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Quantitative analysis of human immunoregulatory cytokines by electrochemiluminescence method
Journal of Immunological Methods 275 (2003) 81 – 88 www.elsevier.com/locate/jim
Quantitative analysis of human immunoregulatory cytokines by electrochemiluminescence method Sergey V. Sennikov *, Sergey V. Krysov, Tatiana V. Injelevskaya, Alexandr N. Silkov, Lyubov V. Grishina, Vladimir A. Kozlov Research Institute of Clinical Immunology, Siberian Branch of Russian Academy of Medical Science, 14, Yadrintsevskaja str., Novosibirsk 630091, Russia Received 6 March 2002; received in revised form 15 November 2002; accepted 16 December 2002
Abstract Quantitative analysis of human immunoregulatory cytokines in physiological media and cell cultures plays an important role in fundamental and clinical research. Here we describe the quantification of interleukin (IL)-2, IL-4, IL-10 and interferon-g (IFN-g) in human serum and peripheral blood mononuclear cell (PBMC)-conditioned medium by electrochemiluminescence method (ECL). We demonstrate that this approach allows to detect cytokine concentration from 1 pg/ml. The high sensitivity in combination with accuracy and wide range of determined concentration indicates that ECL meets the standards of quantitative analysis of cytokines. Simplicity and short time of procedure, small assay volume and high reproducibility make ECL method competitive in practical use with conventional quantitative methods of cytokine detection. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Quantitative analysis; Interleukin; Electrochemiluminescence; Serum; Antibody
1. Introduction The study of immunological mechanisms of development of autoimmune, allergic, hematological diseases and immunodeficiency is impossible without a Abbreviations: ECL, electrochemiluminescence method; rh, recombinant human; Ab, antibodies; IL, interleukin; IFN-g, interferon-g; TAG-NHS Ester, N-hydroxysuccinymidether (NHS) of ruthenium(II) tris-bipiridine chelate; TPA, tripropylamine; BiotinLC-Sulfo-NHS Ester, sulfosuccinimidyl-6-(biotinamido)hecsanoate; CC, calibration curve. * Corresponding author. Tel.: +7-383-2-22-26-74; fax: +7-3832-22-70-28. E-mail address: [email protected] (S.V. Sennikov).
qualitative and quantitative estimation of cytokine production. At a number of diseases of the immune system the quantification of cytokines in blood serum and conditioned medium from stimulated blood cells is essential to determine the immunopathogenetic stage of development of a disease, to choose the proper immunotherapy and to estimate the efficacy of specific immunocorrection (Bienvenu et al., 1993; Cortes and Kurzrock, 1997; Naoumov and Rossol, 1997; Papanicolaou et al., 1998; Sennikov et al., 2001, 2002; Steffen and Ebersole, 1996). Consequently, the development of new methods of estimation of cytokine content in physiologic media and in conditioned medium from cell cultures is
0022-1759/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0022-1759(03)00007-3
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important not only for fundamental research, but also for medical practice. We used electrochemiluminescence method (ECL) developed by IGEN (USA) for quantitative definition of interleukin (IL)-2, IL-4, IL-10 and interferon-g (IFN-g) in human blood serum and in conditioned medium from mitogen-stimulated mononuclear blood cells. The method is based on a reaction of electronic transfer in which molecule precursor tripropylamine (TPA) and ruthenium tris-bipiridine chelate (Ru(bpy32 +) or TAG) are being activated, losing one electron on a surface of an electrode in the process. Therefore, both TPA and ruthenium get the ability to emit a photon of specific length of a wave 620 nm at a moment of restoration of Ru3 + up to Ru2 + (Blackburn et al., 1991; Yang et al., 1994). Ruthenium property to form N-hydroxysuccinymidether (NHS) of ruthenium(II) tris-bipiridine chelate or TAG-NHS Ester allows to use a reaction of electronic transfer with the subsequent photon emission for a quantitative estimation of TAG-labeled molecules in a solution. Pairs of antibodies with different epitopes of binding are labeled either by TAG or biotin. TAG- and biotin-labeled antibodies are added to an assay (1– 4 Ag/ml) that is incubated for 90– 120 min at room temperature and with regular stirring in order to form a complex of labeled antibodies with cytokine molecules. Then paramagnetic streptavidin-coated beads are added to an assay and the following incubation lasts for 10– 30 min to form bead – complex conjugate. Bead –complex conjugate enters luminometer chamber. The mobile magnet pulls in the conjugate to an electrode on a surface of which a reaction of electrochemiluminescence occurs. These reactions result in emission of photon in a wavelength of 620 nm registered by luminometer. The received data are processed by computer. The available quantitative methods for estimation of cytokines in biological and culture media have certain lacks, which concern such parameters as accuracy of definition, sensitivity of a method, use of a radioactive label, complexity of fulfillment of a technique, time for fulfillment of a technique, limited range of determined concentration of cytokines in serum and conditioned medium (Bienvenu et al., 1993). As opposed to the majority of systems widespread for today, such as ELISA, RIA and others, ECL
method of quantitative definition of cytokines has a number of advantages by these parameters. ECL method allows to carry out the analysis of tests with the minimum expenses of time. It does not require a change in temperature of assays. All reactions proceed at room temperature. The method is ecological since a used label is not radioactive. The large range of determined cytokines concentration in the same experiment allows activity with different samples without dissolution. The high sensitivity of a method allows to reveal in assays protein concentrations of less than 1 pg/ml. A method gives an opportunity of working with molecules from 1000 Da and above, as well as with oligonucleotides and nucleotides (e.g. cAMP) with high resolution (Yu and Bruno, 1996).
