Antibodies Against Unusual Forms of Sialylated Glycans.

Previous studies have shown that in the blood of healthy donors (1) there are no natural antibodies against sialylated glycoproteins, which contain Neu5Acα (N-acetylneuraminic acid) as the most widespread form of human sialic acid, and (2) there is a moderate level of antibodies capable of binding unnatural oligosaccharides, where Neu5Ac is beta-linked to a typical mammalian glycan core. In the present study, we investigated antibodies against ßNeu5Ac in more detail and verified the presence of Kdn (2-keto-3-deoxy- D-glycero-D-galacto-nonulosonic acid) as a possible cause behind their appearance in humans, taking into account the expected cross-reactivity to Kdn glycans, which are found in bacterial glycoconjugates in both the α- and ß-forms. We observed the binding of peripheral blood immunoglobulins to sialyllactosamines (where "sialyl" is Kdn or neuraminic acid) in only a very limited number of donors, while the binding to monosaccharide Kdn occurred in all samples, regardless of the configuration of the glycosidic bond of the Kdn moiety. In some individuals, the binding level of some of the immunoglobulins was high. This means that bacterial Kdn glycoconjugates are very unlikely to induce antibodies to ßNeu5Ac glycans in humans. To determine the reason for the presence of these antibodies, we focused on noninfectious pathologies, as well as on a normal state in which a significant change in the immune system occurs: namely, pregnancy. As a result, we found that 2/3 of pregnant women have IgM in the blood against Neu5Acß2-3Galß1-4GlcNAcß. Moreover, IgG class antibodies against Neu5Acß2-3Galß1-4GlcNAcß and Neu5Acß2-6Galß1-4GlcNAcß were also detected in eluates from the placenta. Presumably, these antibodies block fetal antigens.

[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.

[Exosomal surface protein markers in diagnosis of colorectal cancer].

Colorectal cancer (CRC) is one of the most common primary malignancies. Early stages of the disease are asymptomatic in the majority of cases, leading to late detection and high mortality. Available noninvasive diagnostic techniques are limited in sensitivity and specificity, and designing new ones is still a pressing problem. Exosomes are membrane-derived microvesicles secreted into human biological fluids and provide a novel way to assess the course of an oncology disease. The review describes the repertoire of exosomal surface biomarkers found in the blood of CRC patients and the prospects of employing multiplexed tests for exosomal markers in early noninvasive diagnosis of cancer.

[Hydrogel microchip as a tool for studying exosomes in human serum].

Exosomes are cell-derived vesicles that are secreted by both normal and cancer cells. Over the last decade, a few studies have revealed that exosomes cross talk and/or influence major tumor-related pathways such as angiogenesis and metastasis involving many cell types within the tumor microenvironment. The protein composition of the membrane of an exosome reflects that of the membrane of the cell of origin. Because of this, tumor-derived exosomes differ from exosomes that are derived from normal cells. The detection of tumor exosomes and analysis of their molecular composition hold promise for diagnosis and prognosis of cancer. Here, we present hydrogel microarrays (biochips), which contain a panel of immobilized antibodies that recognize tetraspanins (CD9, CD63, CD81) and prognostic markers for colorectal cancer (A33, CD147). These biochips make it possible to analyze the surface proteins of either isolated exosomes or exosomes that are present in the serum samples without isolation. These biochips were successfully used to analyze the surface proteins of exosomes from serum that was collected from a colorectal cancer patient and healthy donor. Biochip-guided immunofluorescent analysis of the exosomes has made it possible for us to detect the A33 antigen and CD147 in the serum sample of the colorectal cancer patient with normal levels of CEA and CA19-9.

[Preparation of recombinant serpins B3 and B4 and investigation of their specific interactions with antibodies using hydrogel-based microarrays].

The objective of this work was to obtain preparations of recombinant squamous-cell carcinoma antigens (serpins B3 and B4) and to investigate their interactions with different monoclonal antibodies using hydrogel-based microarrays (biochips). Two genetic constructs encoding full-length serpin B3 and serpin B4 molecules were created to produce recombinant SPB3 and SPB4 proteins carrying a N-terminal His6-tag. Monoclonal antibodies against serpin B3 (H3, C5, H5, H81, and G9) were also obtained. An experimental gel-based biological microchip was designed to contain gel elements that carry immobilized antibodies against SPB3, immobilized commercial monoclonal SCC107 and SCC140 antibodies against squamous-cell carcinoma antigen (SCCA), and gel elements with immobilized SPB3 or SPB4. Judging by the specificity of recombinant SPB3 and SPB4, which bind to monoclonal antibodies against SCCA and, according to the manufacturer's data, can recognize conformational epitopes of both SPB3 and SPB4, it was concluded that the obtained recombinant serpins had the correct tertiary structure. A biochip-based direct immunoassay showed that SPB4 could bind effectively only to SCC107 and SCC140 antibodies, while SPB3 interacted specifically not only with these antibodies, but also with H3 and C5 monoclonal antibodies. Using biochip-based sandwich immunoassay, a pair of monoclonal antibodies SCC107/C5 that interacted specifically with serpin B3 but did not interact with serpin B4 was identified. Thus, it has been demonstrated that serpin B3 can be selectively determined in the presence of highly homologous serpin B4 using a biochip-based assay.

[The evaluation of ratio of oncomarkers in search of initial focus of tumor: on the occasion of fiftieth anniversary of discovery of alpha-fetoprotein as an oncomarker].

The article presents research data testifying the dominant value of HAFP behavior in diagnostic of oncological diseases. The importance of study of profile of main oncomarkers in patient is demonstrated. The method of hydrohelium biochips, developed in the institute of molecular biology, was used to determine 9 key oncomarkers. The application of this method made it possible to essentially complete the information map in 8 patients according to clinical interpretation of disease. In economically justified variant, this method is able to shorten period of study of patient, to specify character of pathological process and to transfer examination load of patients to the out-patient level.

2-Aminopyridine--a label for bridging of oligosaccharides HPLC profiling and glycoarray printing.

2-Aminopyridine derivatives of oligosaccharides (OS-AP) were printed onto microchips by two different ways. The first method is based on direct covalent insertion of OS-AP in polyacrylamide gel 3D chip. The second method is based on conversion of OS-AP into more reactive OS-aminoalditol followed by covalent printing onto NHS-activated glass slides. This approach extends the range of saccharides suitable for covalent printing due to availability of commercial OS-AP and easy high-performance liquid chromatography separation of glycoprotein N-chains in form of AP derivatives.