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

2018 ◽  
pp. 60-65
Author(s):  
A. A. Kytmanov ◽  
G. D. Elagin ◽  
G. V. Kuklina ◽  
D. V. Pechenkin ◽  
O. O. Fomenkov ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Hybrid Cell ◽  
Cross Reactivity ◽  
Central Research Institute ◽  
Hybridoma Cells ◽  
Central Research ◽  
Test Systems ◽  
Test Kit

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.

scholarly journals Development of Enzyme-Immunoassay for Bacillus anthracis Detection

2018 ◽  
pp. 78-82
Author(s):  
D. V. Pechenkin ◽  
O. O. Fomenkov ◽  
A. V. Eremkin ◽  
G. D. Elagin ◽  
G. V. Kuklina ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Bacillus Anthracis ◽  
Enzyme Immunoassay ◽  
Specific Activity ◽  
Hybrid Cell ◽  
Sandwich Elisa ◽  
Central Research Institute ◽  
Exchange Chromatography ◽  
Central Research ◽  
Test Kit

Objective of the study was to develop enzyme-immunoassay test-kit for the detection of Bacillus anthracis spores. Materials and methods. Microbial cultures from the State Collection of Microorganisms at the premises of Affiliated Branch of the «48th Central Research Institute» of the Ministry of Defense of the Russian Federation and BALB/c mice were used in the research. Hybridization of B-lymphocytes with SP2/0-Ag14 myeloma cells was performed according to G. Kohler and C. Milstein procedure in De St. Fazekas and P. Scheidegger modification. Hybridomas were cultured in the peritoneal cavity of BALB/c mice. Ascitic fluids were isolated from mice, precipitated with ammonium sulfate and purified by means of ion-exchange chromatography for preparation of monoclonal antibodies. Specific activity of hybridoma’s supernatants, ascitic fluids, purified monoclonal antibodies was studied by «sandwich» ELISA. Specific components of test-kit were lyophilized in suitable cryoprotective medium. Results and conclusions.We have obtained new hybrid cell lines producing specific monoclonal antibodies against Bacillus anthracisspore antigens and ascitic fluids from which immunoglobulins were isolated. Optimum combinations of monoclonal antibodies as a sensitizer and a component of immunoperoxidase conjugates have been selected. Monoclonal antibodies 272E10G1-272F7A10 provide the highest sensitivity of ELISA for the detection of anthrax microbe spore antigens. Our enzyme-immunoassay test allows for identification of Bacillus anthracis spores in concentrations up to 5,0·105 spores per milliliter. No cross reaction with closely related saprophytes and other heterologous microorganisms in concentrations of 1,0·108 CFU per milliliter is observed.

scholarly journals Fermented Antler Improves Endurance during Exercise Performance by Increasing Mitochondrial Biogenesis and Muscle Strength in Mice

2021 ◽  
Vol 11 (12) ◽  
pp. 5386
Author(s):  
Seongeun Jung ◽  
Sung-Hwan Kim ◽  
Woonhee Jeung ◽  
Jehyun Ra ◽  
Keon Heo ◽  
...  
Keyword(s):  
Muscle Strength ◽  
Mitochondrial Biogenesis ◽  
Exercise Performance ◽  
Muscle Protein ◽  
Muscle Protein Synthesis ◽  
Central Research Institute ◽  
C2c12 Cells ◽  
Central Research

In this study, we investigated whether antler fermented with lactic acid bacteria (LAB) increases mitochondrial biogenesis and muscle strength in vitro and in vivo. LAB from a strain library were grown in antler extract agar at the Yakult Central Research Institute of Korea. Isolated LAB, named Lactobacillus curvatus HY7602, were used to ferment antlers. Analysis of the effects of fermented antler (FA) revealed that it enhanced the insulin-like growth factor 1 (IGF-I), signaling pathway and mitochondrial metabolic activity in mouse skeletal myotube (C2C12) cells. Next, we evaluated the effect of non-fermented antler (NFA) and FA on exercise performance in C57BL/6J mice. The results showed that HY7602-FA increased treadmill exercise capacity and forced swimming endurance. Furthermore, blood markers associated with muscle fatigue, endurance, and energy supply (e.g., alanine aminotransferase, lactate dehydrogenase, creatinine, creatine kinase, and lactate) in the FA-intake group were lower than in the NFA-intake group. In addition, the expression index of genes associated with muscle protein synthesis, and with mitochondrial energy production and supply, in muscle tissue was remarkably higher in the FA group than in the control and NFA groups. Taken together, these results suggested that HY7602-FA may be an effective functional food and health supplement.

