Serodiagnosis of infectious diseases with antigen microarrays

AIMS: To generate protein microarrays by printing microbial antigens on slides to enable the simultaneous determination in human sera of antibodies directed against Toxoplasma gondii, rubella virus, cytomegalovirus and herpes simplex virus (HSV) types 1 and 2. METHODS AND RESULTS: Antigens were printed on activated glass slides using high-speed robotics. The slides were incubated with serum samples and subsequently with fluorescently labelled secondary antibodies. Human IgG and IgM bound to the printed antigens were detected using confocal scanning microscopy and quantified with internal calibration curves. The microarray assay could detect as little as 0.5 pg of both IgG and IgM bound onto the glass surface. Precision profiles ranged from 1.7 to 18.5% for all the antigens. Microarrays and commercial ELISAs were utilized to detect serum antibodies against the ToRCH antigens in a panel of characterized human sera. Overall >80% concordance was obtained between microarray and ELISA kits in the classification of sera. CONCLUSIONS: These results indicate that the microarray is a suitable assay format for the serodiagnosis of infectious diseases. SIGNIFICANCE AND IMPACT OF STUDY: Antigen microarrays can be optimized for clinical use, their performance is equivalent to ELISA but they offer significant advantages in throughput, convenience and cost.     

Evaluation of surface chemistries for antibody microarrays

Antibody microarrays are an emerging technology that promises to be a powerful tool for the detection of disease biomarkers. The current technology for protein microarrays has been derived primarily from DNA microarrays and is not fully characterized for use with proteins. For example, there are a myriad of surface chemistries that are commercially available for antibody microarrays, but there are no rigorous studies that compare these different surfaces. Therefore, we have used a sandwich enzyme-linked immunosorbent assay (ELISA) microarray platform to analyze 17 different commercially available slide types. Full standard curves were generated for 23 different assays. We found that this approach provides a rigorous and quantitative system for comparing the different slide types based on spot size and morphology, slide noise, spot background, lower limit of detection, and reproducibility. These studies demonstrate that the properties of the slide surface affect the activity of immobilized antibodies and the quality of data produced. Although many slide types produce useful data, glass slides coated with aldehyde silane, poly-l-lysine, or aminosilane (with or without activation with a crosslinker) consistently produce superior results in the sandwich ELISA microarray analyses we performed.     

Immobilization strategies for single-chain antibody microarrays

Sandwich ELISA microarrays have great potential for validating disease biomarkers. Each ELISA relies on robust-affinity reagents that retain activity when immobilized on a solid surface or when labeled for detection. Single-chain antibodies (scFv) are affinity reagents that have greater potential for high-throughput production than traditional IgG. Unfortunately, scFv are typically less active than IgG following immobilization on a solid surface and not always suitable for use in sandwich ELISAs. We therefore investigated different immobilization strategies and scFv constructs to determine a more robust strategy for using scFv as ELISA reagents. Two promising strategies emerged from these studies: (i) the precapture of epitope-tagged scFv using an antiepitope antibody and (ii) the direct printing of a thioredoxin (TRX)/scFv fusion protein on glass slides. Both strategies improved the stability of immobilized scFv and increased the sensitivity of the scFv ELISA microarray assays, although the antiepitope precapture method introduced a risk of reagent transfer. Using the direct printing method, we show that scFv against prostate-specific antigen (PSA) are highly specific when tested against 21 different IgG-based assays. In addition, the scFv microarray PSA assay gave comparable quantitative results (R(2) = 0.95) to a commercial 96-well ELISA when tested using human serum samples. In addition, we find that TRX-scFv fusions against epidermal growth factor and toxin X have good LOD. Overall, these results suggest that minor modifications of the scFv construct are sufficient to produce reagents that are suitable for use in multiplex assay systems.     

