[The detection of level of free light chains of immunoglobulins using enzyme-linked immunosorbent assay techniques for diagnostic of monoclonal gammopathy.]

The content of free light chains of immunoglobulins kappa and lambda and also ratio of their concentrations in blood serum are important diagnostic and prognostic markers in case of monoclonal gammopathy. The technique FreelightTM based on nephelometric detection of free light chains using polyclonal antibodies is one of common modes of detection of free light chains. The actual study was carried out with purpose of validating of national test-system for detection of level of free light chains in blood serum using technique of enzyme-linked immunosorbent assay. The samples of blood serum were taken from 89 healthy donors and 165 patients with monoclonal gammopathy. To detect the level of free light chains enzyme-linked immunosorbent assay testsystem "Polygnost" was used based on application of monoclonal a ntibodies. The number of analytical characteristics of reagents set was determined including limit of detection and range of linearity. The limit of detection of free light chains using enzymelinked immunosorbent assay test-system was two times lower than claimed by manufacturer of nephelometric set "FreelightTM". Hence, analytical characteristics of enzyme-linked immunosorbent assay set make it possible to detect the level of free light chains within range of standard values. The reference limits were established concerning concentration of free light chains kappa (3.25-15.81 mkg/ml), free light chains lambda (3.23-28.05 mkg/ml) and their ratio (0.3-1.9) in blood serum that factually matched the recommended intervals for "FreelightTM" set. In patients with monoclonal gammopathy the level of free light chains was reliably higher (p<0.01) as compared with control group of healthy donors. In case of paraproteinemia reliable alteration (p<0.01) of ratio free light chains kappa/free light chains lambda was observed in comparison with control group. The results of actual study testify that national enzyme-linked immunosorbent assay set has good analytical and diagnostic characteristics and it can be used in laboratory practice.

[PRODUCTION AND CHARACTERIZATION OF THE PANEL OF MONOCLONAL ANTIBODIES AGAINST HUMAN ENDOGLIN].

Endoglin (CD105) is the marker of endothelial and mesenchymal stem cells and the component of TGF-ß, BMP-9 and BMP-10-binding receptor complexes. Its expression is significantly increased on blood vessels endothelium of ischemic tissues and growing tumors. Measurement of concentration of the soluble endoglin in the serum or urine is used as a method for diagnosing cancer and pregnancy disorders. The aim of this work was to create a novel family of monoclonal antibodies recognizing endoglin on the cell surface and in biological fluids. Murine myeloma cells' derived recombinant protein representing the whole extracellular part of endoglin was used as an antigen. F1(SJL/JxBALB/c) mice were the donors of immune splenocytes. Hybridoma screening procedures were performed using E. coli-produced copies of the antigen, endoglin-expressing immortalized human cell lines, and primary cultures of human mesenchymal stromal cells. Ten novel monoclonal antibodies recognizing at least eight distinct epitopes were produced. Eight antibodies bind membrane form of endoglin on the surface of normal and transformed human cells derived from different tissue sources. Two antibodies recognize linear antigenic determinants of the molecule and can be used to detect endoglin by western blot. Sandwich ELISA system was designed in order to measure soluble endoglin in cell culture medium.

[Endoglin (CD105)--a target for visualization and anti-angiogenic therapy for malignant tumors].

Drugs currently used for anti-angiogenic therapy which are based on monoclonal antibodies to VEGF and its receptors are of limited efficiency. Endoglin (CD105) is a protein receptor of TGF-beta superfamily involved in ligand binding and signal transduction regulating VEGF-independent mechanisms of angiogenesis. CD105 is highly expressed on membranes of endothelial cells of vessels in growing tumors. It plays a crucial role in determination the state of activation or quiescence of endotheliocytes. CD105 is present also on membranes of tumor stromal cells (macrophages, fibroblasts, pericytes). High density of CD105-positive microvessels in tumors corresponds with its aggressivness, spreading to regional lymph nodes and poor prognosis. In patients with progressing tumors soluble form of endoglin in peripheral blood may be detected. Monoclonal antibodies to CD105 and their derivatives are regarded as a basis for creation of new generation of anti-angiogenic reagents for visualization of tumor vessels, for direct effect on endothelium or for targeted drugs delivery to growing tumors.

[Effect of alkylresorcinols on thermal denaturation and refolding of bacterial luciferase and synthesis of heat shock proteins revealed in the luminescent molecular and cellular test systems].

Molecular and cellular luminescent biotests were used to reveal the effects of five alkylresorcinol homologues (C7-, C9-, C11-, C12-, and C18-AR) on the thermally induced denaturation and refolding ofbac- terial luciferases, as well as on the synthesis of heat shock proteins. The ARs activities were found to depend on their fine structure and concentration. Direct heat-protective effect of short-chain C7- and C9-AR on the chromatographically pure Photobactrium leiognathii luciferase/oxidoreductase was shown within broad range of concentration (10(-6)-10(-3) M). The long-chain ARs homologues exhibited a similar heat-protective effect at micromolar concentrations only, while their millimolarconcentrations have increased the sensitivity of the model proteins to thermal treatment. The recombinant strain Escherichia coli K12 MG1655 bearing constitutively expressed Vibrio fischieri luxAB genes was used to investigate theARs effect on the intracellular chaperone-independent refolding of bacterial luciferase. The functional activity of heat-inactivated enzyme was restored by micromolar concentrations of short-chain ARs, while long-chain homologues inhibited re- folding in the wide concentration range. The recombinant luminescent E. coli strain bearing the inducible ib- pA'::luxCDABE genetic construction was used to determine the effect of ARs on the synthesis of heat shock proteins (HSP). The preincubation mode of bacterial cells with long-chain alkylresorcinols led to dose-de- pendent stimulation of HSP synthesis (2.7 to 4 times) that confirmed some ARs function as "alarmones". Subsequent thermal treatment resulted in a 5-15-fold decrease of the following HSP induction compared to the control, while the number of viable cells opposite increased 1.5-4-fold. Thus, pretreatment of the bacte- rial cells with long-chain ARs resulted in their preadaptation to subsequent thermally induced stress. Short- chainARs caused less pronounced HSP suppression, although still was accompanied by increased heat resis- tance of the AR-pretreated bacterial cells.