Flow cytometry for the analysis of virusneytralizing activity of antirabies serum and immunoglobulin drug

Here, we discuss development of f low cytometry technique for determining level of rabies antibodies in immune sera and immunoglobulin preparations, which is based on measuring fluorescence level in Vero cell line added with a mix of serially diluted anti-rabies serum or immunoglobulin preparation t...

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Autores principales:	S. V. Generalov, A. L. Kravtsov, V. A. Kozhevnikov, Yu. K. Gavrilova, E. G. Abramova, A. K. Nikiforov
Formato:	article
Lenguaje:	RU
Publicado:	Sankt-Peterburg : NIIÈM imeni Pastera 2019
Materias:	rabies virus rabies immunoglobulin rabies serum neutralizing activity flow cytometry control of biologics Infectious and parasitic diseases RC109-216
Acceso en línea:	https://doaj.org/article/65518a6981a1406e96a069178279cd5f
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spelling	oai:doaj.org-article:65518a6981a1406e96a069178279cd5f2021-11-22T07:09:52ZFlow cytometry for the analysis of virusneytralizing activity of antirabies serum and immunoglobulin drug2220-76192313-739810.15789/2220-7619-2019-1-107-114https://doaj.org/article/65518a6981a1406e96a069178279cd5f2019-05-01T00:00:00Zhttps://www.iimmun.ru/iimm/article/view/797https://doaj.org/toc/2220-7619https://doaj.org/toc/2313-7398Here, we discuss development of f low cytometry technique for determining level of rabies antibodies in immune sera and immunoglobulin preparations, which is based on measuring fluorescence level in Vero cell line added with a mix of serially diluted anti-rabies serum or immunoglobulin preparation together with Moscow 3253 attenuated rabies virus strain, adapted for reproduction in cell lines. For this, rabies sera and immunoglobulin preparation were diluted 1:20 and 1:200 with PBS, respectively, whereas rabies virus was recommended for use at dose of 0.1 ID50/cell. Cell membrane fixation and permeabilization were performed by using a Cytoperm/Cytofix reagent (BD Pharmingen, USA) containing formaldehyde able to inactivate rabies virus, thus complying with biological safety regulations. Anti-rabies FITC-conjugated immunoglobulin (FGBI ARRIAH, Russia) was recommended for staining, that does not limit using similar reagents. Then, a f low cytometer equipped with a 20 mW argon laser (488 nm emission wavelength, throughput — 500 cells per second) was used for analysis. Vero cells displaying a fluorescence intensity exceeding 10 arbitrary units were considered infected. Flow cytometry allows to precisely measure amount of experimental infected cells as well as degree of infection by evaluating cell f luorescence intensity. Amount of antibodies in the samples examined by us was calculated by building a calibration curve based on depicting data for a standard rabies immunoglobulin with activity of 30 IU/ml (NIBSC, Potters Bar, United Kingdom). A high correlation between the data obtained by us and results from other detection methods used to assess activity of anti-rabies preparations such as biological neutralization of rabies virus in white mice (0.92) and modified FAVN test (0.98) was demonstrated. The comparison of the results of the analysis of the level of rabies antibodies obtained using f low cytometry, and the results obtained in tests in vivo, was carried out for the first time. Moreover, it is worth noting that for the first time the level of anti-rabies antibodies assessed by f low cytometry and in vivo tests was compared. An opportunity to perform rapid and easy-to-do analysis on assessing amount of infected cells by measuring fluorescence intensity in f low cytometry assay would allow to apply this approach for quality control while developing and manufacturing immunobiological anti-rabies preparations.S. V. GeneralovA. L. KravtsovV. A. KozhevnikovYu. K. GavrilovaE. G. AbramovaA. K. NikiforovSankt-Peterburg : NIIÈM imeni Pasteraarticlerabies virusrabies immunoglobulinrabies serumneutralizing activityflow cytometrycontrol of biologicsInfectious and parasitic diseasesRC109-216RUInfekciâ i Immunitet, Vol 9, Iss 1, Pp 107-114 (2019)
institution	DOAJ
collection	DOAJ
language	RU
