Continuous copper leaching technology

The parameters of the continuous technological process of leaching copper from fine copper waste using nitric acid as an oxidizer are studied. Optimal conditions for a continuous leaching process were established, in which solutions with a mass concentration of copper ions greater than 25 g/dm3 were obtained

Development of Immune-Chromatographic Monoclonal Test-System for the Detection of <i>Yersinia pseudotuberculosis</i>, Serogroup I

Objective of the study was to develop monoclonal immunoassay for the detection of the pseudotuberculosis agent, serogroup I. Materials and methods. Specific components, that were used for immune-chromatographic test-system development were mouse monoclonal antibodies of hybrid cell lines, obtained to lipopolysaccharide antigen of the outer membrane of the pathogen’s «cold» variant (YP-101N2V4, YP-105S5A10); and rabbit anti-species antibodies against murine immunoglobulins. Particles, (30±2) nm in the diameter, were used to prepare colloidal gold-antibody conjugate. Antibody concentration for conjugation was 10-15 % greater than the D580 exit point on the plateau. For the production of immune-chromatographic test-system a set of membranes - MDI Easypack - manufactured by «Advanced Microdevice», India was deployed. Finished conjugate was applied onto the membrane by means of impregnation. Antibodies in the selected quantities were applied onto the analytical and control membranes via Dispensers. Substrates coated with the conjugate and ready-made working membranes were vacuum dried in a heat cabinet. Assembled immune-chromatographic test-systems were cut off 4.5 mm each and tested for specificity and sensitivity. Results and conclusions. Developed has been immune-chromatographic test-system for the detection of pseudotuberculosis pathogen, serogroup I. Utilized have been monoclonal antibodies of the hybrid cell line YP-105C5A10 in colloidal gold conjugate and monoclonal antibodies of the hybrid cell line YP-101H2B4 in the test line. The test-system allows for the detection of Y. pseudotuberculosis strains, serogroup I, at concentrations varying from 500 ths. m.c.·cm-3 (8 of the 11 strains under study) up to 4 million m.c.·cm-3 and does not identify closely related yersinia and heterologous microorganisms in quantities of 100 million m.c.·cm-3

MANUFACTURING OF HYBRIDOMAS-PRODUCERS OF MONOCLONAL ANTIBODIES TO BRUCELLOSIS AGENT ANTIGENS

Objective of study is to prepare hybridomas-producers of monoclonal antibodies to brucellosis agent antigens. Materials and methods. B. abortus, B. melitensis, B. suis strains from the State collection of microorganisms of the 48th Central Research Institute Affiliated Branch and BALB/c mice. Hybridization was performed as described by G.Kohler and C.Milstein in modification by Fazekas De St. and Scheidegger D. The study of specific activity of immune sera, hybridoma supernatants, ascites fluid, and monoclonal antibody preparations was performed using ELISA. Results and conclusions. Obtained and characterized have been hybridomas-producers of monoclonal antibodies to specific antigens of brucellosis agent. They are active and stable antibody producers in the repeated passaging both, in vitro and in vivo. Obtained have also been the ascites fluid and preparations of monoclonal antibodies of brucellosis agent. Carried out has been substantiated selection of antibodies which could provide for the most sensitive ELISA. It is established that the monoclonal antibodies produced by hybridomas 232B6H7, 232G12F7, 233B2C5 in combination with brucellosis rabbit immunoglobulins allow for the identification of microbial cells of type strains of various Brucella species in concentrations ranging from 0,25·106 mc·sm–3 up to 1,0·106 mc·sm–3 and gave negative results with cultures of heterologous microorganisms in the contents of 1,0·108 mc·sm–3. Hybridomas-producers of monoclonal antibodies are planned to be used for the construction and manufacturing of immunodetection test-systems

Obtaining and Characterization of Hybridomas Producing Monoclonal Antibodies against Coronavirus SARS-CоV-2

