DOI 10.1007/s10527-015-9503-0 Biomedical Engineering, Vol. 49, No. 2, July, 2015, pp. 8589. Translated from Meditsinskaya Tekhnika, Vol. 49, No. 2, Mar.Apr., 2015, pp. 1620. Original article submitted January 19, 2015.
Preliminary Tests of Multiplex Immunoassay for Detection of TORCH Infections in Human Blood Serum Using Flow Cytometry S. A. Dolgushin1*, E. S. Odintsova2, A. Y. Gerasimenko1, A. V. Tronin2, and S. A. Tereshchenko1
Preliminary tests of multiplex immunoassay for detection of TORCH infections in human blood serum using flow cytometry are presented. The immunoassay was intended to provide qualitative detection of the specific human IgG class antibodies to Toxoplasma gondii, rubella virus, and cytomegalovirus in human blood serum. The mul tiplex analysis is performed in vitro on polystyrene microbeads labeled with an organic dye. The diagnostic sen sitivity of the kit is 97%.
The TORCH complex is a group of acute infection diseases consisting of: T – toxoplasmosis; O – others; R – rubella; C – cytomegalovirus; H – herpes simplex virus. TORCH diagnosis should be performed in pregnant women. Specific feature of the TORCH pathogens is the ability to penetrate the placental barrier, thereby exerting pathological effect on all organs and systems of the fetus. In particular, its effect on the nervous system increases the probability of miscarriage, stillbirth, and congenital abnormalities. In many cases, TORCH infection is an indication for interruption of the pregnancy. Possible TORCH infections should also be diagnosed in neonates, because such infections can cause fatal outcome [13]. TORCH infections are now diagnosed using immunoassay and polymerase chain reaction (PCR). These methods have been used for a long time and give reliable results. However, they have substantial disadvan tages: relatively low sensitivity, long duration of analysis, high probability of false results, particularly in the case of PCR.
A new technology of multiplex assay was recently suggested. The technology uses liquid biochips based on a suspension of encoded microcarriers (suspension microparticles or liquid biochips) [47]. This technology uses spectrally encoded microspheres (e.g. polystyrene) with capture molecules (probes) attached to their surface. Each microsphere is an element of the biochip that is identified using a unique spectral code and is character ized by a unique capture molecule (probe) attached to its surface. This biochip element is targeted to the substance of interest, and so the encodedbead technology provides for precise detection of this substance. Automated high performance devices based on flow cytometry are used in this analysis [47]. The advantage of the multiplex analy sis as compared to ELISA is substantial (severalfold) decrease in labor, time, and chemical consumption. The standard method of flow cytometry is based on detection of parameters of optical radiation upon single scattering on a single particle. In contrast to multiplex scattering, this requires a relatively simple detection and processing system [8]. The goal of this work was to test a kit of reagents for multilateral analysis of human humoral immunity with respect to the TORCH infections using the immunoflu orescence method based on polystyrene microbeads labeled with organic dye (OD) for detection of class
1
National Research University of Electronic Technology, Zelenograd, Moscow, Russia; Email:
[email protected] 2 MBSTekhnologiya, Ltd., Novosibirsk, Russia. * To whom correspondence should be addressed.
85
00063398/15/49020085 © 2015 Springer Science+Business Media New York
86
G immunoglobulins (IgG) to Toxoplasma gondii, rubella virus (RuV), and cytomegalovirus (CMV) in human blood serum performed in vitro in clinical diag nostic laboratories, hospitals, and other medical organ izations.
