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 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 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 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.

Sandwich enzyme immunoassay of osteocalcin in serum with use of an antibody against human osteocalcin

1993 ◽  
Vol 39 (6) ◽  
pp. 942-947 ◽  
Author(s):  
D A Monaghan ◽  
M J Power ◽  
P F Fottrell
Keyword(s):  
Monoclonal Antibody ◽  
Enzyme Immunoassay ◽  
Solid Phase ◽  
Sandwich Elisa ◽  
Normal Subjects ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Standard Curve ◽  
Microtiter Plates ◽  
Human Osteocalcin

Abstract We have developed and thoroughly validated a solid-phase sandwich enzyme-linked immunosorbent assay (ELISA) on microtiter plates for osteocalcin in human serum with use of an antibody raised against human osteocalcin. We used a monoclonal antibody against bovine osteocalcin as the capture antibody; the second antibody was a polyclonal antibody against human osteocalcin. The amount of bound second antibody was determined with use of swine anti-rabbit antibody labeled with horseradish peroxidase. We demonstrated independence of volume and determined the recovery of added standard and within- and between-assay precision. The minimal detection limit for osteocalcin was between 1.0 and 1.5 micrograms/L and the midpoint of the standard curve ranged from 14 to 17 micrograms/L. The intraassay CV was < or = 8% in the range 2.7-52 micrograms/L; the interassay CV was usually < or = 15% in the same range. Analytical recovery of human osteocalcin standard added to serum samples was consistently > 90%. Values for osteocalcin measured in serum from 44 normal subjects were similar to those obtained with a competitive enzyme immunoassay (EIA) that used a monoclonal antibody against bovine osteocalcin. There was a good correlation between the two assays [r2 = 0.877, slope and intercept (+/- SE) = 0.88(+/- 0.051) and 0.316(+/- 0.523), respectively]. The range and mean (+/- SD) for the sandwich ELISA and the competitive EIA were 1.7-18.1 micrograms/L [8.7(+/- 4.4) micrograms/L] and 1.9-22.8 micrograms/L [9.1(+/- 4.4) micrograms/L], respectively.

Solid-phase enzyme immunoassay of urokinase using monoclonal antibodies

1983 ◽  
Vol 3 (4) ◽  
pp. 381-388 ◽  
Author(s):  
P. Hérion ◽  
D. Portetelle ◽  
J. -D. Franssen ◽  
J. Urbain ◽  
A. Bollen
Keyword(s):  
Monoclonal Antibodies ◽  
Enzyme Immunoassay ◽  
Solid Phase ◽  
Biological Fluids ◽  
Immunosorbant Assay ◽  
Rapid Procedure ◽  
Preliminary Purification ◽  
Enzyme Linked Immunosorbant Assay

Using two monoclonal antibodies directed against urokinase, we have developed a micro enzyme-linked immunosorbant assay (ELISA) to detect and measure urokinase in biological fluids. The system presents the following characteristics: simple and rapid procedure, reproducibility, sensitivity (urokinase levels down to 1 ng/ml) and evaluation of the enzyme in biological fluids such as urine, pleural elfusions, and ascitic fluids without preliminary purification.

Highly sensitive enzyme immunoassay of proinsulin immunoreactivity with use of two monoclonal antibodies

1993 ◽  
Vol 39 (10) ◽  
pp. 2146-2150 ◽  
Author(s):  
L L Kjems ◽  
M E Røder ◽  
B Dinesen ◽  
S G Hartling ◽  
P N Jørgensen ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Detection Limit ◽  
Human Serum ◽  
Enzyme Immunoassay ◽  
Healthy Subjects ◽  
Sandwich Elisa ◽  
C Peptide ◽  
Peptide Antibody ◽  
Analytical Recovery ◽  
Highly Sensitive

Abstract A highly sensitive two-site sandwich ELISA measuring total proinsulin immunoreactive material in serum or plasma was developed. The assay was based on two monoclonal antibodies, an anti-C-peptide antibody bound to a microtest plate and a biotin-labeled anti-insulin antibody. The detection limit (3 SD above zero value) in buffer was 0.05 pmol/L, corresponding to 0.25 pmol/L in human serum (diluted 1:5). The linear calibrator range was 0.05-20 pmol/L. Interassay CVs were 4.7% at a median (range) of 2.3 pmol/L (1.4-2.8 pmol/L, n = 8), 6.7% at 5.1 pmol/L (3.3-8.0 pmol/L, n = 8), and 8.7% at 10.0 pmol/L (8-12 pmol/L, n = 10). Mean analytical recovery of added human proinsulin (hPI) (2, 5, and 10 pmol/L) to serum was 84% (range 68-128%, n = 9). Human insulin and human C-peptide did not cross-react at 5000 and 10,000 pmol/L, respectively. The four major proinsulin conversion intermediates reacted 65-99%: split(32-33)hPI 74%, des-(31,32)hPI 65%, split(65-66)hPI 78%, and des(64,65)hPI 99%. All serum values from 38 fasting healthy subjects were above the detection limit: median (range) 4.0 (2.1-12.6) pmol/L.

scholarly journals Quantitation of Canine Distemper Virus and Antibodies by Enzyme-Linked Immunosorbent Assays Using Protein A and Monoclonal Antibody Capture

1989 ◽  
Vol 1 (2) ◽  
pp. 110-115 ◽  
Author(s):  
Leon N. D. Potgieter ◽  
Peace A. Ajidagba
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Cell Cultures ◽  
Canine Distemper Virus ◽  
Solid Phase ◽  
Sandwich Elisa ◽  
Protein A ◽  
Canine Distemper ◽  
Clinical Specimens ◽  
Immunosorbent Assays

Monoclonal antibodies produced from 19 cloned hybridomas were selected for this study. Specific canine distemper virus (CDV) antibodies in medium from cloned hybridomas were detected by direct enzyme-linked immunosorbent assays (ELISA) and by indirect immunofluorescence. Three different sandwich ELISA systems were developed either to detect CDV in cell cultures and clinical specimens or to detect specific antibody in canine sera. Protein A and monoclonal antibodies attached in sequence to a solid phase constituted the capture system in the assays. Viral antigens were detected by sandwiching extracts of clinical specimens (or infected cell cultures), monoclonal antibody, and peroxidase-labeled protein A in sequence onto the capture layer. In 1 procedure, biotin-labeled antibody and peroxidase-labeled avidin were used as the last 2 layers in the assay. The CDV antibodies in dog sera were quantitated in a similar manner, but the sequential sandwiching levels consisted of partially purified CDV, serum specimen, and peroxidase-labeled protein A, respectively. The procedures were specific and highly sensitive.

A solid-phase enzyme immunoassay for the detection of tetanus toxin using human and murine monoclonal antibodies

1991 ◽  
Vol 31 (2) ◽  
pp. 135-140 ◽  
Author(s):  
Stephan T. Kiessig ◽  
Christian Hentschel ◽  
Sigbert Jahn ◽  
Michael Mehl ◽  
Roland Starke ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Enzyme Immunoassay ◽  
Tetanus Toxin ◽  
Solid Phase ◽  
Murine Monoclonal Antibodies
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