scholarly journals Preparation and Characterization of a New Monoclonal Antibody Specific Against Lawsonia intracellularis and Its Application in Indirect Immunofluorescence and Immunocytochemistry Assay

2021 ◽  
Vol 8 ◽  
Author(s):  
Ning Xiao ◽  
Jiannan Li ◽  
Minxue Li ◽  
Yuting Hu ◽  
Huixing Lin ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Indirect Immunofluorescence ◽  
Enteric Bacteria ◽  
Cross Reactivity ◽  
Immunofluorescence Assay ◽  
Lawsonia Intracellularis ◽  
Brachyspira Hyodysenteriae

Proliferative enteropathy (PE) is an infectious enteric disease caused by Lawsonia intracellularis (L. intracellularis) and is endemic in pig herds worldwide. However, a L. intracellularis-specific monoclonal antibody plays an important role in the evaluation of L. intracellularis infection in vitro. Therefore, the objective of this study was to produce and identify the characteristics of a new monoclonal antibody against the outer membrane protein (Omp2) of L. intracellularis and apply it in an indirect immunofluorescence assay (IFA) and immunocytochemistry (IHC). The results indicated that three highly specific monoclonal antibodies against the Omp2 protein (4D9, 3G2, and 7G5) of L. intracellularis were obtained by using purified Omp2 as an immunogen, the titers of ascitic fluids of 4D9, 3G2, and 7G5 cells were 1:2,048,000, 1:512,000, and 1:256,000, respectively. IFA analysis showed that the 4D9, 3G2, and 7G5 have no cross-reactivity with other enteric bacteria commonly found in the ilea of pigs or closely related to L. intracellularis, such as Desulfovibrio, Bilophila wadsworthia (B. wadsworthia), Salmonella choleraesuis (S. choleraesuis), Salmonella typhimurium (S. typhimurium), Escherichia coli (E. coli), and Brachyspira hyodysenteriae (B. hyodysenteriae). IFA and IHC results indicated that the monoclonal antibodies can be successfully used as primary antibodies to detect L. intracellularis in infected cells and in the crypt of the ileum from infected tissues of PE. Our findings suggested that the new monoclonal antibody specific against L. intracellularis will be useful for the evaluation of L. intracellularis infection in vivo and in vitro.

Development of enzyme immunoassay for detecting I and II types of shiga-like toxins

2021 ◽  
Vol 29 (5) ◽  
pp. 43-48
Author(s):  
Galina Viktorovna Kuklina ◽  
Denis Valerievich Pechenkin ◽  
Sergei Sergeevich Ipatov ◽  
Andrei Valentinovich Eremkin ◽  
Aleksei Aleksandrovich Kytmanov ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Enzyme Immunoassay ◽  
Solid Phase ◽  
Sandwich Elisa ◽  
Central Research Institute ◽  
Exchange Chromatography ◽  
Hybridoma Cells ◽  
Federal State ◽  
Central Research ◽  
Freeze Dried

Introduction. The aim of the work was development of enzyme immunoassay for detecting I and II types of shiga-like toxins and assessment of it diagnostic properties. Materials and methods. For the research, we used hybridomas producing monoclonal antibodies to shiga-like toxins of types I and II, obtained at the branch of the Federal State Budgetary Institution “48 Central Research Institute” of the Ministry of Defense of Russian Federation (Kirov); BALB/c mice; shiga-like toxins of types I and II. Hybridoma cells were cultured in culture flasks and in the peritoneal cavity of BALB/c mice. Monoclonal antibodies were isolated from ascitic fluids by precipitation with a saturated solution of ammonium sulfate, followed by purification by ion exchange chromatography. The obtained preparations of monoclonal antibodies were used to develop enzyme immunoassay for the detection of shiga-like toxins of types I and II. Specific components of enzyme immunoassay were freeze-dried in a protective environment. Results. As a result of research, preparative quantities of monoclonal antibodies against I and II types of shiga-like toxins were obtained and purified; selection of monoclonal antibodies for sorption on the solid phase and for the synthesis of immunoperoxidase conjugates was carried out. Conclusion. experimental enzyme immunoassay allowing to identify 1 ng/ml I and II types of shiga-like toxins in «sandwich»-ELISA was developed.

