Adaptation of Microarray Assay for Serum Amyloid a Analysis in Human Serum.

Serum amyloid A is an inflammatory biomarker whose concentration changes during infectious and inflammatory diseases. SAA's tendency for aggregation and complex formation makes it difficult to determine its concentration in samples, especially when there is an increased level of it. Immunofluorescence SAA determination on a microarray was adapted for SAA quantification in human serum. Both the procedure and the diluent for the calibrator samples were chosen to obtain a dynamic range between 1 and 100 µg/mL. Mixtures of animal (rabbit, goat, mouse) sera with recombinant antigen diluted in certain concentrations were used for the calibrator samples. The method was tested using serum samples from 15 patients with rheumatoid arthritis or ankylosing spondylitis and 9 healthy donors. The results obtained on the microarray demonstrated a good correlation with the results determined by ELISA (Pearson's correlation coefficient is 0.93). The method developed could be a convenient tool for assessing SAA levels in a number of diseases, such as rheumatoid arthritis or infections of various etiologies, characterized by a significant increase in the level of this protein in the blood. The use of a microarray for the analysis allows the determination of the SAA concentration simultaneously with other inflammatory biomarkers.

[Adaptation of Microarray Assay for Serum Amyloid A Analysis in Human Serum].

Serum amyloid A is an inflammatory biomarker whose concentration changes during infectious and inflammatory diseases. SAA's tendency for aggregation and complex formation makes it difficult to determine its concentration in samples, especially when there is an increased level of it. Immunofluorescence SAA determination on a microarray was adapted for SAA quantification in human serum. Both the procedure and the diluent for the calibrator samples were chosen to obtain a dynamic range between 1 and 100 µg/mL. Mixtures of animal (rabbit, goat, mouse) sera with recombinant antigen diluted in certain concentrations were used for the calibrator samples. The method was tested using serum samples from 15 patients with rheumatoid arthritis or ankylosing spondylitis and 9 healthy donors. The results obtained on the microarray demonstrated a good correlation with the results determined by ELISA (Pearson's correlation coefficient is 0.93). The method developed could be a convenient tool for assessing SAA levels in a number of diseases, such as rheumatoid arthritis or infections of various etiologies, characterized by a significant increase in the level of this protein in the blood. The use of a microarray for the analysis allows the determination of the SAA concentration simultaneously with other inflammatory biomarkers.

Analysis of rheumatoid factor and acute phase proteins using microarrays in patients with rheumatoid arthritis.

One of the biomarkers of biggest clinical importance in rheumatoid arthritis (RA) is rheumatoid factor (IgM RF). The rheumatoid factor has insufficient sensitivity and specificity, therefore, to increase the diagnostic information of the test, acute phase proteins were used as concomitant biomarkers. Using biological microchips, we measured IgM RF, C-reactive protein (CRP) and Serum amyloid protein A (SAA) in patients with RA (n = 60), ankylosing spondylitis (AS) (n=55), systemic lupus erythematosus (SLE) (n=20) and healthy donors (HD) (n=9). It was shown that the medians of IgM RF concentrations are significantly higher (p<0.01) in patients with RA compared to patients suffering from other diseases and healthy donors. CRP and SAA were also significantly increased (p<0.05) in patients with RA and AS compared with SLE and HD. It has been shown that the complex determination of three biomarkers in differentiating RA patients with the comparison group had a higher diagnostic sensitivity than the isolated determination of IgM RF, while the addition of SAA makes the greatest contribution to improving the diagnostic characteristics of the biomarker panel: the use of a logistic regression model based on IgM RF and SAA allowed to increase the diagnostic sensitivity of the analysis from 58.3% to 65%. Thus, the developed microarray-based method can be used to detect and elucidate the diagnostic characteristics of RA biomarkers; however, further use requires validation of the obtained results on an expanded sampling.

[Microarray for Quantitative Determination of Inflammatory Biomarkers in a Culture Medium].

Cytokines and acute phase proteins play an important role in the development of the immune response during inflammatory reactions. Depending on the type of disease, the development of inflammation is accompanied by changes in concentrations (both decrease and increase) of not one, but many inflammatory biomarkers. Here, a quantitative microarray-based method for multiplex immunoassay of eight biomarkers of human inflammation, namely acute phase proteins (C-reactive protein, serum amyloid protein A) and cytokines (IL-6, IL-8, IL-17, IL-18, IP10/CXCL10, TNFα) was developed and the possibility of its use for the detection of inflammatory biomarkers in a culture medium has been demonstrated. The developed method can be used to evaluate changes of the inflammatory biomarker profile induced by different agents or to determine the concentrations of biomarkers after activation of cells while studying different diseases with the help of in vitro models.

[Modification of Anti-Glycan IgG and IgM Profiles in Allergic Inflammation].

Glycans and anti-glycan antibodies (AGAs) are essential for infiltration of inflammatory cells in various allergies. The glycocalyx structure of the cells is modified during disease progression, and this modification is possible to evaluate by assessment of AGAs. A printed glycan array with 55 immobilized glycans and immobilized antibodies to IgG, IgA, and IgM was used to study the changes in AGA profiles in bronchial asthma (BA). Levels of antibodies to certain glycans in BA patients statistically differed from levels in healthy donors (p < 0.0007 by the Mann-Whitney test); the glycan set included 6Su-6`-SiaLec, Sia LeX, Sia6Htype2; Tαα, Manß1-4GlcNAc, and Manα1-4Manß. The obtained results help to better understand the mechanisms of the cell-mediated immune response in bronchial asthma and other types of allergic reactions.

Development of hydrogel biochip for in vitro allergy diagnostics.

A hydrogel biochip was developed for the simultaneous quantitative determination of sIgE for 21 allergens and total IgE in human serum. The biochips are manufactured by photoinduced copolymerization of different molecules (allergens and antibodies) with gel-forming monomers resulting in the formation of three-dimensional hydrogel elements (1nl gel drops). After incubation of the biochip with the serum, the results are visualized using fluorescently labeled anti-IgE antibodies. Using biochips, serum samples from allergic patients and healthy donors were analyzed and good correlation with the results obtained using commercial EIA test systems of generally recognized quality (Dr. Fooke Laboratorien GmbH, Germany) was observed.