[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.

[Development of the quantitative immunochromatography tests for somatic deasease markers detection].

The purpose of work was creation of quantative immunochromatographic tests (ICT) for measuring concentrations of the marker substance associated with somatic diseases: immunoglobulin E (IgE), C-reactive protein (CRP) and fibrin D-dimer in blood serum (plasma), which is carried out with the help of videodigital analyzers of domestic development "Reflecom" and "Zondaj". ICT were designed in sandwich-format, using colloid gold and monoclonal antibodies. It is shown, that calibration curves received with the help of ICTand devices of videodigital registration, are well approximated by exponential dependence Y = Aexp(-x/B)+y0, where Y--device readings, x--analytes concentration, A, B, y0--constants. Samples of blood serum (plasma), under investigation received in conditions of a hospital from patients. Correlation with electrochemiluminescent immunoassay, enzyme linked immunoassay, latex-agglutinations assay and immunochromatographic method was observed. Sensitivity of the developed tests was 7,5 IU/ML for IgE, 5,7 ng/ml for CRP, 500 ng/ml for fibrin D-dimer. The developed analytical complex- videodigital analyzer "Zondaj" and ICT for quantitative measure of concentration IgE, fibrin D-dimer and CRP- can be successfully applied in laboratory practice and clinical laboratory researches alongside with actual immunochemistry methods.

[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.

[Development and optimization of immunoassays for the detection of botulinum toxins].

Monoparametric immunoassay tests for detecting botulinum toxins types A and B and multiparametric assays for simultaneous detection of botulinum toxins type A and B have been developed. It is shown that the sensitivity of assays is affected by the size of nanoparticles of colloidal gold used as a marker of antibodies, load intensity of antibodies of colloidal gold in conjugates, the type of analytical membranes, as well as the chemical composition of buffer solutions used for the storage of conjugates and immunoassay analysis. The detection limit of monoparametric immunoassay tests is 0.5 ng/ml; that of multiparametric assays, 5.0 ng/ml. The developed immunoassay can be used for rapid assay of product quality, for grade control of botulinum toxins in pharmaceuticals, and environmental monitoring.

[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.

[Indication of extremely dangerous infectious pathogens using immunochromatography and digital video analysis].

The use of immunochromatographic indicatory elements based on antibody conjugates and colloidal gold was suggested to detect cells and the antigens of extremely dangerous infectious pathogens. The specificity and specific activity (sensitivity) of the mentioned elements were studied on vaccinal strains of plague, anthrax, and tularemia pathogens. The researchers studied a possibility to increase the sensitivity of immunochromatographic analysis using computed scanning and Reflecom, a specialized digital video recorder of immunochromatogramms. The study demonstrated that the use of electronic devices to record immunochromatogramms increased the sensitivity to microbial cells and antigens 1.5 to 2 times vs. visual registration and simplified documentation of the results.

[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.

[Change in the permeability of liposome phospholipid membranes under the effect of Bacillus thuringiensis delta-endotoxins]. / Izmenenie pronitsaemosti fosfolipidnykh membran liposom pod deistviem preparatov delta-éndotoksinov Bacillus thuringiensis.

We studied release of the fluorescent calcein marker from egg-lecithin liposomes under action of intact delta-endotoxin from Bacillus thuringiensis var. israilensis, and hydrolysed preparation of delta-endotoxin from Bacillus thuringiensis var. kurstaki together with the motional parameters of 5- and 12-doxyl stearic radical probes in the liposomal membrane. The yield of the non-penetrating fluorescent marker was shown to be a sensitive indicator of the disturbance of the membrane barrier function.

[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 use of solid-phase liposomal immunoassay for determining a bacterial antigen of lipopolysaccharide nature]. / Ispol'zovanie tverdofaznogo liposomal'nogo immunoanaliza dlia opredeleniia bakterial'nogo antigena lipopolisakharidnoi prirody.

The solid-phase liposomal immunoassay procedure for the determination of Francisella tularensis lipopolysaccharide (LPS) has been developed. This assay has been made with the use of monolayer liposomes, on the average, 360 nm in diameter with their phospholipid bilayer modified with F. tularensis LPS and their internal space filled with calcein used as fluorescent marker. The assay is based on the principle of the competitive immunosorption of liposomes and antigenic LPS on the surface of polystyrene plates sensitized with specific monoclonal antibodies. The dynamic range of the method is 50-2,500 mg/ml, the variation index being 2.3-11.5%. Optimization of this method and its comparison with the enzyme immunoassay system for the given antigen-antibody pair has been made.

[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.