Multiple biomarker approach for the diagnosis and therapy of rheumatoid arthritis.

The lack of specific clinical symptoms for patients in the early stage of rheumatoid arthritis (RA) has created strong interest in the laboratory diagnosis of RA. The main laboratory markers of RA, rheumatoid factor (RF) and anti-citrullinated protein antibodies (ACPAs), can be found in patients with other pathologies and in healthy donors. Even today, there is no single laboratory test that can diagnosis RA with high sensitivity and specificity. To improve the diagnosis and treatment of RA, alternative biomarkers, including 14-3-3η protein, connective tissue growth factor (CTGF), antibodies against PAD4, antibodies against BRAF, and anti-acetylated and anti-carbamylated protein antibodies have been studied extensively. The use of a multiple biomarker approach, the simultaneous measurement of a set of biomarkers, is an alternative strategy for the diagnosis of RA and for predicting the therapeutic effect of biological disease-modifying antirheumatic drugs (DMARDs). However, despite the large number of studies, only a few biomarker combinations have been validated and can be applied in clinical practice. In this article, results of studies focused on the multiple biomarker approach (both multiplex and combined single-analyte assays) to diagnose RA and to predict response to biological drug therapy are reviewed. Additionally, general factors limiting the use of multiplex analysis in RA diagnostics and therapy are discussed.

Glycan-specific antibodies as potential cancer biomarkers: a focus on microarray applications.

Glycosylation is one of the most common posttranslational modifications of proteins and lipids. In the case of tumors, cell transformation accompanied by aberrant glycosylation results in the expression of tumor-associated glycans that promote tumor invasion. As part of the innate immunity, anti-glycan antibodies recognize tumor-associated glycans, and these antibodies can be present in the bloodstream in the early stages of cancer. Recently, anti-glycan antibody profiles have been of interest in various cancer studies. Novel advantages in the field of analytical techniques have simplified the analysis of anti-glycan antibodies and made it easier to have more comprehensive knowledge about their functions. One of the robust approaches for studying anti-glycan antibodies engages in microarray technology. The analysis of glycan microarrays can provide more expanded information to simultaneously specify or suggest the role of antibodies to a wide variety of glycans in the progression of different diseases, therefore making it possible to identify new biomarkers for diagnosing cancer and/or the state of the disease. Thus, in this review, we discuss antibodies to various glycans, their application for diagnosing cancer and one of the most promising tools for the investigation of these molecules, microarrays.

Biomarkers of Community-Acquired Pneumonia: A Key to Disease Diagnosis and Management.

Community-acquired pneumonia (CAP) is a dangerous disease caused by a spectrum of bacterial and viral pathogens. The choice of specific therapy and the need for hospitalization or transfer to the intensive care unit are determined by the causative agent and disease severity. The microbiological analysis of sputum largely depends on the quality of the material obtained. The prediction of severity and the duration of therapy are determined individually, and existing prognostic scales are used generally. This review examines the possibilities of using specific serological biomarkers to detect the bacterial or viral aetiology of CAP and to assess disease severity. Particular emphasis is placed on the use of biomarker signatures and the discovery of biomarker candidates for a single multiplex analysis.

Allergen-specific IgE and IgG4 patterns among patients with different allergic diseases.

BACKGROUND: In addition to allergen-specific IgE (sIgE), allergen-specific IgG4 (sIgG4) antibodies are also involved in the immune response resulting from an allergen exposure. The aim of our study was to analyze sIgE and sIgG4 patterns in the most common allergic disorders: bronchial asthma, upper airway disorders and atopic dermatitis. METHODS: In this study a screening analysis of blood serum samples from 673 patients aged from 6 months to 17 years with different allergic entities was performed on microarrays. sIgE and sIgG4 levels to the most common allergens were estimated. RESULTS: sIgE response to most pollen allergens is more strongly associated with respiratory diseases than with atopic dermatitis, while sIgE responses to cat and dog dander are more strongly associated with bronchial asthma than with atopic dermatitis and upper airway disorders such as rhinosinusitis and allergic rhinitis. A lower prevalence of sIgG4 to pollen allergens in cases of atopic dermatitis is observed compared with that in cases of asthma and upper airway disorders. Analyzing all the allergic disorders, one can see that sIgG4 response to inhalant allergens is strongly associated with sensitization to the corresponding allergen. CONCLUSION: Allergen-specific IgE and IgG4 patterns that are relevant to concrete allergic diseases differ by sIgE and sIgG4 prevalences to defined allergens.