topic	rabies virus rabies immunoglobulin rabies serum neutralizing activity flow cytometry control of biologics Infectious and parasitic diseases RC109-216
spellingShingle	rabies virus rabies immunoglobulin rabies serum neutralizing activity flow cytometry control of biologics Infectious and parasitic diseases RC109-216 S. V. Generalov A. L. Kravtsov V. A. Kozhevnikov Yu. K. Gavrilova E. G. Abramova A. K. Nikiforov Flow cytometry for the analysis of virusneytralizing activity of antirabies serum and immunoglobulin drug
description	Here, we discuss development of f low cytometry technique for determining level of rabies antibodies in immune sera and immunoglobulin preparations, which is based on measuring fluorescence level in Vero cell line added with a mix of serially diluted anti-rabies serum or immunoglobulin preparation together with Moscow 3253 attenuated rabies virus strain, adapted for reproduction in cell lines. For this, rabies sera and immunoglobulin preparation were diluted 1:20 and 1:200 with PBS, respectively, whereas rabies virus was recommended for use at dose of 0.1 ID50/cell. Cell membrane fixation and permeabilization were performed by using a Cytoperm/Cytofix reagent (BD Pharmingen, USA) containing formaldehyde able to inactivate rabies virus, thus complying with biological safety regulations. Anti-rabies FITC-conjugated immunoglobulin (FGBI ARRIAH, Russia) was recommended for staining, that does not limit using similar reagents. Then, a f low cytometer equipped with a 20 mW argon laser (488 nm emission wavelength, throughput — 500 cells per second) was used for analysis. Vero cells displaying a fluorescence intensity exceeding 10 arbitrary units were considered infected. Flow cytometry allows to precisely measure amount of experimental infected cells as well as degree of infection by evaluating cell f luorescence intensity. Amount of antibodies in the samples examined by us was calculated by building a calibration curve based on depicting data for a standard rabies immunoglobulin with activity of 30 IU/ml (NIBSC, Potters Bar, United Kingdom). A high correlation between the data obtained by us and results from other detection methods used to assess activity of anti-rabies preparations such as biological neutralization of rabies virus in white mice (0.92) and modified FAVN test (0.98) was demonstrated. The comparison of the results of the analysis of the level of rabies antibodies obtained using f low cytometry, and the results obtained in tests in vivo, was carried out for the first time. Moreover, it is worth noting that for the first time the level of anti-rabies antibodies assessed by f low cytometry and in vivo tests was compared. An opportunity to perform rapid and easy-to-do analysis on assessing amount of infected cells by measuring fluorescence intensity in f low cytometry assay would allow to apply this approach for quality control while developing and manufacturing immunobiological anti-rabies preparations.
format	article
author	S. V. Generalov A. L. Kravtsov V. A. Kozhevnikov Yu. K. Gavrilova E. G. Abramova A. K. Nikiforov
author_facet	S. V. Generalov A. L. Kravtsov V. A. Kozhevnikov Yu. K. Gavrilova E. G. Abramova A. K. Nikiforov
author_sort	S. V. Generalov
title	Flow cytometry for the analysis of virusneytralizing activity of antirabies serum and immunoglobulin drug
title_short	Flow cytometry for the analysis of virusneytralizing activity of antirabies serum and immunoglobulin drug
title_full	Flow cytometry for the analysis of virusneytralizing activity of antirabies serum and immunoglobulin drug
title_fullStr	Flow cytometry for the analysis of virusneytralizing activity of antirabies serum and immunoglobulin drug
title_full_unstemmed	Flow cytometry for the analysis of virusneytralizing activity of antirabies serum and immunoglobulin drug
title_sort	flow cytometry for the analysis of virusneytralizing activity of antirabies serum and immunoglobulin drug
publisher	Sankt-Peterburg : NIIÈM imeni Pastera
publishDate	2019
url	https://doaj.org/article/65518a6981a1406e96a069178279cd5f
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Flow cytometry for the analysis of virusneytralizing activity of antirabies serum and immunoglobulin drug

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