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MULTIPLEX DETECTION OF IgG CLASS ANTIBODIES TO TOXOPLASMA GONDII, RUBELLA VIRUS, CYTOMEGALOVIRUS AND HERPES SIMPLEX VIRUS USING A NOVEL MULTIPLEX FLOW IMMUNOASSAY
Sergey Anatolievich DOLGUSHIN1, Ekaterina Mikhaylovna LOSEVA2, Elena Sergeevna ODINTSOVA3, Andrey Vladimirovich TRONIN2, Sergey Vasil'evich SELISHCHEV1
1 National Research University of Electronic Technology 124498, Moscow, Zelenograd, Shokin sq., 1
2MBU-Technology, Ltd.
630090, Novosibirsk, Inzhenernaya str., 16
3 Institute of Chemical Biology and Fundamental Medicine of SB RAS 630090, Novosibirsk, Lavrent'ev av., 8
Preclinical tests of the novel multiplex immunoassay for the detection of TORCH infections in human serum using flow cytometry are presented. The immunoassay is intended for the qualitative detection of specific human IgG class antibodies to Toxoplasma gondii, rubella virus, cytomegalovirus and herpes simplex virus in human serum. Multiplex analysis is performed in vitro on polystyrene microspheres encoded with organic dye. The diagnostic sensitivity of the kit is evaluated to be not less than 95%.
Keywords: bead-based multiplex immunoassay, immunofluorescence, TORCH complex, immunodiagnostics.
TORCH infections are a group of congenitally transmitted infections that cause significant morbidity and mortality in newborns if untreated [1]. TORCH stands for: T - toxoplasmosis, O - other infections, R - rubella, C - cytomegalovirus, H -herpes simplex virus.
The multiplex kits are based on suspensions of encoded microbeads which are spectrally encoded. Molecular probes (antigens) are attached to their surface [1-4]. Each bead represents a finite element of the biochip, so it can be detected by a unique spectral code and characterized by a unique molecular probe attached to its surface. As a result, it becomes possible to carry out sensitive registration of an identifiable chemical compound. The assay is carried out on fully automated high-performance devices that use the principles of a flow cytometry [2-5]. The multiplex analysis if compared with
ELISA requires several times less runtime for analysis; besides, reagent consumption and staff employment time are reduced significantly. Flow cytometry is based on the registration of optical radiation that is scattered on a single particle. This method requires a relatively simple optical system for the registration of the scattered radiation in contrast to the optical systems for the registration of diffusively scattered radiation in time domain [5].
In the present work preclinical tests of a novel multiplex immunoassay for the detection of TORCH infections in human serum using flow cytometry are presented. The kit is based on organic dye-labeled polystyrene beads and is intended to use in clinics for the in vitro detection of blood serum immu-noglobulin G (IgG) to Toxoplasma gondii, rubella virus, cytomegalovirus (CMV) and herpes simplex virus (HSV).
Dolgushin S.A. - candidate ofphysical and mathematical sciences, senior researcher of the chair for biomedical systems, e-mail: dolgushin.sergey@gmail.com
Selishchev S.I. - doctor ofphysical and mathematical sciences, professor, headr of the chair for biomedical systems, e-mail: sersel@miee.ru
Tronin A.V. - director, e-mail: tronin@mbu-tech.com Loseva E.M. - researcher, e-mail: losevaem@gmail.com
Odintsova E.S. - candidate of biological sciences, junior researcher, e-mail: odintsoval@ngs.ru
MATERIALS AND METODS
The assay is based on the the following principles.
(i) An investigated sample (diluted serum at the volume of 20 (l) is mixed with 20 (l of polystyrene beads suspension (QuantumPlex™ 5.5 (im COOH Kit, Bangs Labs, USA). Initial polystyrene beads were at a concentration of ~1 x 108 beads/mL and were encoded by different concentrations of IR organic dye (emission wavelength is 685 nm). The initial beads were chemically bound to antigens of pathogens employing DEPC-Carbodiimide (EDAC) linker and standard binding protocol provided by the beads' supplier. If the serum had IgG that are specific to the antigens of T. gondii, rubella virus, HSV or CMV, in a sample the binding reaction of on the microspheres surface immobilized antigen and specific antibody was occurred. There was not detected any cross reaction among the antigens in the developed multiplex immunoassay.
(ii) The microspheres suspension is washed at the second step. Then, a conjugate is added to the
suspension. The conjugate is specific to human IgG antibodies that labeled by phycoerythrin (PE). Thus, the antigen/IgG-specific antibody/conjugate - complex is formed on the beads' surface.
The control microspheres are present in the mixture, which are intended to control the reaction status of the conjugate. These spheres are loaded with human monoclonal IgG. If control microspheres are mixed with conjugate, then the IgG/conjugate complex is formed on the surface. An absence of the fluorescence from the control microspheres indicates a failure in the test, so the investigated sample should be tested again. Thus, all stages of the reaction can be easily controlled.
The presence of the TORCH infections in the serum sample is indicated by the PE emission peak (575 nm) in a spectrum of fluorescent light emitted from the beads.