Antibody colocalization microarray: a scalable technology for multiplex protein analysis in complex samples

DNA microarrays were rapidly scaled up from 256 to 6.5 million targets, and although antibody microarrays were proposed earlier, sensitive multiplex sandwich assays have only been scaled up to a few tens of targets. Cross-reactivity, arising because detection antibodies are mixed, is a known weakness of multiplex sandwich assays that is mitigated by lengthy optimization. Here, we introduce (1) vulnerability as a metric for assays. The vulnerability of multiplex sandwich assays to cross-reactivity increases quadratically with the number of targets, and together with experimental results, substantiates that scaling up of multiplex sandwich assays is unfeasible. We propose (2) a novel concept for multiplexing without mixing named antibody colocalization microarray (ACM). In ACMs, both capture and detection antibodies are physically colocalized by spotting to the same two-dimensional coordinate. Following spotting of the capture antibodies, the chip is removed from the arrayer, incubated with the sample, placed back onto the arrayer and then spotted with the detection antibodies. ACMs with up to 50 targets were produced, along with a binding curve for each protein. The ACM was validated by comparing it to ELISA and to a small-scale, conventional multiplex sandwich assay (MSA). Using ACMs, proteins in the serum of breast cancer patients and healthy controls were quantified, and six candidate biomarkers identified. Our results indicate that ACMs are sensitive, robust, and scalable.     

Aptamers as detection molecules on reverse phase protein microarrays for the analysis of cell lysates

This study presents and discusses the application of Cy3‐labeled aptamers (where Cy3 is indocarbocyanine) directed against the his‐tag (where his is histidine) for the detection of his‐tagged proteins on microarrays in a so‐called reverse phase assay. These types of assays are widely used tools in protein microarray technology. Up to now antibodies are usually applied as detection molecules. Here, two different spotting techniques, contact and noncontact spotting, as well as different types of slides, aldehyde‐modified glass slides and nitrocellulose membrane coated slides, were examined and compared. Through this study, we validated the importance of a high protein‐binding capacity of the microarray, and the labeling position of the fluorophore within the aptamer. Purified his‐tagged PFEI (Pseudomonas fluorescence esterase I) was used as a model system. Concentrations of PFEI‐his as low as 30 nM were detectable. These results demonstrate the applicability of aptamers as stable detection molecules in protein assays. Additionally, the reverse phase assay was found to be suitable for the detection of PFEI‐his in cell lysates. This might be of further interest in monitoring of protein production and purification processes.     

Protective efficacy of IgM monoclonal antibodies in experimental group B streptococcal infection is a function of antibody avidity

We have produced and characterized six mAb directed against group B streptococci (GBS). All antibodies are IgM. We have previously shown that some of these antibodies are highly protective in the treatment of experimental infections in neonatal rats, whereas others do not appear to have any protective efficacy. Using an ELISA, we demonstrate the specificity of both protective and nonprotective antibodies. Two antibodies, binding different epitopes, are directed against antigenic structures present on all GBS; two are specific for type III carbohydrate determinants; one binds to a protein Ag present on all type I and II GBS; and one appears to bind to type Ia GBS only. Quantitative absorption assays provide evidence that the difference between protective antibodies and nonprotective antibodies is the avidity that the antibody demonstrates for the epitope recognized on the surface of the bacteria; 10 to 15 times as much protective antibody binds to GBS as does nonprotective antibody. Direct binding experiments with radiolabeled antibody confirm this conclusion.     

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.     

Quantitative protein profiling using antibody arrays

Traditional approaches to microarrays rely on direct binding assays where the extent of hybridisation and the signal detected are a measure of the analyte concentration in the experimental sample. This approach, directly imported from the nucleic acid field, may fail if applied to antibody-antigen interactions due to the shortage of characterised antibodies, the significant heterogeneity of antibody affinities, their dependence on the extent of protein modification during labelling and the inherent antibody cross-reactivity. These problems can potentially limit the multiplexing capabilities of protein affinity assays and in many cases rule out quantitative protein profiling using antibody microarrays. A number of approaches aimed at achieving quantitative protein profiling in a multiplex format have been reported recently. Of those reported, the three most promising routes include signal amplification, multicolour detection and competitive displacement approaches to multiplex affinity assays. One in particular, competitive displacement, also overcomes the problems associated with quantitation of affinity interactions and provides the most generic approach to highly parallel affinity assays, including antibody arrays.     

