The correct use and interpretation of antibody microarray data requires proper normalization of the data, which has not yet been systematically studied. Therefore we undertook a study to determine the optimal normalization of data from antibody microarray profiling of proteins in human serum specimens. Forty-three serum samples collected from patients with pancreatic cancer and from control subjects were probed in triplicate on microarrays containing 48 different antibodies, using a direct labeling, two-color comparative fluorescence detection format. Seven different normalization methods representing major classes of normalization for antibody microarray data were compared by their effects on reproducibility, accuracy, and trends in the data set. Normalization with ELISA-determined concentrations of IgM resulted in the most accurate, reproducible, and reliable data. The other normalization methods were deficient in at least one of the criteria. Multiparametric classification of the samples based on the combined measurement of seven of the proteins demonstrated the potential for increased classification accuracy compared with the use of individual measurements. This study establishes reliable normalization for antibody microarray data, criteria for assessing normalization performance, and the capability of antibody microarrays for serum-protein profiling and multiparametric sample classification.     

Carbohydrate microarrays for the recognition of cross-reactive molecular markers of microbes and host cells

We describe here the development of a carbohydrate-based microarray to extend the scope of biomedical research on carbohydrate-mediated molecular recognition and anti-infection responses. We have demonstrated that microbial polysaccharides can be immobilized on a surface-modified glass slide without chemical conjugation. With this procedure, a large repertoire of microbial antigens (approximately 20,000 spots) can be patterned on a single micro-glass slide, reaching the capacity to include most common pathogens. Glycoconjugates of different structural characteristics are shown here to be applicable for microarray fabrication, extending the repertoires of diversity and complexity of carbohydrate microarrays. The printed microarrays can be air-dried and stably stored at room temperature for long periods of time. In addition, the system is highly sensitive, allowing simultaneous detection of a broad spectrum of antibody specificities with as little as a few microliters of serum specimen. Finally, the potential of carbohydrate microarrays is demonstrated by the discovery of previously undescribed cellular markers, Dex-Ids.     

Novel 3-dimensional dendrimer platform for glycolipid microarray

Glycolipids are important biological molecules that modulate cellular recognitions and pathogen adhesions. In this paper, we report a sensitive glycolipid microarray for non-covalently immobilizing glycolipids on a microarray substrate and we perform a set of immunoassays to explore glycolipid-protein interactions. This substrate utilizes a three-dimensional hydrazide-functionalized dendrimer monolayer attached onto a microscopic glass surface, which possesses the characteristics to adsorb glycoliplids non-covalently and facilitates multivalent attributes on the substrate surface. In the proof-of-concept experiments, gangliosides such as GM1, FucGM1, GM3, GD1b, GT1b, and GQ1b, and a lipoarabinomannan were tested on the substrate and interrogated with toxins and antibodies. The resulting glycolipid microarrays exhibited hypersensitivity and specificity for detection of glycolipid-protein interactions. In particular, a robust and specific binding of a pentameric cholera toxin B subunit to the GM1 glycolipid spotted on the array has demonstrated its superiority in sensitivity and specificity. In addition, this glycolipid microarray substrate was used to detect lipoarabinomannan in buffer within a limit-of-detection of 125 ng/mL. Furthermore, Mycobacterium tuberculosis (Mtb) Lipoarabinomannan was tested in human urine specimens on this platform, which can effectively identify urine samples either infected or not infected with Mtb. The results of this work suggest the possibility of using this glycolipid microarray platform to fabricate glycoconjugate microarrays, which includes free glycans and glycolipids and potential application in detection of pathogen and toxin.     

Design of recombinant antibody microarrays for serum protein profiling: targeting of complement proteins

Antibody-based microarrays is a novel technology with great promise for high-throughput proteomics. The process of designing high-performing arrays has, however, turned out to be challenging. Here, we have designed the next generation of a human recombinant scFv antibody microarray platform for protein expression profiling of nonfractionated biotinylated human plasma and serum proteomes. The setup, based on black polymer Maxisorb slides interfaced with a fluorescent-based read-out system, was found to provide specific, sensitive (subpicomolar (pM) range) and reproducible means for protein profiling. Further, a chip-to-chip normalization protocol critical for comparing data generated on different chips was devised. Finally, the microarray data were found to correlate well with clinical laboratory data obtained using conventional methods, as demonstrated for a set of medium abundant (micromolar (microM) to nanomolar (nM) range) protein analytes in serum and plasma samples derived from healthy and complement-deficient individuals.     

Development of a fluorescent and colorimetric detection methods-based protein microarray for serodiagnosis of TORCH infections

We developed a protein microarray methodology that has the ability of serodiagnosis of IgM antibodies directed against TORCH pathogens. Six chemical surface modifications were validated by a dimension atomic force microscope (AFM) and contact angle measurement, agarose modified surface of which offered an appropriate platform for detecting IgM antibody. Further, signal amplification sensitivities on agarose modified microarrays were detected by Cy3-labeled biotin-streptavidin and immunogold-based assays. The detection limits of IgM antibody on the microarrays were 0.48 and 0.24mug/ml, quantitatively equal to 0.25 and 12.5pg, respectively, on each spot as ascertained by the two assays. Satisfactory linear correlations between the signal intensity and the logarithm of the IgM concentration were obtained. Finally, 60 serum samples characterized by a commercial ELISA were evaluated by the protein microarray. There were good concordances between the results of the protein microarray and ELISA assay for sorting of the TORCH infected sera (95.0% by fluorescence-based assay and 96.7% by immunogold-based assay). Clearly, the potential application of this protein microarray format facilitates clinical detection of not only the antibodies directed against TORCH pathogens but also other autoimmune diseases.     

