ABSTRACT
Antibody responses to purified protein derivate PPD of tuberculin and to antigens MPB63 and MPB83 of Mycobacterium bovis were determined in bovine herd (94 adult animals). Statistical approach based on approximation by multiple Gaussians with Levenberg-Marquardt algorithm for analysis of antibody level distribution against antigens examined was provided. Our results confirm that indirect ELISA with recombinant MPB83 and MPB63 as well as conventional PPD could be used for test-systems development for detection of cow tuberculosis infection at the herd level.
Subject(s)
Antibodies, Bacterial/blood , Antigens, Bacterial/immunology , Cattle Diseases/diagnosis , Immunoenzyme Techniques/statistics & numerical data , Mycobacterium bovis/immunology , Statistical Distributions , Tuberculosis, Bovine/diagnosis , Animals , Cattle , Cattle Diseases/microbiology , Immunoenzyme Techniques/methods , Recombinant Proteins/immunology , Tuberculosis, Bovine/microbiologyABSTRACT
Screening for candidate reassortants is an important step in the development of live influenza vaccine (LIV). The temperature-sensitive (ts) and cold-adapted (ca) phenotypes of vaccine strains are generally determined, by employing chicken embryos, and used as ts and ca attenuation markers. However, it is difficult to use the egg-determined ts phenotypes of vaccine candidate reassortants as an attenuation marker due to a wide circulation of natural ts epidemic influenza viruses. This study used two new alternative ts and ca attenuation markers in MDCK cells. The MDCK cell line was shown to be able to differentiate cold-adapted influenza viruses from any epidemic strains whereas they were undistinguishable when using eggs. The reduced ability of influenza type A vaccine viruses to grow in the MDCK cell culture at temperatures above 37 degrees C can be successfully used as a "cell-culture" ts marker. The similar marker for influenza B viruses may serve their reduced activity in the MDCK cells at 38 degrees C. The high reproductive activity of cold-adapted viruses in the MDCK cells at 26 degrees C was shown to be a suitable ca attenuation marker. The presented attenuation markers may be included into the standard scheme of primary screening of ts reassortant candidates for commercial live influenza vaccine as additional selection factors and may be used as basic markers in the design of culture vaccine.
Subject(s)
Alphainfluenzavirus/physiology , Betainfluenzavirus/physiology , Influenza Vaccines , Animals , Biomarkers/analysis , Cell Line , Chick Embryo , Dogs , Influenza Vaccines/genetics , Reassortant Viruses , Temperature , Vaccines, Attenuated/genetics , Virus ReplicationABSTRACT
Phage display technology is an effective approach to the development of the next generation of immunodiagnostic reagents. Naive murine phage display a library of single-chain variable antibodies (scFv) was used to isolate scFv recognizing the diphtheria toxin, an important diagnostic antigen of diphtheria. The diphtheria toxin B subunit-binding clone with affinity constant of 1.13 x 10(7) M(-1) was selected. scFv preserved activity on storage in the course of 8 months.
Subject(s)
Antibodies, Monoclonal/immunology , Diphtheria Toxin/immunology , Immunoglobulin Variable Region/immunology , Peptide Library , Recombinant Fusion Proteins/immunology , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/genetics , Diphtheria/diagnosis , Diphtheria/immunology , Drug Stability , Immunoglobulin Variable Region/chemistry , Immunoglobulin Variable Region/genetics , Mice , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/geneticsABSTRACT
Reassortant strains for modern live influenza vaccines are prepared using growing chicken embryos. It is very important to switch manufacture of influenza vaccines from chicken embryos to cell cultures, especially due to the threat of future pandemic, when there will be need of big quantities of vaccine for immunization of all age groups. Efficacy of production of reassortant strains with 6:2 vaccine formulation of genome (6 internal genes from the donor of attenuation and 2 genes coding external antigens--hemagglutinin and neuraminidase--from epidemic strain) in MDCK cell culture, using standard techniques employed for production of the vaccine in chicken embryos, was studied. It was shown that yield frequency of aforementioned reassortants of influenza A viruses did not exceed 5.7% whereas in chicken embryos vaccine 6:2 reassortants were isolated with frequency of 4%. For influenza B viruses, yield of 6:2 reassortants in growing chicken embryos exceeded 67% whereas in MDCK cell culture we were unable to produce clones with required genome composition. Thus, existing method while effective for production of vaccine reassortants in chicken embryos is low effective for isolation of 6:2 reassortants in MDCK cell culture. Fundamentally new techniques are needed for production of reassortant strains for live influenza vaccine in cell culture.