Development of miniaturized immunoassay: Influence of surface chemistry and comparison with enzyme-linked immunosorbent assay and Western blot

Protein microarray technology provides a useful approach for the simultaneous serodetection of various antibodies in low sample volumes. To implement functional protein microarrays, appropriate surface chemistry must be designed so that both the protein structure and the biological activity can be retained. In the current study, two surface chemistries for protein microarrays and immunofluorescent assays were developed. Glass slides were functionalized with N-hydroxysuccinimide (NHS) ester via a monofunctional silane or maleic anhydride-alt-methyl vinyl ether (MAMVE) copolymer to allow covalent grafting of histone proteins. Analytical performance of these microarrays was then evaluated for the detection of anti-histone autoantibodies present in the sera of patients suffering from a systemic autoimmune disease, namely systemic lupus erythematosus (SLE), and the results were compared with those of the classical enzyme-linked immunosorbent assay (ELISA) and Western blot. The detection limit of our MAMVE copolymer microarrays was 50-fold lower than that of the classical ELISA. Furthermore, 100-fold less volume of biological samples was required with these miniaturized immunoassays.     

Normal human sera contain antibodies directed at Fab of IgA

Serum samples from 26 normal volunteers were evaluated by isotype-specific ELISA for the presence of IgG and IgM antibodies directed at IgA. Although there were wide variations in antibody levels, anti-IgA antibodies of both isotypes were found in all individuals tested. The anti-IgA activity was detected against a variety of polymeric and monomeric IgA1 and IgA2 myeloma proteins containing both kappa and lambda light chains. By using Fab and Fc fragments generated by incubation of an IgA1 myeloma protein with IgA1 protease, it was shown that the anti-IgA activity was specific for the Fab portion of the IgA molecule. It was also demonstrated that the serum of two individuals contained both IgG and IgM activity directed at autologous affinity-purified IgA. IgM antibody levels against both whole IgA and Fab of IgA were significantly higher than IgG antibody levels. Cells producing anti-IgA antibodies of both isotypes were detected in lipopolysaccharide-stimulated human spleen.     

A simple, rapid, and sensitive integrated protein microarray for simultaneous detection of multiple antigens and antibodies of five human hepatitis viruses (HBV, HCV, HDV, HEV, and HGV)

Protein microarrays for parallel detection of multiple viral antigens and antibodies have not yet been described in the field of human hepatitis virus infections. Here, we describe a simple, rapid and sensitive integrated protein microarray with three different reaction models. The integrated protein microarray could simultaneously determine in human sera two viral antigens (HBsAg, HBeAg) and seven viral antibodies (HBsAb, HBcAb, HBeAb, HCVAb, HDVAb, HEVAb, HGVAb) of human hepatitis viruses within 20 min. The results of the protein microarray were assessed directly by the naked eye but can also be analyzed by a quantitative detector. The detection limit of this protein microarray was 0.1 ng/ml for HBsAg. Overall, >85% concordance was observed between the integrated protein microarrays and an enzyme-linked immunosorbent assay for above hepatitis viral antigen and antibody detections in human sera. This integrated protein microarray can be easily optimized for clinical use and epidemiological screening for multiple hepatitis virus infections.     

Development of immunohistochemistry and enzyme-linked immunosorbent assays for the detection of circulating antibodies against Enteromyxum scophthalmi (Myxozoa) in turbot (Scophthalmus maximus L.)

