[The identification of the AB0 blood type system by means of the single nucleotide polymorphisms analysis]. / Opredelenie gruppovoi prinadlezhnosti po sisteme AV0 metodom SNP-analiza.

The authors describe a domestically produced test-system for the determination of the AB0 blood type by means of the single nucleotide polymorphisms (SNP) analysis. The results of the trials indicate that the proposed test-system can be employed for the investigation of DNA specimens of individual origin obtained from any objects of expertise including micro-objects containing human nuclear DNA.

[The detection of RhD antigen of the rhesus system by the real time polymerase chain reaction].

A method for the detection of RhD using a DNA preparation and the newly-developed test-system based on the real time polymerase chain reaction is described. The study including a large variety of specimens has demonstrated the applicability of the novel system to forensic medical examination.

Effect of producer cell line on functional activity of anti-D monoclonal antibodies destined for prevention of rhesus sensitization.

The ability of anti-D antibodies to cause antigen-specific immunosuppression depends on their interaction with low-affinity Fcgamma-receptors. Human monoclonal antibodies to D antigen of the rhesus system were investigated by antibody-dependent cytotoxicity assay in order to estimate their ability to induce hemolysis mediated by low-affinity Fcgamma receptors. We demonstrate that affinity of monoclonal antibodies to receptors of this type does not depend on primary structure of Fc-fragment, but depends on the producer cell line which expresses the antibodies. Monoclonal IgG1 antibodies interacting with FcgammaRIIa and FcgammaRIII lost this property, if they were secreted by human-mouse heterohybridoma, but not by human B-cell line. On the opposite, monoclonal antibodies that could not activate low-affinity Fcgamma receptors were highly active after human cells fusion with rat myeloma YB2/0. Hemolytic activity of IgG3 remained unchanged after fusion of human cells with rodent cells.

[Detection of ABH-Lewis antigens in trace discharges using one-stage immunoenzyme assay with dot modification].

A one-step modification ofimmunoenzyme assay (dot variant) is proposed for the detection of ABH-Lewis antigens in human discharges (saliva, sperm, vaginal discharge). Sensitivity and specificity of the new method is higher than those of other methods currently used in forensic medicine for the detection of antigens in human body discharges; its other advantages over routine techniques include procedural simplicity, high performance, and the possibility of conducting series measurements with minimal labour inputs. Moreover, it provides information not only about group characteristic but also about category of discharge. The analysis is carried out in supernatant fractions which permits to reserve cellular material for molecular-genetic studies.

[The development and application of an immunoenzyme assay kit for the detection of compounds of the opiate family in human biological liquids].

Monoclonal antimorphine antibodies both free and conjugated with horse- radish peroxidase have been raised and used to develop an assay kit for the detection of narcotic opiate-based drugs by an immuno-enzyme assay (IEA). The kit contains all ingredients necessary for the enzymatic reaction. A total of 215 urine and blood samples were analysed using the new kit. The results were compared with the data obtained by thin layer chromatography and high-performance liquid chromatography. False negative results were absent while false positive (inconclusive) results were recorded in three cases, probably due to the fact that sensitivity of IEA is higher than that of control methods. It is concluded that the kit may be used in laboratory screening studies for detecting opiates in biological fluids.

Regeneration of the liver in mice treated with a mixture of hepatotoxins in delayed periods after bone marrow transplantation.

Possible effect of bone marrow cells on liver regeneration was studied in mice injected with a mixture of hepatotoxins (allyl alcohol and CCl4) in a dose equal to LD50. The mixture of hepatotoxins was used to minimize the restitution regeneration of the liver. The dose of allyl alcohol causing (in combination with CCl4) maximum liver damage was selected beforehand. Increasing the dose of allyl alcohol in the two-component mixture resulted in more severe necrosis of the liver. The maximum dose of alcohol (50 mg/kg) in combination with CCl4 caused irreversible injury to the liver leading to 100% mortality after 2-4 days. In radiation chimeras reconstituted by bone marrow cell transplantation, in which liver damage was induced by a mixture of hepatotoxins containing the maximum dose of allyl alcohol, we observed normalization of liver tissue structure and function. The mechanism of this effect is not clear.

Lymphocyte antibody-dependent cytotoxicity test for evaluation of clinical role of monoclonal anti-D-antibodies for prevention of rhesus sensitization.

Monoclonal antibodies to D antigen were studied in the reaction of antibody-dependent cytotoxicity for evaluation of the possibility of using these antibodies for preventing rhesus sensitization. High hemolytic activity of four anti-D-monoclonal antibodies in the antibody-dependent cytotoxicity test, mediated by their interaction with FcgammaRI, and the capacity to accelerate elimination of D+ erythrocytes from circulation did not provide the immunosuppressive effect. It was hypothesized that monoclonal antibodies for prevention of rhesus sensitization should interact with FcgammaRIII on lymphocytes. These monoclonal antibodies are extremely rare: only 4 of 125 studied antibodies mediated hemolysis in the antibody-dependent cytotoxicity test with lymphocytes, while all polyclonal anti-D-preparations exhibited this activity.