2. Materials and methods 2.1. Equipment Quantitative protein detection was made on ORIGENR Analyser provided by IGEN. 2.2. Antibodies Polyclonal and monoclonal antibodies (Ab) were purchased at R&D System (UK). Monoclonal Ab MAB202 and polyclonal Ab AB-202-NA were used against recombinant human (rh) IL-2; monoclonal MAB204 and polyclonal AB-204-NA were used against rhIL-4; monoclonal MAB217 and polyclonal AB-217-NA were used against rhIL-10; monoclonal Ab MAB284 and polyclonal AB-284-NA were used against rhIFN-g. All lyophilized Ab was reconstituted at 1 mg/ml in PBS with 0.1% BSA. Labeled Ab were dissolved to 1 or 2 Ag/ml in PBS with 1.5% Tween 20 for detection in serum, and in PBS with 1% Tween 20 + 1% BSA for detection in conditioned medium, directly before use. 2.3. Cytokines Recombinant cytokines IL-2, IL-4 and IL-10 purchased at R&D System were used for calibration curve (CC). IFN-g was purchased at Thomae-Biberach/Riss (Germany). All recombinant proteins were
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dissolved in PBS, pH 7.4, with 0.05% NaN3 and BSA 0.1% to 2 Ag/ml concentration; aliquots were stored at 70 jC before use.
CO2. Cells were stimulated by 10 Ag/ml ConA (Sigma, USA). For construction of calibration curve we used the same medium.
2.4. Gelfiltration
2.9. Antibody biotinylation procedure
Gelfiltration was performed at PD-10 Column Sephadex G-25M (Pharmacia, Sweden).
ORIGENR Biotin-LC-Sulfo-NHS Ester or sulfosuccinimidyl-6-(biotinamido)hecsanoate was donated by IGEN. Polyclonal Ab was dissolved in PBS pH 7.8 (amine-free) at 1 mg/ml. Biotin was dissolved in sterile distilled water at 2 mg/ml. An optimum molar ratio of biotin – antibody 20:1 was used. The mixture of biotin – antibodies was incubated for 60 min at room temperature. The reaction was stopped by addition of 20-Al 2 M glycine. The mixture of labeled Ab was cleared from nonbinding label on PD-10 Column (Pharmacia). Biotinylated Ab was stored at + 4 jC before use.
