[The developoment of total complement activity assay based on complement-dependent immune liposome lysis].

BACKGROUND: The purpose of work was development of a fast and reproduced procedure for measurement of the total complement activity (TCA) in human or animal blood serum. MATERIALS AND METHODS: Steady at storage liposomes preparations, which surface sensitized 2,4-DNP haptens, and the internal volume contains calceine or sulforhodamine 101 are obtained. Complement-dependent immune lysis of liposomes at presence of the anti-2,4-DNP immunoglobulines and complement preparations from animals are investigated. RESULTS: It is shown that the degree of liposomes immune lysis depends on complement concentration in a wide range that can be used for definition of TCA level. Research of blood sera from patients has revealed correlation (r = 0.793) between data received with the help of liposome immunolytic systems, and the data of nephelometric analysis with application of suspension sheep erythrocytes. CONCLUSION: The method allows to define total complement activity in blood serum in 15 minutes without separation of reaction components. This might be useful for measurement TCA level at patients with various diseases and realization of scientific researches.

[The use of hydrosol hexacianferrate (II) ferrum (III) for developing diagnostic lateral flow tests].

On the basis of synthesized negatively charged hydrosol hexacianferrate (II) ferrum (III) (HCFF) by diameter 10-20 HM are received stable conjugates with antibodies and antigens glycoprotein and lipopolysaccharide (LPS) nature. Synthesized hydrosol (HCFF) is new type of a disperse phase in the lateral flow assay. Conjugates mentioned above were applied for construction lateral flow tests-systems for revealing cholera toxin, the rabbit antibodies to recombinant glycoproteine complex from Micobacterium tuberculosis H37 Rv, human immunoglobulin, LPS antigens S. typhimurium, S. enteritidis. Developed lateral flow tests-systems had high analysis speed (5-7 min), good specificity and sensitivity: on cholera toxin of 2.0 ug/ml, on LPS antigens S. typhimurium, S. enteritidis 0.5 ug/ml.

[Experimental verification of a model of complement-dependent immune lysis of liposomes].

The quantitative dependences of the immune complement-dependent lysis of a monodisperse suspension of 200-nm liposomes whose membrane was sensitized by monovalent hapten (2,4-DNP-epsilon-caproyl-DPPE) or a polyvalent antigen (LPS from F. tularensis) on the initial concentration of specific antibodies (IgG) to hapten and the antigen have been investigated. The quantity of antibodies binding sites on the surface of liposome was evaluated. The difference between the complement-dependent immune lysis of poly- and monodisperse suspensions of liposomes was shown. The experimental results are well described by the direct binding model.

[An experimental luminescent immunochromatographic system].

Luminescent immunochromatographic assay for antigens of microorganisms was developed to detect antigens and cells of microorganisms, toxins. Luminescent submicrone latex particles, quantum labels, ganglioside-containing liposomes with sulforhodamine-01 in the internal volume were used as a luminescent mobile phase for conjugation with antibodies. Devices for registration of luminescent immunochromatograms were developed. Submicrone latexes were shown to have advantages over quantum labels, ganglioside-containing liposomes with sulforhodamine-101 and colloid gold for immunochromatographic procedures. Sensitivity of luminescent latex-based immunochromatographic indicator element was 3-30 that elements based on colloid gold conjugates for identical microorganisms and elements.

[The use of fluorescent liposomal immunoanalysis for the diagnostics and indication of glanders and melioidosis].

The authors have developed a homogenous method for the detection of the cells, surface antigen, and Ag8 of glanders and melioidosis pathogens and antibodies to them, based on the compliment-dependent lysis of Ag8-sensitized liposomes. The method is highly specific; its sensitivity to antibodies to glanders and melioidosis pathogens is 24 to 440 times higher than that of RID: its sensitivity to Ag8 is 100 ng/ml corresponding to 1.25x10-10 M, and its sensitivity to cells is 10(5) to 10(6) cells/ml).

[The use of liposomes for detection of the surface lipopolysaccharide antigen, Vibrio cholerae cells, and antibodies against them].

A test system for determination of Vibrio cholerae cells, surface O-antigen, and antibodies against them was developed on the basis of complement-dependent lysis of liposomes sensitized by the lipopolysaccharide-dependent antigen from Vibrio cholerae 569B. The factors that affect the function of the liposomal reagent were studied, and the conditions for detecting antibodies and antigenic material were optimized. This system is highly specific and sensitive to be used for the determination of anticholeraic antibodies (30-50 times as effective as agglutination tests), lipopolysaccharide antigen (100 ng/ml, which corresponded to 3.0 ng of lipopolysaccharide in the sample studied), and Vibrio cholerae cells (3.3 x 10(7) m.b./ml, which corresponded to 10(6) m.b. in sample). It takes 30-40 min to detect the lipopolysaccharide antigen and 90 min to detect V. cholerae cells.

