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Aim. Experimental substantiation of possibility to determine the rabies virus-infected Vero cell line portion in culture by flow cytometry (FC) and FITC labeled diagnostic anti-rabies immunoglobulin (DAI), manufactured in Russia. Materials and methods. Fixation and permeabilization of Vero cells, infected by rabies virus strain «Moscow 3253», was carried out by means of Суtofix/Cytoperm reagent (BD Biosciences, USA) according the Vengatesan D. et al. method (2006) and then intracellular rabies antigen was stained by DAI. Percentage of infected cells was determined by FC in 24, 48 and 72 h and as well in 48 h when the cell cultures were infected with tenfold dilutions of virus-containing fluid from 10-1 to 10-8. Results. There was a significant increase in the percentage of infected cells on average from 30 to 70% in time interval from 24 to 48 h. With 1000-fold dilution of viral-containing fluid the FC method detected the 6,9±0,21% of infected cells in Vero cultures (P<0,001, n=3). Conclusion. FC has proved to be a fast, sensitive and reliable method for determining the relative number of virus- infected Vero cells in cultures. The drug DAI had a sufficient activity for its effective use in the automated version of MFA based on the FC method. The use of FC is possible at various stages of anti-rabies drug production and control, and is also promising in terms of further improving of the rabies diagnosis.

The development and use of new methods of quality control of medicines involve the use of a lot of reference materials in quality control testing. Specialists of the Russian Research Antiplague Institute “Microbe” have proposed an alternative methodological approach to determination of potency of anti-rabies immunoglobulin in cell culture, which requires the development of an in-house reference standard (RS) certified against the biological reference preparation (BRP) of the European Pharmacopoeia¾human rabies immunoglobulin.The aim of the study was to develop and evaluate the metrological characteristics of an in-house RS for anti-rabies immunoglobulin potency, to be used in the virus neutralisation test in a Vero cell culture.Materials and methods: The following materials were used in the study: equine rabies immunoglobulin, Vero continuous cell culture, fixed rabies virus (Moscow 3253Vero strain), human rabies immunoglobulin BRP of the European Pharmacopoeia. The potencies of the candidate in-house RS and rabies immunoglobulin samples were determined in the neutralisation test in cell culture. The results were recorded using a fluorescent microscope. Statistical processing was carried out in accordance with general chapter 1.1.0014.15 of the State Pharmacopoeia of the Russian Federation, 14th edition.Results: the certified value of the in-house RS potency was 180.8±18.8 IU/mL. The confidence limits were determined at the 0.95 probability level. The shelf life of the in-house RS is 1.5 years (when stored according to the sanitary regulation SanPiN 3.3686-21). The certified in-house RS was assigned with the number 41-01-20. A set of technical and operational documentation was developed and approved for the in-house RS. The developed in-house RS can be used for in vitro determination of anti-rabies immunoglobulin potency, expressed in international units, to confirm its compliance with the product specification file.Conclusions: the authors developed an in-house RS for anti-rabies immunoglobulin potency, to be used in the virus neutralisation test in cell culture, certified against the human rabies immunoglobulin BRP of the European Pharmacopoeia.