抗CD20嵌合抗体片段F(ab′)2突变体在大肠杆菌中的高效分泌表达

构建抗CD20嵌合抗体片段F(ab′)₂ 突变体 ,研究其在大肠杆菌中的高效表达及其表达产物的生物学活性。采用PCR法构建抗CD20嵌合抗体片段F(ab′)₂ 突变体 ,并用双脱氧终止法测定DNA序列 ;采用 19L发酵罐高密度发酵抗CD20嵌合抗体片段F(ab′)₂ 突变体 ,采用亲和色谱和分子筛色谱法纯化表达产物 ,并用SDS-PAGE和薄层激光扫描鉴定纯化产物 ;采用活细胞间接免疫荧光法测定纯化产物与靶细胞的结合活性 ;MTT法测定纯化产物对Raji细胞的生长抑制作用 ,并研究其作用机理。DNA序列测定结果表明 ,抗CD20嵌合抗体片段F(ab′)₂ 突变体已成功构建 ,表达可溶性产物的产量达 360mg L ,具有与Raji细胞 (CD20⁺)结合的活性 ,并抑制Raji细胞的生长 ,其作用机理为诱导Raji细胞凋亡。此突变体有望成为治疗非何杰金氏B细胞淋巴瘤的药物。     

Polyethylene glycol modification of the monoclonal antibody A7 enhances its tumor localization

The F(ab')2 fragment of murine monoclonal antibody A7 was covalently bonded to polyethylene glycol (PEG, molecular weight: 5000) and the conjugate was compared to the parent F(ab')2 fragment by in vitro and in vivo studies. PEG-conjugated antibody fragment retained its antigen-binding activity in a competitive radioimmunoassay. The conjugate had a longer half-life and showed increased accumulation in tumors. Although the tumor: blood ratio for parent F(ab')2 fragment was higher than that for the conjugate, it showed higher value than whole MAb A7. The tissue: blood ratios were kept low with the conjugate, indicating that the conjugate was uptaken to normal organ with lesser extent, as compared with parent F(ab')2 fragment. Our findings indicate that this PEG-conjugated F(ab')2 fragment could be a promising carrier for use in targeting cancer chemotherapy.     

Profiling of differential protein expression in angiogenin-induced HUVECs using antibody-arrayed ProteoChip

ProteoChip has been developed as a novel protein microarray technology. So far it has been applied in new lead screening and molecular diagnostics and we expect its role to grow in the field of biology. Here, we investigated the application of ProteoChip for the study of differential protein expression profiles in angiogenin-induced human umbilical vein endothelial cells (HUVECs). Antibody microarrays constructed by immobilizing 60 distinct antibodies against signal-transducing proteins on ProteoChip base plates were used to analyze the expression pattern of cell-signaling proteins in HUVECs treated with angiogenin. The antibody microarray approach showed that angiogenin induced the up- and down-regulation of several cellular regulators related with cell proliferation. Changes in the expression of signaling proteins determined by antibody microarray were validated by Western blot analysis. In this experiment, ten up-regulated proteins and six down-regulated proteins were identified and confirmed by immunoblot analysis. Taken together, these data suggest that antibody microarrays using ProteoChip technology can be a powerful tool for high-throughput analysis of proteomes in biological samples.     

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.     

Integral protein microarrays for the identification of lung cancer antigens in sera that induce a humoral immune response

The identification of biomarkers (both molecules and profiles) in patient sera offers enormous interest for the diagnosis of cancers. In this context, the detection of antibodies to tumor cell autologous antigens possesses great potential. The humoral immune response represents a form of biological amplification of signals that are otherwise weak because of very low concentrations of antigen, especially in the early stages of cancers. Herein we present the use of integral microarrays spotted with tumor-derived proteins to investigate the antibody repertoire in the sera of lung cancer patients and controls. The use of two-dimensional liquid chromatography allowed us to separate proteins from the lung adenocarcinoma cell line A549 into 1760 fractions, which were printed in duplicate, along with various controls, onto nitrocellulose coated slides. The sensitivity and specificity of the microarrays to detect singular antibodies in fluids were first validated through the recognition of fractions containing a lung marker antigen by antibody probing. Twenty fractions were initially selected as highly reactive against the anti-PGP9.5 antibody, and subsequent mass spectrometry analyses confirmed the identity of PGP9.5 protein in four of them. As a result, the importance of neighboring fractions in microarray detection was revealed due to the spreading of proteins during the separation process. Next, the microarrays were individually incubated with 14 serum samples from patients with lung cancer patients, 14 sera from colon cancer patients, and 14 control sera from normal subjects. The reactivity of the selected fractions was analyzed, and the level of immunoglobulin bound to each fraction by each serum sample was quantified. Eight of the 20 fractions offered p values < 0.01 and were recognized by an average of four reacting patients, whereas no serum from normal individuals was positive for those fractions. Protein microarrays from tumor-derived fractions hold the diagnostic potential of uncovering antigens that induce an immune response in patients with certain types of cancers.