[Change in the dynamics of a spin probe in complement-dependent lysis of a liposome]. / Izmenenie dinamiki spinovogo zonda pri komplementzavisimom immunnom lizise liposom.

The mobility of phospholipid chains in membranes of liposomes consisting of egg lecitin, cholesterol, dicetylphosphate, sensitized by the lipopolysaccharide antigen F. tularensis by the action of a homologous antiserum and a rabbit complement preparation was studied using 5- and 16-doxylstearate spin probes. It was shown that, during the immune lysis of liposome membranes, changes in the dynamics of spin probes occur, which correlate with the formation of transmembrane channels and exit of the fluorescent marker from the interior of liposomes. It was found that the ratio of the intensities I1/I2 of two low-field extrema in the ESR spectrum is most sensitive to changes in the liposome membrane that are induced by immune components.

[Sulforodamine 101: novel effective marker in liposomal immunoassay]. / Sul'forodamin 101 - novyi éffektivnyi marker v liposomal'nom immunoanalize.

The new fluorescence marker Sulforodamine 101 that allows one to avoid a labour-consuming stage of purification of a marker substance (for example, calceine, other fluorescein derivatives) from hydrophobic admixtures that reduce the storage of a liposomal reagent was used in liposomal immunoassay. Sulforodamine 101 shows the maximum increase in a fluorescence signal when liposomes are destroyed with encapsulated marker, which is 45 times greater than does Sulforodamine B (8 times) and comparable with calceine (30 times). The comparative studies using liposomes containing calceine and Sulforodamine 101 have indicated that they are similar storage and sensitivity (50 ng/ml) of liposomal test system as determined by the concentration of F. tularensis polysaccharides in solutions.

[Liposomal immunoassay as a method for detecting microorganism antigens and their antibodies]. / Liposomal'nyi immunoanaliz kak metod obnaruzheniia antigenov mikroorganizmov i antitel k nim.

The paper presents experimental data on the use of the liposomal immunoassay (LIA) with a fluorescence marker to detect lipopolysaccharide antigens (LPS-AG) of the causative agents of infectious diseases (S. typhimurium, S. typhi, F. tularensis) and antibodies to them in the model systems and human serum. The sensitivity of determination of specific antibodies to LPS-AG is shown to be 15-160 times as high as that of RPGA and the sensitivity of determination of LPS-AG is comparable to that of solid-phase enzyme immunoassay. The stability and storage of diagnostic immunoliposomal test systems are dealt with. It is shown that the liposomal diagnostic agents can be stable without losing their properties for years. Whether LIA is of diagnostic value in detecting salmonellosis in children in the clinical setting is discussed and the value of this assay is compared with that of other laboratory methods. The data on how LIA can be automated are presented. Its analytical advantages in using in laboratory diagnosis are discussed.

[Use of monoclonal antibodies in the diagnosis of ophthalmic chlamydiosis]. / Ispol'zovanie monoklonal'nykh antitel dlia diagnostiki oftal'mokhlamidioza.

A total of 168 patients with various ocular diseases were tested for Chlamydia. Scrapings off the conjunctiva were examined by the fluorescent antibody technique with monoclonal antibodies. A high diagnostic efficacy of the method in patients with acute and, which is even more important, with the subacute form of ophthalmic chlamydiasis was demonstrated. Moreover, the method effectively diagnosed ophthalmic chlamydiasis in patients with inflammations of the anterior and median segments of the eye of obscure etiology.

[Assessing the efficacy of DNA hybridization for diagnosis of adenoviral and chlamydial diseases of eyes]. / Izuchenie éffektivnosti metoda DNK-gibridizatsii v diagnostike adenovirusnykh i khlamidiínykh zabolevanií glaza.

Analysis of scrapings off the conjunctiva of patients with suspected adenoviral infection (n = 60) and ophthalmochlamydiasis (n = 132) by different diagnostic tests showed good prospects of using DNA hybridization: the efficacy of detecting adenoviral diseases of the eyes was 35% improved in comparison with the fluorescent antibody method with polyclonal serum and the diagnosis of ophthalmochlamydiasis was 22.8% improved in comparison with enzyme immunoassay.