Patterns of sensitization to inhalant and food allergens among pediatric patients from the Moscow region (Russian Federation).

The immunological profiles of human specific IgE (sIgE) and specific IgG4 (sIgG4) vary by genetic predisposition, living conditions in different geographical locations and patient's age. The aim of our study was to analyze sIgE and sIgG4 patterns and their age-dependent changes in patients from the Moscow region. For identifying sIgE and sIgG4 profiles the blood samples from 513 patients aged 6 months to 17 years who were showing symptoms of allergic diseases were analyzed using microarrays containing 31 allergens. The highest sIgE prevalence was observed for birch pollen (32%) among pollen allergens, cat dander (24%) among indoor allergens, and egg whites (21%) among food allergens. The most common sIgG4 response was developed toward egg whites (80% of patients). Age-related elevation was identified for patients with increased sIgE to pollen allergens and indoor allergens (cat or dog dander and house dust mites). For each allergen, the proportion of cases with significant levels of sIgG4 appeared to increase with patients' age. The data on allergen-specific sIgE and sIgG4 prevalence show both general trends and some local special aspects that are indicative for the Moscow region. This information should be useful in terms of epidemiology of allergic diseases.

Microarray analyzer based on wide field fluorescent microscopy with laser illumination and a device for speckle suppression.

A microarray analyzer was developed to obtain images and measure the fluorescence intensity of microarrays at three wavelengths from 380 nm to 850 nm. The analyzer contains lasers to excite fluorescence, barrier filters, optics to project images on an image detector, and a device for suppressing laser speckles on the microarray support. The speckle suppression device contains a fibre-optic bundle and a rotating mirror positioned in a way to change the distance between the bundle butt and mirror surface during each mirror revolution. The analyzer provides for measurements with accuracy within ± 5%. Obtaining images at several exposure times allowed a significant expansion in the range of measured fluorescence intensities. The analyzer is useful for high throughput analysis of the same type of microarrays.

Development of a microarray-based method for allergen-specific IgE and IgG4 detection.

BACKGROUND: sIgE and sIgG4 detection is necessary for more accurate and effective type I hypersensitivity diagnostics and the estimation of disease development. Typically, the analyses of these antibodies are performed separately with the help of various specialized systems. The aim of this study was to develop a microarray-based method for the simultaneous quantitative detection of sIgE and sIgG4 to the most common allergens in a single sample. METHODS: A quantitative method for the simultaneous detection of sIgE and sIgG4 was developed based on the technology of hydrogel microchips previously designed at Engelhardt Institute of Molecular Biology, Russian Academy of Sciences (EIMB RAS). The microarray contained gel pads with immobilized allergens and gel pads that allow for the obtaining of sIgE and sIgG4 internal calibration curves for each allergen during the assay. The possibility of the simultaneous detection of sIgE and sIgG4 was developed using the corresponding Cy5 and Cy3 fluorescent dyes. RESULTS: The multiplex immunoassay method using hydrogel microarrays developed in this study allowed the quantitative detection of sIgE and sIgG4 to 31 allergens from different groups in a single assay. A comparison of the microarray with the existing plate-based analogues (i.e., ALLERG-O-LIQ and sIgG4 ELISA) was performed by analysing 152 blood serum samples and by evaluating Pearson correlation coefficients, ROC analysis, and Passing-Bablok linear regression results. CONCLUSION: The implementation of this method in allergy diagnostics will provide the possibility of simultaneously performing primary patient screening and obtaining additional information concerning the severity of the allergies and the choice of an appropriate therapy.

Allergen extracts and recombinant proteins: comparison of efficiency of in vitro allergy diagnostics using multiplex assay on a biological microchip.

