title: A simplified ATP method for the rapid control of cell viability in a freeze-dried BCG vaccine.
abstract: We propose a simple and cost-effective ATP method for controlling the specific activity of a freeze-dried BCG vaccine. A freeze-dried BCG vaccine is reconstituted with 1ml saline and incubated for 15min at room temperature and then for 1h at 37°C. The vaccine is then treated with apyrase to remove extracellular ATP. After that, the cells are lysed with DMSO and the ATP content in the lysate is measured by the bioluminescence method. To implement the method, we developed a kit that requires no time-consuming preparation before the analysis. We demonstrated the linear relationship between the experimental values of the specific activity (106CFU/mg) and intracellular ATP content (ATP, pmol/mg) for different batches of the studied BCG vaccines; the proportionality coefficient was К=0.36±0.02. We proposed a formula for calculating the specific activity from the measured content of intracellular ATP (ATP, pmol/mg). The comparison of the measured and calculated values of the specific activity (106CFU/mg) shows that these values are similar; their differences fall within the allowable range of deviations for the specific activity values of the BCG vaccine.
authors: Faculty of Chemistry, Lomonosоv Moscow State University, Moscow 119991, Russia; LLC Lumtek, Vorobievy Gory 1/77, Moscow 119992, Russia. Electronic address: nugarova@gmail.com.
authors: Natalia N
authors: NN
authors: Ugarova
authors: Faculty of Chemistry, Lomonosоv Moscow State University, Moscow 119991, Russia; LLC Lumtek, Vorobievy Gory 1/77, Moscow 119992, Russia.
authors: Galina Yu
authors: GY
authors: Lomakina
authors: Faculty of Chemistry, Lomonosоv Moscow State University, Moscow 119991, Russia; LLC Lumtek, Vorobievy Gory 1/77, Moscow 119992, Russia.
authors: Yulia
authors: Y
authors: Modestova
authors: FSUC SIC Microgen, MOH RF, 10, Vtoroi Volkonsky per., Moscow 127473, Russia.
authors: Sergey V
authors: SV
authors: Chernikov
authors: FSUC SIC Microgen, MOH RF, 10, Vtoroi Volkonsky per., Moscow 127473, Russia.
authors: Natalia V
authors: NV
authors: Vinokurova
authors: FSUC SIC Microgen, MOH RF, 10, Vtoroi Volkonsky per., Moscow 127473, Russia.
authors: Elena V
authors: EV
authors: Оtrashevskaya
authors: FSUC SIC Microgen, MOH RF, 10, Vtoroi Volkonsky per., Moscow 127473, Russia.
authors: Vyacheslav Y
authors: VY
authors: Gorbachev
mesh_terms: D000255:Adenosine Triphosphate / Q000032:analysis* / Q000378:metabolism; D001081:Apyrase / Q000378:metabolism; D001500:BCG Vaccine / Q000737:chemistry; D001431:Bacteriological Techniques / Q000191:economics / Q000379:methods*; D015169:Colony Count, Microbial; D005612:Freeze Drying / Q000379:methods; D008163:Luminescent Measurements / Q000379:methods; D050296:Microbial Viability; D009163:Mycobacterium bovis / Q000254:growth & development*; D011786:Quality Control; D013696:Temperature; D013997:Time Factors
chemical_list: D001500:BCG Vaccine; D000255:Adenosine Triphosphate; D001081:Apyrase
keywords: ATP assay; BCG viability; Colony forming units; Freeze-dried BCG vaccine; Tuberculosis