RESULTS AND DISCUSSION
The assay of the microspheres was carried out using a flow cytometer BD FACS Canto II. A fluores-
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Fig. Multiplex assay of negative (-) and positive (+) serum samples for HSV (b+, c+, d+), T. gondii (b -, c -, d+), rubella virus (b-, c-, d+) and CMV (b-, c+, d+). The clouds correspond to the following beads populations: 1 - control (K+), 2 - HSV, 3 - rubella virus, 4 - cytomegalovirus, 5 - T. gondii. Results prior to incubation with the serum sample (a) and after the incubation (b, c, d)
cence excitation is carried out with the argon laser (power 15 mW, wavelength 488 nm). The filter FL-3 (Per-CP-H) was used for detection of microspheres. Fluorescence was recorded for PE and PerCP/PE-Cy5 fluorescence channels by second (FL2) and third (FL3) photomultiplier tube, respectively.
HSV, rubella virus, CMV and T. gondii antigen was covalently attached to the microspheres N 2, 3, 4 and 5, respectively. Human monoclonal IgG was covalently attached to the microspheres N 1. All individual sera previously examined on the presence of antibodies to respective antigens using corresponding ELISA kits. As Figure shows, a negative serum cloud of data is located in the first decade of the abscissa (for T. gondii, rubella virus (b, c) and CMV (b)). For the analysis of positive samples displacement of clouds in the higher values of the fluorescence channel FL2 PE occurs (for HSV (b, c, d), T. gondii, rubella virus (d) and CMV(c, d)). Each serum was analyzed three times and the reproducible results for each infection were obtained.
There were investigated 94 different human serum samples totally. Each serum was tested using a multiplex kit and controlled by corresponding IgG ELISA kit. The results obtained with multiplex kit were in agreement with ELISA for 89 samples and varied for 5 serum samples. Thus, the diagnostic sensitivity of the kit is evaluated to be not less than 95 %. The results of preclinical tests for reagents kit satisfy the requirements for this class of test systems. It was found that a diagnostic sensitivity of the developed reagent kit is close to the sensitivity of commercial products.
ACKNOWLEDGEMENTS
This work was financially supported by the Ministry of Education and Science of the Russian Federation (agreement № 14.575.21.0090, identifier RFMEFI57514X0090).
REFERENCES
1. BinnickerM.J., Jespersen D.J., Harring J.A. Evaluation of three multiplex flow immunoassays compared to an enzyme immunoassay for the detection and differentiation of IgG class antibodies to herpes simplex virus types 1 and 2 // Clin. Vaccine Immunol. 2010. 17. 253-257.
2. Binnicker M.J., Jespersen D.J., Harring J.A. Multiplex detection of IgM and IgG class antibodies to Toxoplasma gondii, rubella virus, and cytomegalovi-rus using a novel multiplex flow immunoassay // Clin. Vaccine Immunol. 2010. 17. (11). 1734-1738.
3. Dhiman N., Jespersen D.J., Rollins L.O. et al. Detection of IgG-class antibodies to measles, mumps, rubella, and varicella-zoster virus using a multiplex bead immunoassay // Diagn. Microbiol. Infect. Dis. 2012. 67. (4). 346-349.
4. Guigue N., Menotti J., Hamane S. et al. Performance of the BioPlex 2200 flow immunoassay in critical cases of serodiagnosis of toxoplasmosis // Clin. Vaccine Immunol. 2014. 21. (4). 496-500.
5. Tereshchenko S.A., Dolgushin S.A., Titenok S.A. An imperfection of time-dependent diffusion models for a determination of scattering medium optical properties // Opt. Commun. 2013. 306. 26-34.
МНОГОПАРАМЕТРОВАЯ ДЕТЕКЦИЯ IGG АНТИТЕЛ К TOXOPLASMA GONDII, RUBELLA VIRUS, CYTOMEGALOVIRUS И HERPES VIRUS ПРИ ПОМОЩИ НОВОГО МУЛЬТИПЛЕКСНОГО НАБОРА РЕАГЕНТОВ
Сергей Анатольевич ДОЛГУШИН1, Екатерина Михайловна ЛОСЕВА2, Елена Сергеевна ОДИНЦОВА3, Андрей Владимирович ТРОНИН2, Сергей Васильевич СЕЛИЩЕВ1
1 Национальный исследовательский университет «МИЭТ» 124498, г. Москва, Зеленоград, площадь Шокина, д. 1
2ЗАО «МБС-Технология»
630090, г. Новосибирск, ул. Инженерная 16
3Институт химической биологии и фундаментальной медицины СО РАН 630090, г. Новосибирск, пр. Ак. Лаврентьева, 8
В работе представлены результаты доклинических испытаний нового мультиплексного набора реагентов для выявления инфекций группы TORCH при помощи метода проточной цитофлуориметрии. Набор предназначен для количественного выявления в сыворотке крови человека иммуноглобулинов класса G, специфичных к антигенам Toxoplasma gondii, Rubella virus, Cytomegalovirus и Herpes simplex virus. Мультиплексный анализ выполняется in vitro на полистирольных микросферах, меченных органическим красителем. Установлено, что диагностическая чувствительность разработанного набора реагентов составляет не менее 95 %.
Ключевые слова: мультиплексный иммуноанализ, микросфера, иммунофлуоресцентный анализ, TORCH комплекс, иммунодиагностика
Долгушин С.А. - к.ф.-м.н., старший научный сотрудник кафедры биомедицинских систем, dolgushin.sergey@gmail.com
Селищев С.И. - д.ф.-м.н., профессор, заведующий кафедрой биомедицинских систем, sersel@miee.ru Тронин А.В. - директор, tronin@mbu-tech.com Лосева Е.М. - сотрудник, losevaem@gmail.com Одинцова Е.С. - к.б.н., м.н.с., odintsoval@ngs.ru