BACKGROUND: Immunological test systems for diagnostics of type I hypersensitivity involve the following types of antigens: whole allergen extracts, individual highly purified proteins and their recombinant analogues. The goal of this study was to compare the results obtained with whole allergen extracts (birch pollen, cat dander, and timothy grass pollen) and their respective recombinant proteins in biochip-based immunoassay. METHODS: Multiplex fluorescent immunoassay of 139 patients' blood serum samples was carried out using biological microchips (biochips). sIgE concentrations for the chosen allergens and their recombinant components were measured. ROC analysis was used for comparison of the results and determination of diagnostic accuracy. RESULTS: The results for the birch pollen extract and its recombinant allergens have shown that the diagnostic accuracy of the methods utilizing the whole allergen extract, its major component Bet v 1 and the combination of major and minor components (Bet v 1 and Bet v 2) was the same. Values for diagnostic accuracy for the cat dander extract and its major recombinant component Fel d 1 were equal. In contrast with birch pollen and cat dander allergens, using of recombinant components of timothy grass pollen (Phl p 1, Phl p 5, Phl p 7 and Phl p 12) did not allow reaching the diagnostic accuracy of using natural extract. CONCLUSIONS: Multiplex analysis of samples obtained from patients with allergy to birch pollen and cat dander using biological microchips has shown that comparable accuracy was observed for the assay with natural extracts and recombinant allergens. In the case of timothy grass allergen, using the recombinant components may be insufficient.

Multiplex determination of serological signatures in the sera of colorectal cancer patients using hydrogel biochips.

Colorectal cancer (CRC) is the third most common malignancy in industrialized countries. Despite the advances in diagnostics and development of new drugs, the 5-year survival remains only 60-65%. Our approach to early diagnostics of CRC is based on the determination of serological signatures with an array of hemispherical hydrogel cells containing immobilized proteins and oligosaccharides (glycochip). The compounds immobilized on the glycochip include tumor-associated glycans (SiaTn, Tn, TF, Le(C) , Le(Y) , SiaLe(A) , and Manß1-4GlcNAcß) and antibodies against human immunoglobulins IgG, IgA, and IgM. The glycochip detects antibodies against tumor-associated glycans in patients' sera. The simultaneous measurement of the levels of immunoglobulins enhances the diagnostic impact of the signatures. In this work, we found previously unreported increase in antibodies against oligosaccharide Manß1-4GlcNAcß in patients with CRC. In parallel with these experiments, we determined the levels of oncomarkers carcinoembryonic antigen (CEA), cancer antigen (CA) 19-9, CA 125, CA 15-3, human chorionic gonadotropin (HCG), and alpha-fetoprotein (AFP) using another gel-based biochip with immobilized antibodies (oncochip) developed earlier in our laboratory. In total, 69 samples from healthy donors, 33 from patients with colorectal carcinoma, and 27 from patients with inflammatory bowel diseases were studied. The use of combined signatures of antiglycan antibodies and oncomarkers provides much better predictive value than the conventional measurement of oncomarkers CEA and CA 19-9. Positive predictive value of CRC diagnoses using together glycochip and oncochip reached 95% with the sensitivity and specificity 88% and 98%, respectively. Thus, the combination of antibody profiling with detection of conventional oncomarkers proved to be a promising tool in diagnostics of CRC.

UV fluorescence of tryptophan residues effectively measures protein binding to nucleic acid fragments immobilized in gel elements of microarrays.

Microarrays allow for the simultaneous monitoring of protein interactions with different nucleic acid (NA) sequences immobilized in microarray elements. Either fluorescently labeled proteins or specific fluorescently labeled antibodies are used to study protein-NA complexes. We suggest that protein-NA interactions on microarrays can be analyzed by ultraviolet (UV) fluorescence of tryptophan residues in the studied proteins, and this approach may eliminate the protein-labeling step. A specialized UV microscope was developed to obtain fluorescent images of microarrays in the UV wavelengths and to measure the fluorescence intensity of individual microarray elements. UV fluorescence intensity of BSA immobilized in microarray gel elements increased linearly with increased BSA amount with sensitivity of 0.6 ng. Real-time interaction curves between the DNA-binding domain of the NFATc1 transcription factor (NFATc1-DBD) and synthetic hairpin-forming oligodeoxyribonucleotides immobilized within 0.2 nL microarray gel elements at a concentration 5 × 10(-5) M and higher were obtained. The UV fluorescence intensities of microarray gel elements containing NFATc1-DBD-DNA complexes at equilibrium allowed the estimation of the equilibrium binding constant for complex formation. The developed method allows the protein-NA binding to be monitored in real time and can be applied to assess the sequence-specific affinity of NA-binding proteins in parallel studies involving many NA sequences.

Gel-based microarrays in clinical diagnostics in Russia.