2.5. Streptavidin-coated beads DynabeadsR M-280 streptavidin-coated beads were received at IGEN. Polystyrene beads, which have a magnetic basis, diameter 2.8 Am, are covered with streptavidin, and covalently connected with a bead surface. Beads in 10 mg/ml concentration are dissolved in PBS pH 7.4 with 0.1% BSA and 0.02% NaN3 and stored at 4– 8 jC. Before use the beads were dissolved at 1 mg/ml in PBS with 1.5% Tween 20 or in PBS with 1% Tween 20 + 1% BSA for detection in serum or conditioned medium, respectively. 2.6. Buffers Buffers were donated by IGEN. The cleaning buffer or ORIGENR Cell Cleaner contains KOH and Triton X-100. The analytical buffer or ORIGENR Assay Buffer is a phosphate buffer containing tripropylamine (TPA). The buffers are stored at room temperature. 2.7. Serum For calibration we used the normal human serum purchased at Rockland (UK). For analysis we used serum of healthy volunteers, prepared by standard protocol. 2.8. Conditioned medium Peripheral blood mononuclear cells (PBMC) were isolated by standard method on ficoll-urografin gradient. The mononuclear cells were incubated in RPMI-1640 medium supplemented with 10% heat inactivated FCS (ICN), 2 mM/l L-glutamin, 100 mg/ l ampicillin and 50 mg/l gentamicin at 1 106 cells/ml in 24-well plates (1 ml/well) for 48 h at 37 jC and 5%
2.10. Antibody TAG labeling procedure ORIGENR TAG-NHS Ester or N-hydroxysuccinimide ether (NHS) of a ruthenium(II) tris-bioyridine chelate was donated by IGEN. Optimum molar ratio for TAG/Ab binding with maximum labeling of Ab was 5:1. ORIGENR TAGNHS Ester (75 Ag) was dissolved in 50-Al DMSO and 1.5 Ag/Al was obtained. Ab mixture with TAG label is incubated for 60 min at room temperature at constant shaking. The reaction is stopped by addition of 20-Al 2 M glycine. To remove nonreacting label, the solution was subjected to gelfiltration on PD-10 Column (Pharmacia). 2.11. Statistics The Wilcoxon pair test was used to compare results from unstimulated cells with that from stimulated cells.
3. Results We recommend to optimize the conditions of interaction Ab, cytokine, streptavidin and TPA for each Ab pair. The optimization of conditions is carried out on such parameters as antibody concentration
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ratio, incubation duration, the number of streptavidin beads added in assay and TPA amount. In the present work the selection of antibody ratio was done by the following scheme: TAG/biotin ratio—1:1, 1:2, 2:1, 2:2 Ag/ml at a range of standard dilutions of recombinant cytokine from 0 to 10 000 pg/ml. Fig. 1 shows the selection of the antibody ratio given on IL-4 as an example. We tested 30, 90 and 120 min of antibody and assay incubation to find the optimum time. One hundred and twenty minutes is usually enough for the first incubation of antibodies with assay, and 30 min for the second incubation of immune complex with streptavidin beads. This protocol is efficient while working with assay of donors blood serum, but the procedure should be corrected while making measurements in conditioned medium. In this case it is better to make biotin – streptavidin reaction prior to incubation of streptavidin beads with assays, thus preventing binding of free biotin of conditioned medium. The protocol for conditioned medium: 20 – 30-min incubation of antibodies and streptavidin beads mixture, and 90 – 120-min incubation with assay. Fig. 2 illustrates the selection of the protocol for definition IL-4 in conditioned mediums. According to ORIGENR manufacturer’s protocol, effective kinetics of formation of immune complexes can be reached with 30 Ag of beads for a test.
Fig. 1. Selection of the antibody ratio for detection of IL-4 in human serum. Ratio of labeled antibody (TAG/biotin) were (n) 1—1:1; (o) 2—1:2; (z) 3—2:1; (D) 4—2:2. rhIL-4 were diluted in Rockland serum in concentrations 0, 10, 100, 1000 and 10 000 pg/ ml. Optimal CC were obtained for ratio 1:1.
Fig. 2. Selection of the optimal protocol for detection of IL-4 in conditioned medium. Ratio of labeled antibody (TAG/biotin) 1:1. rhIL-4 were diluted in culture medium in concentrations 0, 10, 100, 1000 and 10 000 pg/ml. Protocol 1: 25 Al biotinylated antibody (1 Ag/ml) + 25 Al streptavidin-coated beads (1 mg/ml)—incubation of 30 min. Addition of 25 Al sample + 25 Al ruthenylated antibody (1 Ag/ml)—incubation of 120 min. Dilution with 200 Al of assay buffer. Protocol 2: 25 Al sample + 25 Al ruthenylated antibody (1 Ag/ ml) + 25 Al biotinylated antibody (1 Ag/ml)—incubation of 120 min. Addition of 25 Al streptavidin-coated beads (1 mg/ml)—incubation of 30 min. Dilution with 200 Al of assay buffer. Optimal CC were obtained for protocol 2.