scholarly journals Development of Elisa Test-Systems and Immune-Chromatographic Reagent Panel Designed for the Detection of Staphylococcal Enterotoxins of A and B Types

2021 ◽  
pp. 94-99
Author(s):  
A. V. Eremkin ◽  
S. S. Ipatov ◽  
G. V. Kuklina ◽  
D. V. Pechenkin ◽  
A. A. Kytmanov ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Elisa Test ◽  
Food Samples ◽  
Central Research Institute ◽  
Exchange Chromatography ◽  
Lateral Flow ◽  
Hybridoma Cells ◽  
Central Research ◽  
Staphylococcal Enterotoxins ◽  
The Russian Federation

Objective of the study was the development of experimental ELISA tests and lateral flow immunoassays for detection of staphylococcal enterotoxins, A and B types.Materials and methods. Hybridomas, producing monoclonal antibodies against staphylococcal enterotoxins A and B from State Collection of the Affiliated Branch of the “48th Central Research Institute” of the Ministry of Defense of the Russian Federation, BALB/c mice and staphylococcal enterotoxins A and B were used in the research. Hybridoma cells were incubated in culture flasks and in the peritoneal cavity of BALB/c mice. Monoclonal antibodies were isolated from ascitic fluids through precipitation with saturated ammonium sulfate subsequently purified using ion-exchange chromatography. Obtained preparations of monoclonal antibodies were used for the construction of ELISA tests and immune-chromatographic reagent panels for the detection of staphylococcal enterotoxins A and B. Specific components of ELISA tests were lyophilized in protective media.Results and discussion. ELISA tests and lateral flow immunoassays which allow for detecting staphylococcal enterotoxins A and B at concentrations of 0.5 ng/ml and higher, including in food samples, have been constructed. 

scholarly journals Obtaining and Characterization of Hybridomas Producing Monoclonal Antibodies to Shiga-Like Toxins of I and II Types

2021 ◽  
pp. 83-88
Author(s):  
G. V. Kuklina ◽  
S. S. Ipatov ◽  
D. V. Pechenkin ◽  
A. V. Eremkin ◽  
A. A. Kytmanov ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Cell Lines ◽  
Hybrid Cell ◽  
Sandwich Elisa ◽  
Minimum Detectable Concentration ◽  
Myeloma Cells ◽  
Hybrid Cell Lines

Objective – obtaining and characterization of hybrid cell lines producing monoclonal antibodies against I and II types of shiga-like toxins.Materials and methods. Shiga-like toxins obtained in “48thCentral Research Institute” of Ministry of Defense of Russian Federation (Kirov), BALB/c mice, myeloma cells SP2/0-Ag14 were used in research. Immune splenocytes and SP2/0-Ag14 myeloma cells were fused according to G. Kohler and C. Milstein method in De St. Fazekas and D. Scheidegger modifcation using 50 % polyethylene glycol. Hybrid cell lines producing specifc monoclonal antibodies were cloned by limited dilutions. Hybridomas growth and producing properties were studied in vitro and in vivo. Specifc activity of immune sera, culture and ascitic fluids were studied by indirect ELISA. Monoclonal antibodies from ascitic fluids were precipitated by saturated ammonium sulfate, followed by ion exchange chromatographyResults and discussion. 8 hybridomas producing monoclonal antibodies against I and II types shiga-like toxins were obtained. Hybridomas are characterized by stable proliferation and antibody-producing activity during 10 passages in vitro and 3 passages in vivo (observation period). Obtained monoclonal antibodies can be used for ELISA detection of I and II types shiga-like toxins. Minimum detectable concentration of shiga-like toxins in sandwich ELISA is 1 ng/ml. The possibility of detecting shiga-like toxins without typical differentiation was shown when using in the enzyme immunoassay a polyreceptor mixture of monoclonal antibodies for sensitizing the plate and a polyspecifc mixture of immunoperoxidase conjugates.