Immobilization of molecular probes in 3D hydrogel elements provides some essential advantages compared with conventional flat surfaces. In this article, an integrated technology based on the use of low-density microarrays comprised of hemispherical gel elements, developed at the Engelhardt Institute of Molecular Biology (Moscow, Russia) for various applications will be reviewed. The structure of the gel can be adapted for immobilization of virtually any biological molecules in a natural hydrophilic environment. The discrimination between matching and mismatching duplexes of nucleic acids in these conditions is more reliable than on conventional flat surfaces, minimizing the number of elements needed to detect specific sequences. Protein molecules immobilized in hydrogel-based biochips better preserve their biological properties. As described in this article, such biochips were successfully applied for laboratory diagnostics in a wide variety of clinical conditions involving the identification of bacterial and viral pathogens, cancer-related mutations and protein tumor markers.

Quantification of target proteins using hydrogel antibody arrays and MALDI time-of-flight mass spectrometry (A2M2S).

Mass spectrometry-based analysis techniques are widely applied in proteomics. This study presents a novel method for quantitative multiplex candidate protein profiling. It applies immunocapture of differentially labeled protein complements on hydrogel antibody arrays and subsequent quantification by MS. To make this approach quantitative a labeling approach was devised. The impact of labeling on the antibody/antigen interaction was assessed in detail by surface plasmon resonance. Owing to there solution by mass more than two protein samples can be compared simultaneously. Direct labeling of crude samples such as sera was developed and so enables the absolute quantification of target proteins straight from crude samples without a protein purification step. It was used to measure the concentration of apolipoprotein A-1 in serum. This method has been termed A2M2S for Affinity Array sand MALDI Mass Spectrometry.

Why 3-D? Gel-based microarrays in proteomics.

Gel-based microarrays (biochips) consisting of nanoliter and sub-nanoliter gel drops on hydrophobic substrate are a versatile technology platform for immobilization of proteins and other biopolymers. Biochips provide a highly hydrophilic environment, which stabilizes immobilized molecules and facilitates their interactions with analytes. The probes are immobilized simultaneously with gel polymerization, evenly distributed throughout individual elements, and are easily accessible because of large pores. Each element is an isolated nanotube. Applications of biochips in the studies of protein interactions with other proteins, nucleic acids, and glycans are described. In particular, biochips are compatible with MALDI-MS. Biochip-based assay of prostate-specific antigen became the first protein microarray approved for clinical use by a national regulatory agency. In this review, 3-D immobilization is compared with mainstream technologies based on surface immobilization.

Clinical screening of gene rearrangements in childhood leukemia by using a multiplex polymerase chain reaction-microarray approach.

PURPOSE: Currently, many forms of leukemia are considered potentially curable, with prognosis and clinical outcome strongly dependent on the underlying molecular pathophysiology. A substantial number of leukemia patients harbor nonrandom karyotypic abnormalities that define subgroups with unique biological and clinical features. For detection of these types of gene rearrangements, a combination of multiplex RT-PCR with hybridization on oligonucleotide gel array was presented previously, which identified five chromosomal translocations with fusion variants. In the present study, additional clinically relevant translocations were included in our analysis using a second generation of microarrays. We also expanded significantly on the clinical correlation of our findings. EXPERIMENTAL DESIGN: An oligonucleotide microarray was designed for hybridization with products of a multiplex RT-PCR to identify the following translocations: t(9;22)p190, t(4;11), t(12;21), t(1;19), typical for acute lymphoblastic leukemia; t(9;22)p210 for chronic myeloid leukemia; and t(8;21), t(15;17), inv16, typical for acute myeloblastic leukemia. RESULTS: To demonstrate the potential clinical application of the method, 247 cases of childhood leukemia were screened, and the above-mentioned gene rearrangements were found in 30% of cases. The sensitivity and specificity of the assay is comparable with the RT-PCR technique, so that it can be used to follow minimal residual disease. The feasibility of an additional refinement of the method, on-chip-multiplex PCR, has been successfully demonstrated by identifying a common translocation, t(9;22), in chronic myeloid leukemia. CONCLUSIONS: Our data suggest that the microarray-based assay can be an effective and reliable tool in the clinical screening of leukemia patients for the presence of specific gene rearrangements with important diagnostic and prognostic implications. The method is amenable for automation and high-throughput analysis.