Immunohistochemistry and enzyme-linked immunosorbent assays were developed for the detection of specific antibodies against the myxosporean parasite Enteromyxum scophthalmi in turbot (Scophthalmus maximus L.). Fish which had survived a previous epizootic were exposed to the parasite by cohabitation with infected animals, and 83 days later the plasma was tested for the presence of antibodies. Plasma of non-exposed fish was used as negative control. Immunohistochemistry (IHC) using rabbit anti-turbot IgM antibody was first used to detect these antibodies, and to study to which parasite structures they were directed against. Also, an antibody-ELISA using whole cell lysates of the parasite as antigen, and a monoclonal antibody anti-turbot IgM, was developed. All the exposed fish were found to have specific antibodies against the parasite, and none of them developed signs of disease or died during the experiment. Primary cells were the main parasite stage immunolabelled, and the staining was distinctly located on the cytoplasm and the cytoplasmic membrane. IHC was more sensitive than ELISA, as the endpoint was two to four fold higher with the former technique. Although there was great individual variation, the antibody titres found can be considered high, reaching up to 1:32,000 with ELISA and 1:64,000 with IHC. The results suggest that turbot showing acquired immunity against E. scophthalmi, could develop resistance against new infections.     

A novel antibody microarray format using non-covalent antibody immobilization with chemiluminescent detection

To date, protein and antibody microarrays have been used in reverse-phase and sandwich-based methods in order to detect known proteins such as biomarkers in samples. Our group developed "libraries" of antibodies against unknown proteins, referred to as mKIAA proteins, and we attempted to discover candidate novel biomarkers by protein expression profiling.To profile mKIAA protein expression using these antibodies, we established an antibody microarray system using chemiluminescent detection. A number of techniques for protein-antibody microarrays have been reported; however, no entirely suitable protocol for crude protein samples has been established. To address this issue, we immobilized purified antibodies on hydrophilic surface polymer slides (Maxisorp, Nunc). Although our system is based on the direct labeling of crude protein samples, we achieved sufficient sensitivity (detection limit: 50 pg mL(-1)) and low backgrounds. This sensitivity is on a level with the sandwich immunoassay-based antibody array system. Using our protocol, we developed an antibody microarray spotted with 960 anti-mKIAA antibodies (total: 3888 spots for quadruplicate assessments), and we carried out protein expression profiling of mKIAA proteins. In this study, we generated an expression profile of 960 mKIAA proteins and compared the present results with those obtained via cDNA microarray.     

An antibody-based microarray assay for the simultaneous detection of aflatoxin B<Subscript>1</Subscript> and fumonisin B<Subscript>1</Subscript>

Advances in microsystem technology have enabled protein and nucleic acid-based microarrays to be used in various applications, including the study of diseases, drug discovery, genetic screening, and clinical and food diagnostics. Analytical methods for the detection of mycotoxins, however, remain largely based on thin layer chromatography (TLC), high pressure liquid chromatography (HPLC), or enzyme-linked Immunosorbent assay (ELISA) . The aim of our work, therefore, was to transfer an immunological assay from microtitrr plates into microarray format, in order to develop a multiparametric, rapid, sensitive and inexpensive method for the detection of mycotoxins for use in food safety applications. Microarray technology enables the fast and parallel analysis of a multitude of biologically relevant parameters. Not only nucleic acid-based tests but also peptide, antigen, and antibody assays, using different formats of microarrays, have evolved within the last decade. Antibody-based microarrays provide a powerful tool that can be used to generate rapid and detailed expression profiles of a defined set of analytes in complex samples and are potentially useful for generating rapid immunological assays of food contaminants. In this paper, we report a feasibility study of the application of antibody microarrays for the simultaneous (or independent) detection of two common mycotoxins, Aflatoxin B₁ and Fumonisin B₁. We present the development of microarray detection of aflatoxin B1 and fumonisin B1 in standard solutions with detection limits of 3 ng/ml of AFB1 and 43 ng/ml for FB1, and have developed a competitive immunoassay in microarray format for simultaneous analyses. The quality of the microarray data is comparable to data generated by microplate-based immunoassay (ELISA), but further investigations are needed in order to characterise our method more fully. We hope that these preliminary results might suggest that further research is warranted in order to develop hapten microarrays for the immunochemical simultaneous analysis of mycotoxins, as well as for other small molecules (e.g. bacterial toxins or biological warfare agents).     