[A highly sensitive, homogeneous method for determining antibodies and antigens by using liposomes, monoclonal antibodies and complement]. / Vysokochuvstvitel'nyi, gomogennyi metod opredeleniia antital i antigenov s pomoshch'iu liposom, monoklonal'nykh antitel i komplementa.

A simple and sensitive method for the determination of Francisella tularensis antigen and antibodies to this antigen by means of the complement lysis of liposomes sensitized with F. tularensis lipopolysaccharide (LPS) is proposed. The possibility of participation of monoclonal antibodies (McAb) in the complement-dependent lysis of liposomes has been studied. The method permits the detection of 50-100 ng/ml of soluble LPS antigen in the presence of antiserum or McAb IgG F-B11-x and of 50-10 ng/ml of it in the presence of IgG F-B11-x and anti-IgG within 90 minutes.

[The quantitative analysis of lipopolysaccharide antigen in the blood serum of patients with salmonellosis by using complement-bound liposome lysis]. / Kolichestvennyi analiz lipopolisakharidnogo antigena v syvorotke krovi bol'nykh sal'monellezom s pomoshch'iu komplementzavisimogo lizisa liposom.

Titration of group B Salmonella O-antigen in the blood sera of patients and donors was carried out by means of the complement-dependent lysis of liposomes sensitized with S. typhimurium LPS. Good correlation (r = 0.95) of the levels of S. typhimurium somatic O-antigen in the patients' sera determined by liposomal immunoassay and aggregate hemagglutination test was established. The concentration of the antigens in the tested samples was within 0.5-50 micrograms/ml. Statistical analysis of the results obtained by liposomal immunoassay techniques demonstrated differences in the distribution functions for the blood sera of patients with different diseases and of donors.

[The determination of the antibodies and the somatic O-antigen of Salmonella group B by using the complement-bound lysis of liposomes]. / Opredelenie antitel i somaticheskogo O-antigena sal'monell gruppy B s pomoshch'iu komplementzavisimogo lizisa liposom.

A simple and sensitive method for the determination of group B Salmonella O-antigen and specific antibodies to group B Salmonella by means of the complement-dependent lysis of liposomes sensitized with S. typhimurium lipopolysaccharide (LPS) is proposed. The factors affecting the sensitivity of the method during the determination of antibodies and free antigen have been studied. The method permits the determination of soluble LPS antigen in concentrations of 0.5-200 micrograms/ml.

[Dynamics of accumulation of extracellular lipoteichoic acid in Bacillus cereus]. / Dinamika nakopleniia vnekletochnoi lipoteikhoevoi kisloty u Bacillus cereus.

The dynamic of accumulation of extracellular lipoteichoic acid (LTA) was studied depending on the growth stage of Bacillus cereus st. 96. A maximum amount of extracellular LTA was detected in the middle of the exponential growth. The quantity of the biopolymer present in the culture medium at the beginning of the stationary growth under conditions of catabolite repression of sporulation and without repression was found to be different. Experimentally increased concentrations of LTA inhibited B. cereus sporulation. Besides, dormant spores of B. cereus st. 96 were found to contain LTA.

[Isolation, purification and identification of protective substances from a culture broth of germinating Bacillus cereus spores]. / Vydelenie, ochistka i identifikatsiia protektiruiushchikh veshchestv iz kul'tural'noi zhidkosti prorastaiushchikh spor Bacills cereus.

A fraction increasing the resistance of resting spores to UV-irradiation and high temperature has been isolated from the culture medium at the stage of B. cereus st. 96 spore initiation. Amino acid analysis, gas chromatography, electrophoresis, and TLC of the products of acidic and alkaline hydrolysis of the isolated fraction demonstrated that the active component of the fraction was the lipoteichoic acid.

[Osmotic activity and ionic permeability of membrane vesicles from Streptococcus faecalis and Micrococcus lysodeikticus cells]. / Osmoticheskaia aktivnost' i ionnaia pronitsaemost' membrannykh puzyr'kov iz kletok Streptococcus faecalis i Micrococcus lysodeikticus.

Membrane fractions containing osmotically active vesicles with sufficiently low membrane permeability for K+, Na+ and Cl- ions typical for the intact cell membrane were isolated from the cells of the glycolyzing bacterium Streptococcus faecalis. In their osmotic properties and ionic permeability the membrane fractions of S. faecalis were found similar to those of the respiring bacterium Micrococcus lysodeikticus, which are capable of the energy-dependent potassium transport. It may be thus assumed that the S. faecalis fractions obtained may be used to study ionic transport. The removal of proton-dependent ATPase of the S. faecalis membrane preparations did not affect the permeability of membranes for K+ ions which is indicative of different mechanisms of proton and potassium translocation.