Materials and Methods Principle of analysis. Preliminarily diluted blood serum is mixed with a suspension of polystyrene microbeads labeled with the OD in different concentra tions and chemically bound with pathogenic antigens. In the presence in blood serum of IgG specific to T. gondii, RuV, and CMV antigens, the antigen specifically binds with antibody on the surface of the microbeads. The sus pension of microbeads is then washed and mixed with conjugate of antibodies specific to human IgG labeled with fluorescence marker (phycoerythrin, PE). Thus, the complex antigen/specific IgGantibody/conjugate is formed. In addition to the microbeads bound to specific anti bodies, the mixture also contains microbeads controlling the working state of the conjugate. The beads contain human monoclonal IgG. Their interaction with conju gate gives rise to IgG/conjugate complex. Thus, all stages of the reaction are controlled. The assay should be repeat ed in the absence of fluorescence. The fluorescence indicates the presence of the spe cific IgG to the infections of interest in the blood serum. The fluorescence is detected in the peak of the OD and in the peak of phycoerythrin. The following agents and materials were used: casein (SigmaAldrich, USA), proclin (SigmaAldrich), tris (hydroxymethyl)aminomethane (Helicon, Russia), ethanolamine (SigmaAldrich), RuV antigen, Toxoplasma gondii antigen, CMV antigen (MBSTechnology, Ltd., Russia), 2(Nmorpholino)ethanesulfonic acid (M2933 500G; SigmaAldrich), and PolyLink Protein Coupling Kit (Bangs Laboratories Inc., USA). Other materials used were of domestic production, especially pure grade: hydrochloric acid, ethanol, acetone, sodium chloride, sodium hydroxide, disodium phosphate, sulfuric acid, hydrogen dioxide, acetonitrile, reagent kits available from MedicoBiological Soyuz, Ltd., cat. No. TG11 (RU No. FSR 2011/10510), VG114 (RU No. RZN 2014/1512), RG11 (RU No. RZN 2013/436). Quantum Plex Carboxyl polystyrene microbeads (Cat. No. 250; Bangs Laboratories Inc., USA) with diam eter 6 μm labeled with OD (Starfire Red, excitation wave length 488 nm, fluorescence wavelength 685 nm) were used for analysis in different concentrations.
Dolgushin et al.
Various buffer solutions were also used: phosphate buffered saline with Tween 20 (FBST): 150 mM NaCl, 40 mM Na2HPO4, pH 7.4; solution for sera dilution (SSD): 1.5 M arginine (Panreac, USA), 2.5 mM merthi olate (thimerosal), 1× FBST; solution for preliminary sera dilution (SPSD): 1× FBST, 0.025% casein. Serum samples were supplied by MedicoBiological Soyuz, Ltd. PolyLink Protein Coupling Kit was used for covalent binding of proteins to the microbeads according to the manufacturer’s manual. Sera tests by ELISA were also performed according to the instruction given in the man ufacturer’s manual.
Results Assay procedure. The wells of a plate contained blood serum, 90 μl SPSD, and 10 μl of tested samples. The solu tion was mixed by pipetting, and 60 μl of SSD was added. Vortex mixing was followed by addition of 20 μl of specif ic and control microbeads and 20 μl of blood serum. The contents of wells were stirred, and the plate was incubated for 30 min at 37°C in the dark. After incubation, 200 μl of FBST solution was added to the wells, and the plate was triple washed using a vacuum filter. Then 100 μl of conju gate working solution was added into the wells and stirred. The filterplate was incubated for 20 min at 37°C in the dark. After incubation, the filterplate was triple washed with FBST. Then 100 μl of FBST solution was added to each well, stirred, and the filterplate was placed into a special module for automatic supply of samples for further analysis using the flow cytometer. Bead population study in flow cytometer. The proper ties of microbeads labeled with OD in different concen trations were tested using a mixture of five populations with BD FACS CantoII flow cytometer with BD FACS Diva software. The fluorescence was excited with an air cooled argon laser (power, 15 mW; wavelength, 488 nm). The fluorescence was filtered using an FL3 (PerCPH) filter. The fluorescence was detected for PE and PerCP/PECy5 using FL2 and FL3 photomultipliers (PM), respectively. The data of forward side scatter (FSC) and side scat ter (SSC) were obtained, as well as microbead fluores cence intensity (Fig. 1). The FSC versus SSC data for sin gle microbeads is shown in Fig. 1. PE microbead fluores cence intensity was assayed using curves FL3 versus FL2. The fluorescence intensity is illustrated in Fig. 2. Interaction between microbead populations carrying antigens of various infections and antibodies from human blood serum. The interaction of blood serum IgG with
Multiplex Immunoassay of TORCH Infections in Human Blood Serum
Fig. 1. Data for forward side scatter (FSC) and side scatter (SSC) of microbead population mixture.