A Molecular Approach to Immunoscintigraphy: A Study of the T-Antigen Conformation on the Surface of Tumors

1987 ◽  
Vol 26 (01) ◽  
pp. 1-6 ◽  
Author(s):  
S. Selvaraj ◽  
M. R. Suresh ◽  
G. McLean ◽  
D. Willans ◽  
C. Turner ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Tumor Cell ◽  
T Antigen ◽  
Human Carcinomas ◽  
Animal Tumor ◽  
Synthetic Antigens

The role of glycoconjugates in tumor cell differentiation has been well documented. We have examined the expression of the two anomers of the Thomsen-Friedenreich antigen on the surface of human, canine and murine tumor cell membranes both in vitro and in vivo. This has been accomplished through the synthesis of the disaccharide terminal residues in both a and ß configuration. Both entities were used to generate murine monoclonal antibodies which recognized the carbohydrate determinants. The determination of fine specificities of these antibodies was effected by means of cellular uptake, immunohistopathology and immunoscintigraphy. Examination of pathological specimens of human and canine tumor tissue indicated that the expressed antigen was in the β configuration. More than 89% of all human carcinomas tested expressed the antigen in the above anomeric form. The combination of synthetic antigens and monoclonal antibodies raised specifically against them provide us with invaluable tools for the study of tumor marker expression in humans and their respective animal tumor models.

scholarly journals In vitro and in vivo inhibition of malaria parasite infection by monoclonal antibodies against Plasmodium falciparum circumsporozoite protein (CSP)

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Merricka C. Livingstone ◽  
Alexis A. Bitzer ◽  
Alish Giri ◽  
Kun Luo ◽  
Rajeshwer S. Sankhala ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Monoclonal Antibodies ◽  
Disease Burden ◽  
Vaccine Candidate ◽  
Specific Binding ◽  
Circumsporozoite Protein ◽  
Target Epitope ◽  
Selection Of

AbstractPlasmodium falciparum malaria contributes to a significant global disease burden. Circumsporozoite protein (CSP), the most abundant sporozoite stage antigen, is a prime vaccine candidate. Inhibitory monoclonal antibodies (mAbs) against CSP map to either a short junctional sequence or the central (NPNA)n repeat region. We compared in vitro and in vivo activities of six CSP-specific mAbs derived from human recipients of a recombinant CSP vaccine RTS,S/AS01 (mAbs 317 and 311); an irradiated whole sporozoite vaccine PfSPZ (mAbs CIS43 and MGG4); or individuals exposed to malaria (mAbs 580 and 663). RTS,S mAb 317 that specifically binds the (NPNA)n epitope, had the highest affinity and it elicited the best sterile protection in mice. The most potent inhibitor of sporozoite invasion in vitro was mAb CIS43 which shows dual-specific binding to the junctional sequence and (NPNA)n. In vivo mouse protection was associated with the mAb reactivity to the NANPx6 peptide, the in vitro inhibition of sporozoite invasion activity, and kinetic parameters measured using intact mAbs or their Fab fragments. Buried surface area between mAb and its target epitope was also associated with in vivo protection. Association and disconnects between in vitro and in vivo readouts has important implications for the design and down-selection of the next generation of CSP based interventions.

scholarly journals Previous Humoral Immunity to the Endemic Seasonal Alphacoronaviruses NL63 and 229E Is Associated with Worse Clinical Outcome in COVID-19 and Suggests Original Antigenic Sin

Life ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 298
Author(s):  
Daniele Focosi ◽  
Angelo Genoni ◽  
Ersilia Lucenteforte ◽  
Silvia Tillati ◽  
Antonio Tamborini ◽  
...  
Keyword(s):  
Humoral Immunity ◽  
Cross Reactivity ◽  
Clinical Severity ◽  
World Health ◽  
Laboratory Findings ◽  
Cellular And Humoral Immunity ◽  
Rate Ratios ◽  
Original Antigenic Sin

Antibody-dependent enhancement (ADE) of severe acute respiratory syndrome coronavirus-2 (SARS CoV-2) infection has been hypothesized. However, to date, there has been no in vitro or in vivo evidence supporting this. Cross-reactivity exists between SARS CoV-2 and other Coronaviridae for both cellular and humoral immunity. We show here that IgG against nucleocapsid protein of alphacoronavirus NL63 and 229E correlate with the World Health Organization’s (WHO) clinical severity score ≥ 5 (incidence rate ratios was 1.87 and 1.80, respectively, and 1.94 for the combination). These laboratory findings suggest possible ADE of SARS CoV-2 infection by previous alphacoronavirus immunity.

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