[Comparative study of membranes of Streptococcus faecalis and Micrococcus lysodeikticus]. / Sravnitel'noe izuchenie membran Streptococcus faecalis i Micrococcus lysodeikticus

A comparative study of ultrastructure and IR-spectroscopy of osmotic shock membranes from cells of glycolyzing (Streptococcus faecalis) and respiring (Micrococcus lysodeikticus) bacteria, was made. The S. faecalis and M. lysodeikticus membranes differ in their cross-section. Treatment of the preliminary washed membranes of S. faecalis and M. lysodeikticus with a low ionic strength solution removes 40% and 70% of their proteins respectively, decreases the membrane cross-section but does not change their fracture faces. Pre-cooling of the membrane suspensions within the temperature range of +5 degrees-10 degrees results in the appearance of large smooth areas on S. faecalis membrane fracture faces, but does not affect the ones of M. lysodeikticus membrane. Treatment of the washed suspensions with Triton X-100 results in the appearance of drastic changes of S. faecalis membrane fracture faces and does not change the fracture faces of M. lysodeikticus membranes; treatment by the detergent does not alter the IR-spectroscopy of membranes of both bacteria. Treatment of S. faecalis and M. lysodeikticus membranes with high temperature irreversibly changes the structure of 20% and 40% of protein components respectively,, but does not affect the distribution of the subparticles on their fracture faces. It is assumed that the differences found are determined by the composition of lipid components of the membranes studied and that the amount of proteins closely bound with lipids in the membranes of S. faecalis is likely to be greater than that of M. lysodeikticus membranes.

[Energization of membrane vesicles from the cells of glycolyzing bacterium Streptococcus faecalis]. / Energizatsiia membrannykh pyzyr'kov iz kletok glikoliziruiushchei bakterii Streptococcus faecalis.

Membrane fractions were isolated from Streptococcus faecalis cells of a glycolyzing microorganism, devoid of the respiratory chain, using the methods of osmotic shock of the protoplasts, ultrasonic treatment of the cells and ultrasonic treatment of the protoplasts. All fractions possessed the ATPase activity, the highest activity being observed in the fraction isolated by ultrasonication of the protoplasts. All preparations were estimated with respect to the presence of vesicles, formed by the "inside-out" and "inside-in" membranes, using ATPase as a marker of the membrane orientation. In the membrane fractions obtained by ultrasonication of the protoplasts, the "inside-out" vesicles were prevalent. ATP-dependent energization of the membranes, sensitive to the action of dicyclohexylcarbodiimide and tetrachlorotrifluoromethyl benzimidazole, was demonstrated by measuring the transport of the lipophylic anion of phenyldicarbaundecaborane and aniline naphthalene sulfonate fluorescence.

[Biochemical and ultrastructural characteristics of the membranes of Streptococcus faecalis]. / Izuchenie biokhimicheskikh i ul'trastrukturnykh kharakteristik membran Streptococcus faecalis

It is demonstrated by thin-section that S. faecalis cells (Strain BKM-B6) have no inner membranes, and their plasma membranes do not show any invagination into the cytoplasm. It is established that B and A fracture faces of the membranes in the intact bacteria, protoplasts, osmotic shock and sonicated membrane fractions contain uniformly distributed particles with a density of 200--400 and 4000--5000 particles/mu m2, respectively. Washing of the osmotic shock membrane fraction with a buffer solution of 0.25 M tris-SO4 with 2 M LiCl removes 35--40% of non-membrane proteins. Further washing (repeated 3 times) of the fraction with 0.001 M tris-SO4 removes additionally 40% of the protein, decreases the ATPase activity by 80%, and slightly decreases the cross-section of the membranes, but it does not affect of their A and B fracture faces. The membrane fracture faces A in all fractions exhibit small areas with tetragonally packed particles (with a tetragon size of 150 A X 150 A and a density of 20000--22000 particles/mu m2). These areas seem to be responsible for changing in the isolated membrane properties. Treatment of the washed membrane fractions with 0.2% triton X-100 results in a decrease of the vesicle sizes and appearance of drastic changes of their fracture faces. Changes of the membrane fracture faces under the action of triton X-100 are likely to be due to a breakdown in the hydrophobic interaction between the protein and lipid components. As it is shown by freez-fracturing technique, osmotic shock and sonicated membrane fractions contain 95% and 50% of rightside-out vesicles. It correlates with biochemical data on the membrane orientation of vesicles in different fractions.