microbeads covalently bound to different antigens was studied. Microbeads No. 3 were bound to antigens of T. gondii. Then the assay was done according to the proce dure described above. Individual sera were tested using ELISA for corresponding antibodies. The microbeads with covalently bound RuV (microbeads No. 1) and CMV (microbeads No. 3) were tested by a similar proce
a
87
dure. The results of the tests are shown in Fig. 3. In neg ative tests, the cloud of the data is located in the first decade of the abscissa (Fig. 3, b, e, and h), as well as in the control of a given microbead population not incubat ed with serum (Fig. 3, a, d, and g). Positive samples demonstrated a shift of the cloud toward positive fluores cence amplitudes in the FL2 РЕ channel (Fig. 3, c, f, and i). These results show that the conjugate interaction is specific. It interacts only with microbeads containing pathogen antigens and serum antibodies specifi cally bound to them. The data cloud shift along the abscissa is observed only after incubation of the beads with sera. Studies of interaction of specific microbeads with sera led to the conclusion that a multiplex system could be constructed to detect three types of specific antibod ies. Sera tests with multiplex system. The WP AB(+/–) BiolumixToRC working panel of sera was constructed to run preliminary tests of antibody–antigen interaction of T. gondii, RuV, and CMV. The group contained 10 sam ples. In specific and control microbead suspensions (Fig. 4), microbeads No. 5 contained antigen to RuV; microbeads No. 4, to CMV; microbeads No. 3, T. gondii; microbeads No. 2, human monoclonal IgG. The No. 1 microbeads were omitted (Fig. 2) because of superposi tion of peaks No. 1 and 2.
b
Fig. 2. a) Fluorescence intensity of mixture of five microbead populations with respect to each other. A BD FACS CantoII cytofluorimeter was used for detection. Maximums correspond to microbead populations 15, from left to right; b) microbead population fluorescence inten sity in coordinates (FL2, FL3); clouds correspond to microbead populations 15, from the bottom to the top.
88
Dolgushin et al.
a
c
b
d
e
f
g
h
i
Fig. 3. Results of tests of negative (b, e, h) and positive (c, f, i) sera to the T. gondii (b, c), RuV (e, f), and CMV (h, i) of human blood serum. The fluorescence data were obtained using microbead populations incubated with the conjugate or without incubation with sera (a, d, g).
The results obtained after multiplex assay of working panel sera are consistent with the ELISA data for nine sera. Fifty samples of blood sera were tested using the ELISA method and the multiplex assay. A total of 150
individual assays were tested (i.e. 50 samples of sera were tested for the presence of three analytes); of them 146 results were correct (coinciding with the ELISA result). Thus, the diagnostic sensitivity of the kit was be 97%.
Multiplex Immunoassay of TORCH Infections in Human Blood Serum
a
89
b
5 4 3 2
Fig. 4. Multiplex assay of the serum positive to IgG of T. gondii. The clouds correspond to microbead populations 2, 3, 4, 5, from the bottom to the top: 2) conjugate control (C+); 3) T. gondii; 4) CMV; 5) RuV. a) Before incubation with serum; b) after incubation with serum.
Conclusion
REFERENCES
Preliminary tests of the multiplex immunoassay for the detection of IgG antibodies to antigens of Toxoplasma gondii, rubella virus, and cytomegalovirus in human blood serum were performed in this work using the ELISA method with polystyrene microbeads labeled with organ ic dye. The results of the preliminary tests of the kit of reagents are consistent with the requirements for such test systems. The diagnostic sensitivity of the kit was found to be on par with the leading international models.
1. Kishkun A.A., Laboratory Diagnosis Technique Manual [in Russian], GEOTARMedia, Moscow (2014). 2. Kitsak V.Ya., Viral Infections in Pregnant Women: Fetus and Neonate Pathologies [in Russian], RMAPO, Koltsovo (2004). 3. Vasil’ev A.N., Fedorova N.E., Klimova R.R., Adieva A.A., Klin. Lab. Diagn., No. 6, 5255 (2012). 4. Braeckmans K., De Smedt S.C., Leblans M., Pauwels R., Demeester J., Nat. Rev. Drug Discov., 1, 447456 (2002). 5. Smits G.P., van Gageldonk P.G., Schouls L.M., van der Klis F.R.M., Berbers G.A.M., Clin. Vaccine Immunol., 19, 396400 (2012). 6. Binnicker M.J., Jespersen D.J., Harring J.A., Clin. Vaccine Immunol., 17, 253257 (2010). 7. Binnicker M.J., Jespersen D.J., Harring J.A., Clin. Vaccine Immunol., 17, 17341738 (2010). 8. Tereshchenko S.A., Dolgushin S.A., Titenok S.A., Optics Commun., 306, 2634 (2013).
This work was supported by the Russian Ministry of Education and Science (project No. 14.575.21.0090, identifier RFMEFI57514X0090).
