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Express-Diagnostic Test-Kits

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Development of Immunofiltration and Immunoenzyme Express-Diagnostic Test-Kits for Determination of Infectious Diseases

Tech Area / Field

  • MED-DID/Diagnostics & Devices/Medicine

Status
8 Project completed

Registration date
23.03.1998

Completion date
13.12.1999

Senior Project Manager
Kondratenkov Yu B

Leading Institute
Research Center of Molecular Diagnostics and Theraphy, Russia, Moscow

Collaborators

  • Battelle Memorial Institute, USA, VA, Arlington\nPacific Northwest National Laboratory, USA, WA, Richland\nDepartment of the Navy / Naval Research Laboratory, USA, DC, Washington\nBattelle Memorial Institute, USA, OH, Columbus\nUS Department of Health & Human Services / National Institute of Health / National Institute of Allergy and Infectious Diseases, USA, MD, Bethesda

Project summary

The state of the art in the modem biomedical sciences sets forth new goals in the field of immunodiagnostics, which are to increase sensitivity and compress time of the assay. For the purposes of achieving these goals at the present moment monoclonal antibodies (MoAb), non-equilibrium conditions for antigen-antibody complex-formation, especially flow-through, and polymer indicator dyes are widely used. The aim of the project is to develop a highly sensitive immunofiltration and immunoenzyme methods and on the base of these methods to create express-diagnostic test-kits for the determination of infectious diseases. Description and merits of the work:

In the recent years in RCMDT a new technology of express-diagnostics of infectious agents has been developed using the methods mentioned above. To reach the effect of immuno-assay time compressing there have been offered large-porous polymer filters or non-porous glass microspheres, which, when packed in the microcolumns, give the liquid flow-through rate not less than 0.1 ml/min. The assay specificity is maintained on two levels: first- by using MoAb for immobilization on solid phase and antigen capturing, second - by means of utilisation of secondary biotin-labeled MoAb, which are introduced to the assay after the tested sample has flown-through the column. High sensitivity of the method is guaranteed by 3 preconditions: (1) large capacity of immunosorbent; (2) possibility of obtaining of an excess of secondary biotinilated MoAb; (3) use of polymer conjugates streptavidin-albumin-dye for visualization of immune complexes with the following photometric detection.

In addition to photometric detection fluoroscein-labelled secondary antibodies are planned to be used. This will cut the time of assay off one stage and increase sensitivity 10-fold, but at the same time require a more sophisticated instrumental provision. There is a potential of further cutting the assay time by means of application of portable photometers and fluorimeters, which allow to measure the signal directly on the column without the elution of the dye. Summing up all the merits of the suggested technology the following characteristics is worth marking:

time of assay from the moment of the sample administration till the obtaining of the result

15 - 20 min

number of stages including sample preparation

4 - 6

assessment of the results

visual/instrumental

instrumental detection

photometry/fluorimetry

type of assay

quantitative/qualitative

sensitivity

0.1 - 10 ng/ml

sample volume

0.25 - 10 ml

specificity

not less than 98%

variation coefficient

8 - 15%

number of tests

1 - Ґ

type of sample

whole blood, serum, plasma, urine, feces, nasal washings, cell culture supernatants, water-salt and organic extracts and washings, drinking water, industrial drainage waste water

storage period

min 12 months at +4°C

As it follows from the above description of the immunofiltration method its specificity is determined by MoAb and Ag used. During the last 10 years in RCMDT there have been obtained genetically stable cell lines of murine hybridomas, producing MoAb to different viral and microbial antigens, i.e:
Yersmia pestis, F-I antigen
LPS Francesella tularensis
LPS Chlamydia trachomatis
LPS Legionella pneumophila
Salmonella A, B, D group specific O-antigens
Toxoplasma gondii P-30 membrane antigen
St. Aureus enterotoxin (SEA, SEB)
Hepatitis В surface antigen (HBsAg)
Hepatitis A virus (HAV)
Rota virus group-specific antigen
Adenovirus, hexon
Herpes simplex virus (HSV-2), glycoproteid D
Influenza virus A hemagglutinin, nucleoproteid
RSV, F protein
Rabies virus, glycoproteid
Rubella virus, glycoproteids (E1, E2, C)

Most of the suggested MoAb were used for the development of diagnostic test kits on the base of EIA or immunofluorescence analysis with the required sensitivity and specificity, therefore the development of immunofiltration kits for the express-diagnostics of extremely dangerous and dangerous infections, including the final stage of the pilot batches manufacture, their trials and registration will require not more than 30-36 months. Taking into account the demands of the sanitary-epidemiological service and clinical diagnostics of infectious diseases in the Russian Federation as well as the goals of ISTC, we consider it reasonable to develop the following kits in the frames of the present agreement:

A. Based on imunofiltration method:


Immunofiltration monoclonal test-kit for Yersinia pestis, F-I antigai express-determination
Immunofiltration monoclonal test-kit for Adenovirus gastroenteritis express-diagnostics
Immunofiltration monoclonal test-kit for IgG/lgM Toxoplasma gondii express-determination

B. It is also planned to develop a three-compound complex of test-kits based on EIA for the diagnostics of Rota-virus diarrhea (gastroenteritis) and indication of Rota-virus group A in samples and environmental objects:

1. Immunoenzyme monoclonal test-kit for detection of antigens and virus particles of human Rota-virus group A in complex with confirmation test.

Kit characteristics:

time of assay from the moment of the sample administration till the obtaining of the result

2 hours

number of stages including sample preparation

3

assessment of the results

visual/instrumental

instrumental detection

photometry

type of assay

quantitative/qualitative

sensitivity

1-3 ng/ml or 100000 virus particles/ml

sample volume

100 ml

specificity

not less than 98%

enzyme label

horseradish peroxidase

number of tests

20-40

type of sample

urine, feces, nasal washings, cell culture

supematants, water-salt and organic extracts and washings, drinking water, industrial drainage waste water

storage period

6 months

2. Immunoenzyme

test-kits for differential detection of human IgG/lgM antibodies in diagnostics of Rota-virus diarrhea.

Kit characteristics:



time of assay from the moment of the sample administration till the obtaining of the result

2 hours

number of stages including sample preparation

3

assessment of the results

visual /instrumental

instrumental detection

photometry

type of assay

quantitative/qualitative

sensitivity

not less than 96%

sample volume

100 ml

specificity

not less than 98%

enzyme label

horseradish peroxidase

number of tests

20

type of sample

blood serum and plasma

storage period

6 months


Scientific novelty of the present project is a new method of fast immunoassay, with the factors influencing the time of assay, its specificity and sensitivity, investigated. These factors are: the nature of matrix, kinetics of antigen-ant body interaction, assay format, label type etc. In the course of work novel methods of EIA with all the factors of time compressing and specificity lifting investigated, clinical data for candidate test-kits for Public Health Care System obtained. Applied aspect of the work is the final development of new kits for express-diagnostics of extremely dangerous and infectious diseases, their trials and registration and pilot batches manufacture for the use in the Russian Federation.


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