Streptavidin has high affinity to biotin (Kd = 10 15 M) and is characterized by a low degree of nonspecific binding. One milligram of DynabeadsRM280 binds at least 300 pmol of free biotin or 5– 10 Ag of biotinylated antibodies. The selection of optimum quantity of beads for the most effective kinetics of formation of immune complex can be performed by dilutions in 20, 30, 40 and 60 Ag for an assay. In our experiments the amount of streptavidin beads in reaction did not effect such parameters as sensitivity and range of determined concentration (data not shown). While preparing standard dilutions, it is necessary to take into account what matrix is to be used. In case the quantification of cytokines is made in blood serum of donors, it is necessary to prepare standard dilutions of recombinant cytokines in normal human serum. It will help to lower ‘‘matrix effects’’ as much as possible. It is better, but not necessary, to use the serum previously cleared from admixture of determined protein. Such approach will raise ECL sensitivity (Collins, 1988).
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Working with samples of conditioned medium, we prepared the standard in identical culture medium with identical contents of FCS, antibiotics, etc. Calibration curve (CC) built for rhIL-2 defined in blood serum is shown in Fig. 3. The ratio of TAG/ biotin Ab against rhIL-2, chosen by us at optimization of conditions, was 2:2 Ag/ml. The range of standard rhIL-2 dilutions for CC was 0 – 3000 pg/ml. The sensitivity of the method enabled to detect up to 1 pg/ml IL-2 in samples of blood serum of donors. CC appropriate to standard dilution in culture medium is shown in Fig. 3. For this CC by us the same range of the standards 0– 3000 pg/ml was used. Sensitivity to IL-2 in culture medium was higher (0.9 pg/ml). The reaction condition:
25 Al sample + 25 Al ruthenylated antibody (2 Ag/ ml)—incubation of 20 min. 25 Al streptavidin-coated beads (1 mg/ml) + 25 Al biotinylated antibody (2 Ag/ml)—incubation of 20 min. Mixing of both cocktails and incubation of 90 min. Dilution with 150 Al of assay buffer. Read assay.
Fig. 4 shows an example of CC for IL-4 built on standard rhIL-4 dilutions in human serum. The most
Fig. 3. Calibration curves for hIL-2. rhIL-2 were diluted in Rockland serum (n) and culture medium (o) in concentrations 0, 12, 37, 111, 333, 1000 and 3000 pg/ml. The measurement of ECL signal for each point of the calibration curve was measured in doublet. The calibration curves were plotted on average values of two measurements.
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Fig. 4. Calibration curves for hIL-4. rhIL-4 were diluted in Rockland serum (n) and culture medium (o) in concentrations 0, 12, 37, 111, 333, 1000, 3000 and 9000 pg/ml. The measurement of ECL signal for each point of the calibration curve was measured in doublet. The calibration curves were plotted on average values of two measurements.
optimum ratio of labeled antibodies to IL-4 was 1:1 Ag/ml. The sensitivity for IL-4 defined in a serum was 2 pg/ml. The range of standard dilutions used for CC was 0– 9000 pg/ml. As well as in the case with IL-2, at measurement of IL-4 in conditioned medium (Fig. 4), the sensitivity was higher and was equal to 1.8 pg/ml.
Fig. 5. Calibration curves for hIL-10. rhIL-10 were diluted in Rockland serum (n) and culture medium (o) in concentrations 0, 12, 37, 111, 333, 1000, 3000 and 9000 pg/ml. The measurement of ECL signal for each point of the calibration curve was measured in doublet. The calibration curves were plotted on average values of two measurements.
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in culture medium. In both cases, the dilution range was 0 –9000 pg/ml; the sensitivity of the method at working with conditioned medium assays was 2 pg/ ml. The reaction condition:
25 Al sample + 25 Al ruthenylated antibody (1 Ag/ ml) + 25 Al biotinylated antibody (2 Ag/ml)— incubation of 120 min. Addition of 25 Al streptavidin-coated beads (1 mg/ ml)—incubation of 30 min. Dilution with 200 Al of assay buffer. Read assay. Fig. 6. Calibration curves for hIFN-g. rhIFN-g were diluted in Rockland serum (n) and culture medium (o) in concentrations 0, 12, 37, 111, 333, 1000, 3000 and 9000 pg/ml. The measurement of ECL signal for each point of the calibration curve was measured in doublet. The calibration curves were plotted on average values of two measurements.