[Preparation, evaluation of specificity and use of anti-Le monoclonal antibodies]. / Poluchenie, opredelenie spetsifichnosti i primenenie monoklonal'nykh anti-Le-antitel.

Anti-Le(a) and anti-Le(b) monoclonal antibodies were obtained and attempts at evaluating their epitope specificity have been made. A method for identification of Lewis phenotypes in salivary specimens by dot-I enzyme immunoassay has been developed. Analyses of salivary samples from 72 donors detected donors with phenotypes Le(a-b-) and Le(a+b+), which was confirmed by hemagglutination test.

[Immunoglobulin G monoclonal human anti-rhesus Rho(D) to prevent rhesus-incompatibility]. / Immunoglobulin G monoklonal'nyi chelovecheskii anti-rezus Rho(D) dlia profilaktiki rezus-nesovmestimosti.

RhD immunisation which follows pregnancy can be prevented by administration to the rhesus-negative mother of 20 micrograms anti-D immunoglobulin per 1 ml of D-positive fetal red cells in the maternal circulation. With the aim of substitution of polyclonal anti-D with monoclonal one we developed human-mouse cell lines producing anti-RhD IgG1 by fusing EBV-transformed human immune lymphocytes with murine myeloma. After several clonings and passages the EBV genome was eliminated from the cell lines. The antibodies were purified from culture supernatants using protein A affinity chromatography and tested for sterility, virus contamination, pyrogenecity, toxicity and DNA content. The monoclonals were compared with the standard polyclonal anti-RhD in in vitro and in vivo assays. In antibody-dependent cell cytotoxicity (ADCC) 2 of 4 studied monoclonals promoted greater RBS lysis than polyclonal anti-D at equivalent concentrations. Detection of binding site number of monoclonals anti-D revealed about 10,000/RBS D-determinants on DCe/dce erythrocyte which agrees with the data for polyclonal anti-D. Best in ADCC monoclonal anti-D sharply increased human autologous 51Cr-labelled rbc sensitised in vitro as well as accelerated clearance of RhD RBS from circulation of Rh-negative volunteers injected with 150 micrograms monoclonal anti-D. After clinical trial using of monoclonal anti-D is permitted in Russia.

[Monoclonal anti-D immunoglobulin for prevention of hemolytic disease of the newborn]. / Monoklonal'nyi anti-D immunoglobulin dlia profilaktiki gemoliticheskoi bolezni novorozhdennykh.

Stable human-mouse heterohybridoma producing human monoclonal anti-D IgG1 has been derived by fusing Epstein-Barr virus (EBV) transformed human immune lymphocytes with mouse myeloma. The absence of EBV DNA in heterohybridoma was demonstrated by the polymerase chain reaction. The antibody was purified by affinity chromatography on protein A Sepharose and tested for sterility, pyrogenicity and toxicity according to the Pharmacopoeia XI and the ability to clear from circulation RhoD-positive red cells. The time of clearance of sensitized RBC was about 3 hrs. Since it is believed to be an association between the rate of RBC clearance by anti-D and the ability of the antibody to suppress the immunization, the monoclonal immunoglobulin may prove suitable for immunoprophylaxis of RhD hemolytic disease.

[The use of immunoenzyme analysis for determining ABH antigens in traces of saliva and sperm]. / Primenenie immunofermentnogo analiza dlia opredeleniia ABH-antigenov v sledakh sliuny i spermy.

The authors suggest a simple sensitive technique for enzyme immunoassay (EIA) of ABH antigens in saliva and semen. A two-staged dot blot solid-phase EIA on nitrocellulose membranes was employed with anti-ABH monoclonal antibodies obtained in immunization of mice with human red cells. 4-chloro-1-naphthol substrate solution was used to visualize the peroxidase label. The results of analysis of salivary and spermatic samples obtained from donors of various groups evidence that this EIA variant may be useful in forensic medicine.

[Ultrasonic and x-ray parallels in the diagnosis of cholelithiasis]. / Ul'trazvukovye i rentgenologicheskie paralleli v diagnostike kholelitiaza.

The sensitivity and specificity of ultrasound investigation (USI) and x-ray methods of indirect and direct contrast studies of the biliary system in the diagnosis of cholecysto- and choledocholithiasis were compared by a blind control method. USI was shown to be superior over all x-ray contrast methods in the diagnosis of cholecystolithiasis. The diagnosis of choledocholithiasis usually requires the use of x-ray methods among which preference should be given to endoscopic retrograde cholangiography as the most informative method permitting simultaneous intervention on the major duodenal papilla.