The aim of the work was to obtain and characterize hybridomas producing monoclonal antibodies to antigens of coronavirus SARS‑CoV‑2, promising for the construction of diagnostic immunochemical tests. Materials and methods. Recombinant nucleocapsid and receptor binding fragment of spike protein of SARS‑CoV‑2 were used for immunization of BALB/c mice. Antigens were absorbed on aluminium hydroxide gel and injected subcutaneously to BALB/c mice at a 7-day-interval. Immune splenocytes and myeloma cells SP2/0-Ag14 were fused by polyethylene glycol 1450. Cell cultures producing specific antibodies against nucleocapsid and receptor binding fragment were selected applying indirect ELISA in 96-well plates sensitized by desired antigens. Clones of hybridomas were obtained using the method of limiting dilutions. Production properties were studied through in vitro cultivation in 24-well culture plates. Immune-ascitic fluids were collected during the cultivation of hybrid cells in peritoneal cavities of BALB/c mice. Monoclonal antibodies were purified by affinity chromatography on protein A sepharose sorbent, conjugated with horseradish peroxidase, and tested for the possibility to be used in sandwich ELISA for detection of inactivated SARS‑CoV‑2 coronavirus strain “Isolate B”. Results and discussion. As a result of hybridization and selection of clones, hybridomas producing monoclonal antibodies to nucleocapsid and receptor binding fragment of SARS‑CoV‑2 have been obtained. During the in vitro and in vivo cultivation the clones maintained the consistent proliferative and antibody producing activity. The application of monoclonal antibody 415D12 as a capture one and 411D12 antibody conjugated with horseradish peroxidase as a detector antibody in ELISA allows for identifying SARS‑CoV‑2 coronavirus at a minimum concentration of 1·103 PFU per ml

Development of Immuno-Enzymatic Monoclonal Tests-Systems for the Detection of Glanders and Melioidosis Agents

Objective of the study was the development of immune-enzymatic monoclonal test-kit for detecting glanders and melioidosis agents. Materials and methods. We used microbial cultures and hybrid cell lines obtained from the collection of the «48th Central Research Institute» of the Ministry of Defense of the Russian Federation. Hybridoma cells were incubated in the peritoneal cavity of BALB/c mice. Preparations of glanders and melioidosis monoclonal antibodies were isolated from the ascetic fluids through precipitation with ammonium sulfate and purification by means of ion-exchange chromatography. Specific components of the test-kits were subjected to freeze drying in corresponding protective media. Study of diagnostic properties of the developed test systems was performed using ELISA. Results and conclusions. We have obtained preparations of monoclonal antibodies in vivo, as well as isolated and purified immunoglobulins from ascetic fluids. We also selected the pairs of monoclonal antibodies for manufacturing specific components. Experimental series of immune-enzymatic monoclonal test-systems allowing for specific detection of glanders and melioidosis causative agents in concentrations ranging from 0.5·106 CFU/ml and higher were made. The absence of cross-reactivity with closely related saprophytes and heterologous microorganisms in concentrations of 1,0·108 CFU/ml was shown. Demonstrated was the possibility in principle to differentiate between Burkholderia malleiand Burkholderia pseudomallei using ELISA. Test systems are promising for follow up state registration as medical products for in vitro diagnostics

Manufacturing of Hybridomas-Producers of Monoclonal Anti-Bodies to Tularemia Agent Antigens

Carried out have been two experimental studies on hybridization of mouse myeloma cells and lymphocytes of BALB/c mice immunized with inactivated microbe Francisella tularensis cultures. As a result obtained have been hybridomas-producers of monoclonal antibodies (MAb) specific to the antigens of tularemia agent. Evaluated have been the prospects of its application for the detection of the agent under discussion using enzyme-linked immunoassay. Established is the fact that monoclonal antibodies produced by 31G1F10, 32E5D3, 35B11C8, 36C2F11 hybridomas make it possible to identify microbe cells of various tularemia agent strains when concentrated up to 0.5·106 mc/sm3, and do not interact with cultures of heterologous microorganisms when concentrated to 1.0·108 mc/sm3, which testifies to their specificity. These MAb are planned to be used for the construction of immune-enzyme and immune-chromatographic test-systems designed for tularemia agent detection