The reaction condition:
The measurement of IFN-g concentrations in human serum has shown that for this cytokine, ECL method also has a high sensitivity (1 pg/ml) at 2:1 Ag/ ml Ru/biotin Ab ratio. The dilution range for CC shown in Fig. 6 was 0 – 9000 pg/ml. CC received in culture medium was also shown in Fig. 6. The sensitivity for assays of conditioned medium was 1 pg/ml. The reaction condition:
25 Al sample + 25 Al ruthenylated antibody (1 Ag/ ml) + 25 Al biotinylated antibody (1 Ag/ml)— incubation of 120 min. Addition of 25 Al streptavidin-coated beads (1 mg/ ml)—incubation of 30 min. Dilution with 200 Al of assay buffer. Read assay. Fig. 5 shows CC for IL-10 defined in blood serum. The minimum IL-10 concentration, detected by ECL method, was 1.23 pg/ml. Such sensitivity was achieved at 1:2 Ag/ml ratio of antibodies labeled by ruthenium and biotin, respectively. Fig. 5 shows CC for IL-10 built on standard dissolved
25 Al sample + 25 Al ruthenylated antibody (2 Ag/ ml) + 25 Al biotinylated antibody (1 Ag/ml)— incubation of 90 min. Addition of 25 Al streptavidin-coated beads (1 mg/ ml)—incubation of 30 min. Dilution with 200 Al of assay buffer. Read assay. The results of the cytokine level measurement in the serum and conditioned medium from 10 donors were shown in Table 1. The definition of IL-10 concentration in conditioned mediums was not carried out.
Table 1 Cytokine levels in serum and conditioned medium from PBMSa Fluids and treatment
Serum Medium conditioned by unstimulated PBMC Medium conditioned by ConA-stimulated PBMC
Mean F standard error (pg/ml) IL-2
IL-4
IL-10
IFN-g
13.6 F 4.01 33.1 F 8.42 113.0 F 32.79*
24.8 F 5.74 379.3 F 102.18 1208.5 F 211.85*
41.1 F 6.76 – –
12.9 F 2.33 234.3 F 73.29 1155.2 F 420.14*
a Supernatants were harvested at 48 h from 106 PBMCs. Data from 10 donors are shown. * The cytokine level was significantly higher as compared to unstimulated cells ( p < 0.01).
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4. Discussion Growing clinical interest to this parameter causes the improvement of methods of quantitative definition of cytokine contents in blood serum and conditioned medium from cell culture. The estimation of cytokine content in physiologic media and conditioned medium from cell cultures is important not only for fundamental research, but also for clinical use in making diagnosis and for immunocorrective therapy of various diseases of the immune and hematopoietic systems. The main benefits of this method are the following: accuracy of detection, sensitivity, simplicity of fulfillment of a technique, nonuse of radioactive label, reproducibility of measurements and big range of determined concentrations of cytokines in serum and conditioned medium. As of opposed to the majority of systems widespread today, such as ELISA, RIA and others, ECL method of quantitative definition of cytokines has a number of advantages by these parameters. The washing procedure is not required while using this method. The reaction is made at room temperature and only a small volume of assay is needed for analysis. The main parameters of ECL method of definition of IL-2, IL-4, IL-10 and IFN-g content in blood serum and conditioned medium from cell culture are shown in comparison with similar parameters shown by the firms that manufactured the commercial sets for quantification of cytokine contents. Large range of concentrations defined by ECL method allows definition of the cytokine concentration within the limits of CC in almost any studied assay. ECL method of definition of the content of cytokines in blood serum and conditioned medium from cell culture provides good reproducibility of the results at correct storage of standards and reagents. The sensitivity of ECL method is higher or comparable to sensitivity of submitted sets. The kit of some companies for ultra-exact measurements up to hundreds of femtograms (fg) has a small range of standard dilutions in CC that limits the area of their application. The use of chemiluminescence label provides ecological purity of the method fulfillment. The duration of incubation of assays with labeled antibodies makes 2 – 2.5 h, which together with the absence of washing procedures considerably reduces the time of the method fulfillment.
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The short time of incubation depends on the kinetics of antigen –antibody reaction passing and is provided in ECL method by immunomagnetic method of antigen capture by means of mobile magnet microbeads. Immunomagnetic method (in comparison with other test systems) provides more free and effective antigen binding in various complex biological liquids. Magnetic beads provide delivery of immune complexes directly on a surface of electrode (anode) where the reaction of electrochemiluminescence at the TPA presence takes place. The obtained results on the use of ECL method for definition of the level of various cytokines in blood serum and conditioned medium allow to conclude that the parameters of the method are comparable to the main parameters shown for commercial kits and even surpassing them by some parameters. These parameters are the following: simplicity of method fulfillment, time of technique, absence of washing procedures, small volume of assay, large range of determined concentration of cytokines in serum and conditioned medium, and reproducibility of the method. ECL can be used for both scientific and clinical studies.
Acknowledgements We are very grateful to IGEN for the equipment and consultation on ECL method.
