The selection and optimization of the detection system for self-contained multiplexed dot-immunoassay.

This study performed a comparative estimation of the detection systems with the use of conjugates based on peroxidase, alkaline phosphatase, colloidal gold, and the amplification system "Super-CARD" for multiplex dot-immunoassay of antibodies. The results of the study show that the sensitivity of the detection system with colloidal gold was approximately 8 times higher than that of the system with amplification "Super-CARD", 30 times higher than that of the system with conjugate of alkaline phosphatase, and 250 times higher than the sensitivity of the system with the peroxidase conjugate. Gold immunosols limit the direct detection of human IgG of 10 pg with dynamic range of optical signal change from 5 ng to 10 pg. This limit corresponds to a range of concentrations of IgG from 2.5 µg/mL to 5 ng/mL and covers the range of specific IgG concentrations, to which a typical natural immune response leads. The most typical reasons for aggregate formation during obtainment of colloidal gold and the binding of colloidal gold with biocomponents were explored. The method of minimization of particle clusters formation was suggested as well as the method for increasing stability of diluted preparations of probe by means of sedimentation of aggregates from the ready-made product.

Interaction mechanism of anabolic steroid hormones with structural components of erythrocyte membranes.

The interaction of testosterone, androsterone, dehydroepiandrosterone (DHEA), and dehydroepiandrosterone sulfate (DHEAS) with erythrocyte membranes was studied. It was shown that testosterone and androsterone have a high constant of binding to the membranes (K(b) ≈ 10(6) M(-1)), whereas K(b)'s for DHEA and DHEAS are 2 orders of magnitude lower. Hydrogen bonds and hydrophobic interactions play an important role in binding of anabolic steroids. Hydrogen bonds form with CO and NH groups both of membrane proteins and phospholipids. This results in the formation of complex domains rising above the surface of membranes. Strengthening of hydrophobic interactions in the domains promotes the displacement of water dipoles to adjacent regions, thus loosening the phospholipid bilayer. Overall, microviscosity of erythrocyte membranes strongly increases, which decreases the plasticity of erythrocytes and hampers their motion in blood capillaries. This mechanism may underlie the development of diffusion myocardial hypoxia and hypoxic cardiac arrest.

Interaction mechanism of cortisol and catecholamines with structural components of erythrocyte membranes.

Nonspecific mechanisms of the stress hormones interaction with erythrocyte membranes were studied by means of atomic force microscopy, fluorescence analysis, and IR spectroscopy. It was shown that stress hormones (cortisol, adrenaline, noradrenaline) can bind to erythrocyte membranes with high affinity (K(b) approximately 10(6) M(-1)). The binding mechanism involves hydrogen bonds and hydrophobic and electrostatic interactions. Active groups of the hormones (NH(2), NHCH(3), keto, and hydroxy groups) interact simultaneously with CO and NH groups both of proteins and phospholipids. This leads to the formation of complex protein-lipid domains that distort the surface of the erythrocyte membrane. Water dipoles are displaced from the domains to adjacent regions and facilitate membrane loosening. The interaction of hormones with the membrane is accompanied by structural transitions of disorder --> order (tangle --> alpha-helix, tangle --> beta-structure) in membrane proteins and structural transitions of order --> order in phospholipids. Formation of large domains (clusters) of the lipid-protein and lipid nature leads to distortion of membranes and deteriorates their elasticity and rheological properties.