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Test Kits for Plague Diagnostics

#2426


Development of Test Kits to Diagnostically Significant Antigens of Yersinia Pestis with the Use of Combinatorial Phage Libraries of Miniantibodies

Tech Area / Field

  • MED-DID/Diagnostics & Devices/Medicine
  • BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology
  • BIO-SFS/Biosafety and BioSecurity/Biotechnology

Status
8 Project completed

Registration date
19.02.2002

Completion date
26.01.2010

Senior Project Manager
Genisaretskaya S V

Leading Institute
State Research Center for Applied Microbiology and Biotechnology, Russia, Moscow reg., Obolensk

Supporting institutes

  • Institute of Bioorganic Chemistry, Russia, Moscow\nInstitute of Biochemistry and Physiology of Plants and Microorganisms, Russia, Saratov reg., Saratov

Collaborators

  • Universite de Paris-Sud / Institut de Génétique et Microbiologie, France, Orsay\nInstitut National de la Santé et de la Recherche Médicale / Immunilogie Cellulaire et Clinique, Unité INSERM 255, France, Paris

Project summary

At present, the laboratory clinical diagnostics and environmental detection of plague are performed using isolation of pure culture of Y. pestis, detection of its antigens by means of homologous antibodies (immunoserological diagnostics), or disclosure of specific for Y. pestis DNA sequences in samples for analysis. Methods of serological diagnostics are used at the stages of making a provisional diagnosis, identification of isolated cultures, and for the purposes of retrospective diagnostics. All the immunoserological diagnosticums are pided into two groups: (i) diagnosticums, constructed on the base of Y. pestis species-specific antigens, and (ii) - on the base of antibodies raised to these antigens. At present, substantially sera of hyperimmune animals or monoclonal antibodies are used as primary products for construction of immunoglobulin diagnosticums.

Recently a fundamentally novel method for in vitro producing of fragments of antibodies with the high affinity and specificity was developed. The method empowers to forego the use of laboratory animals, long-term immunization procedures, expensive media and cell cultures. This technology involves construction of miniantibody libraries exposed on filiform bacteriophages or phagemids. In these libraries recognizing antibody fragments may be integrated into phage capsid or excreted as separate soluble molecules.

Phages, having certain peculiarity, are selected by dint of binding with target antigen on a solid phase or in solution. Picking of such phage clones is technically easier, inexpensively and takes less time as compared with animal immunization and hybridoma technology. The yield of soluble fragments of phage miniantibodies is on the average 500 mg/l when cultivation is performed in a fermenter. In the making immunodiagnostic preparations the technology of phage display will allow to make a clean sweep of the use of laboratory animals, as well as to improve fundamentally standardization and control of manufacturing of primary products for a new generation of immunodiagnosticums to Y. pestis antigens.

Another disadvantage of the modern immunodiagnosticums is that fact that all of them are based on the antibodies against capsular antigen Caf1 (capsular antigen fraction I), the main component of the forming at 37 °C Y. pestis capsule. So, the usage of these diagnosticums has a number of restrictions.

- As a rule, isolation of Y. pestis cultures from the field or clinical samples are performed on the solid media at 28 °C. Under the circumstances Caf1 protein synthesis decreases by approximately 800-1000-fold as compared with 37 °C. Therefore the modern immunodiagnosticums may be used for identification of isolated cultures only after additional cultivation of bacteria at 37 °C.

- It is well known, that Y. pestis do not synthesize Caf1 in flea organism.

- Our data and publications of other researchers indicate that elimination of pFra or mutations in caf1 operon, causing cessation of capsule formation, and, correspondingly, making Y. pestis cells undetectable, sometimes do not decrease virulence of the mutants.

These peculiarities of Y. pestis Caf1 antigen serve as a reason for development of several groups of test kits for detection of Y. pestis antigens:

- for detection of Y. pestis antigens being synthesized in sufficient for diagnostic purposes amounts both at the host temperature (і 37 °C), and at flea temperature (~ 28 °C): Ymt, Pla, Pst, LPS, etc.;

- for detection of Y. pestis antigens (Yops, V, Psa, and components of system for siderophore-dependent acquisition of iron, Ybt, Psn, etc.), which are necessary for manifestation of virulence in the cases of "peripheral" routes of infection (subcutaneous, respiratory, alimentary);

- for detection of Y. pestis antigens, which are necessary for effective transmission through flea (Ymt, Hms).

Some results of attempts to develop such diagnosticums were already published. However we do not know any cases of bringing of such works up to development of commercial preparations.

Previously the authors of the project had cloned genes and constructed producers of the main Y. pestis antigens, and optimized methods for preparative isolation of these antigens.

Protein antigens and antigens of other nature are also successfully studied with the help of combinatorial libraries of variable fragments of mouse antibodies.

Selection of clones, which carry recognizing antibody fragments against a number of high- and low-molecular antigens, was also performed with the use of sheep phage display.

The authors of the project had developed methods for gold-sol synthesis and its conjugation with biospecific macromolecules (Russian patent and Laboratory-Technological Standing Orders), quality control of the constructed preparations and different techniques for their application in biological researches, including solid-phase assay, light and electron microscopy (transmission and scanning) as well as photometry and spectrophotometry.

The methods for employment of biospecific markers, conjugates of colloidal gold, in dot-blot assay of soluble and corpuscular antigens including detection of infecting agents were developed.

Within the framework of the project we propose to press technology of phage display into the service of development and construction of the new generation of immunologic test kits. One of the main pre-eminence of the last is its ability to detect conformational differences between antigens from the same family. Systematic application of research knowledge concerning construction of phage displays of antibody fragments on purpose to prove technology of construction of the new generation of immunoglobulin test kits to diagnostically significant Y. pestis antigens, including development of non-specific application prototypes and processes, field tests, testing and evaluation, and other efforts will be instrumented.

Scientific novelty of the project consists in ascertainment of comparative efficiency of the proposed to development the new generation of immunoglobulin test kits to diagnostically significant Y. pestis antigens in the laboratory diagnosis of plague. In particular, bacterial producers of fragments of antibodies to immunodominant epitopes of diagnostically significant Y. pestis antigens (Caf1, Ymt, V, Pla, Pst, Psa, LPS) will be constructed; new antigens may be looked for and panels of miniantibodies to different epitopes of several antigens, which are perspective for use as components of plague vaccine will be created.

The goals of the project will be solved by means of a complex of modern research techniques, including gene engineering, electron microscopy, and spectroscopy. The study of the properties of constructing preparations will be performed with the use of a complex of biochemical, immunologic, and immunochemical (enzymoimmunoassay and immunogold) methods.

The project realization will be an essential contribution into development of applied biology to perform environmental detection of plague and medicine for clinical diagnostics as well as into solution of significant social problem, control for bioterrorism, being of serious danger for world community. Moreover, performance of the project will allow the scientists and engineers of the Stare Research Center for Applied Microbiology, engaged previously in studies in the area of biodefense, to reorientate the circle of their scientific interests and to use the gained experience to carry out fundamental and applied studies, connected with solution of international scientific and technical problems in the fields of biology and medicine, to create long-term prospects for fruitful activity within the framework of International Scientific Association.


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