Measuring proteins on microarrays

A prerequisite of proteomics is the ability to quantify many selected proteins simultaneously. Immunoassays on microarrays are an attractive solution, as equipment and antibodies are available and assays are simple, scalable and reproducible. Recently, considerable progress has been made in this area as evidenced by increased sensitivity and coverage (degree of multiplexing). Routine use of antibody microarrays in research and diagnostic settings will require increased availability of binding reagents, novel signal amplification procedures, inexpensive and robust platforms for microarray production and detection, and turn-key systems for running high-throughput assays.     

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.     

Cloning and expression of partial Japanese flounder (Paralichthys olivaceus) IgD

The cDNA sequence of the Japanese flounder (Paralychthys olivaceus) IgD has been previously reported (GenBank accession no. AB052658) and this was followed by the detection of IgD mRNA expression in some flounder organ tissues. However, it has not been determined whether the flounder IgD gene is virtually expressed into IgD protein. To characterize the flounder immunoglobulins utilized in elucidating the mechanism, evolution and diversity of the flounder immune system, antibodies specific to IgD and IgM were necessary. In the present study, partial flounder recombinant IgD (rIgD), IgM (rIgM) and the conserved regions of IgD and IgM (rCIg) were produced by cloning the cDNA sequence using isotype specific primers which were designed to produce unique fragments of IgD and IgM specific amino acid sequences. The production of recombinant Igs was ascertained by SDS-gel electrophoresis and immunoblot analysis using anti-T7 d Taq antibody. The produced recombinant Igs were purified using affinity columns, and used as immunogens. Antibodies specific to the isotype of flounder Igs were generated by immunizing rabbits with rfIgs and the antibodies produced were identified by enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Specificities of the generated antibodies were evaluated by testing cross-reactivity between recombinant IgM and IgD. By ELISA, rabbit antibodies against the rfIgD fragment (anti-rfIgD) failed to recognize any kind of flounder serum Igs, whereas respective antibodies against rfCIg (anti-rfCIg) and rfIgM fragments (anti-rfIgM) reacted with serum Igs. Likewise, in immunoblot assays, though anti-rfIgD did not, both anti-rfCIg and anti-rfIgM bound with the ~85 kd flounder IgM heavy chain. By flow cytometry analysis, anti-rfCIg, anti-rfIgD and anti-rfIgM reacted with 6%, 3% and 6.5% of cells, respectively, suggesting that flounder IgD is not secreted in serum but expressed on flounder B-like cell surfaces as in mammals. Antibodies produced against recombinant flounder Igs could be used to develop sandwich assay systems for detecting flounder Igs and for further investigating the flounder immune system.     

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.     

Protein A-based antibody immobilization onto polymeric microdevices for enhanced sensitivity of enzyme-linked immunosorbent assay

Highly efficient antibody immobilization is extremely crucial for the development of high-performance polymeric microdevices for enzyme-linked immunosorbent assay (ELISA). In this article, a site-selective tyrosinase (TR)-catalyzed protein A strategy for antibody immobilization was developed to enhance the sensitivity of ELISA in poly-(methyl methacrylate) (PMMA) microchannels for interferon-gamma (IFN-gamma) assay. To effectively immobilize the target antibodies, oxygen plasma was first used to activate the inert PMMA. This is followed by poly(ethyleneimine) (PEI) coating, an amine-containing functional polymer. For comparison, protein A was also immobilized through the commonly used amine-glutaraldehyde (GA) chemistry. Oxygen plasma treatment effectively increased the amount of PEI attachment and subsequent binding efficiency of the primary antibody. The antibody immobilized via TR-catalyzed protein A was able to provide much better specific antigen capture efficiency than GA chemistry due to the optimal spacing and orientation. Consequently, by using this new method, the detection signal and the signal-to-noise ratio of the ELISA immunoassay in microdevices were all significantly improved. In comparison to the standard assay carried out in the 96-well microtiter plate, the treated microchannels exhibited a broader detection range and a shorter detection time. And the detection limit was also decreased to 20 pg/mL, much lower than that obtained in other microdevices.     

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.