References Bienvenu, J., Coulon, L., Doche, C., Gutowski, M.-C., Grau, G.E., 1993. Analytical performances of commercial ELISA-kits for IL-2, IL-6 and TNF-a. A WHO study. Eur. Cytokine Netw. 4 (6), 447 – 451. Blackburn, G.F., Shah, H.P., Kenten, J.H., Leland, J., Kami, R.A., Link, J., Peterman, J., Powell, M.J., Shah, A., Talley, D.B., Tyagi, S.K., Wilkins, E., Wu, T., Massey, R.J., 1991. Electrochemiluminescence detection for development of immunoassays and DNA probe assay for clinical diagnostics. Clin. Chem. 37, 1534 – 1539. Collins, W.P. (Ed.), 1988. Complementary Immunoassays. John Wiley & Son Ltd., London, UK. Cortes, J., Kurzrock, R., 1997. Interleukin-10 in non-Hodgkin’s lymphoma. Leuk. Lymphoma. 26 (3 – 4), 251 – 259. Naoumov, N.V., Rossol, S., 1997. Studies of interleukin-12 in chronic hepatitis B virus infection. J. Viral Hepatitis. 4 (Suppl. 2), 87 – 91.
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Papanicolaou, D.A., Wilder, R.L., Manolagas, S.C., Chrousos, G.P., 1998. The pathophysiologic roles of interleukin-6 in human disease. Ann. Intern. Med. 15; 128 (2), 127 – 137. Sennikov, S.V., Krysov, S.V., Injelevskaya, T.V., Silkov, A.N., Kozlov, V.A., 2001. Production of cytokines by erythroid cells of human embryonal liver. Eur. Cytokine Netw. 12 (2), 274 – 279. Sennikov, S.V., Krysov, S.V., Silkov, A.N., Injelevskaya, T.V., Kozlov, V.A., 2002. Production of IL-10, TNF-a, IFN-g, TGFh1 by different populations of erythroid cells derived from human embryonal liver. Cytokine. 17 (4), 221 – 225.
Steffen, M.J., Ebersole, J.L., 1996. Sequential ELISA for cytokine level in limited volumes of biological fluids. BioTechniques. 21, 504 – 509. Yang, H., Leland, G.K., Yost, D., Massey, R.J., 1994. ECL: a new diagnostic and research toolBio/Technology. vol. 12. Yu, H., Bruno, J.G., 1996. Immunomagnetic-electrochemiluminescent detection of Echerichia coli O157 and Salmonella typhimurium in food and environmental water samples. Appl. Environ. Microbiol. 587 – 592.
Quantitative analysis of human immunoregulatory cytokines by electrochemiluminescence method Sergey V. Sennikov *, Sergey V. Krysov, Tatiana V. Injelevskaya, Alexandr N. Silkov, Lyubov V. Grishina, Vladimir A. Kozlov Research Institute of Clinical Immunology, Siberian Branch of Russian Academy of Medical Science, 14, Yadrintsevskaja str., Novosibirsk 630091, Russia Received 6 March 2002; received in revised form 15 November 2002; accepted 16 December 2002
Abstract Quantitative analysis of human immunoregulatory cytokines in physiological media and cell cultures plays an important role in fundamental and clinical research. Here we describe the quantification of interleukin (IL)-2, IL-4, IL-10 and interferon-g (IFN-g) in human serum and peripheral blood mononuclear cell (PBMC)-conditioned medium by electrochemiluminescence method (ECL). We demonstrate that this approach allows to detect cytokine concentration from 1 pg/ml. The high sensitivity in combination with accuracy and wide range of determined concentration indicates that ECL meets the standards of quantitative analysis of cytokines. Simplicity and short time of procedure, small assay volume and high reproducibility make ECL method competitive in practical use with conventional quantitative methods of cytokine detection. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Quantitative analysis; Interleukin; Electrochemiluminescence; Serum; Antibody
1. Introduction The study of immunological mechanisms of development of autoimmune, allergic, hematological diseases and immunodeficiency is impossible without a Abbreviations: ECL, electrochemiluminescence method; rh, recombinant human; Ab, antibodies; IL, interleukin; IFN-g, interferon-g; TAG-NHS Ester, N-hydroxysuccinymidether (NHS) of ruthenium(II) tris-bipiridine chelate; TPA, tripropylamine; BiotinLC-Sulfo-NHS Ester, sulfosuccinimidyl-6-(biotinamido)hecsanoate; CC, calibration curve. * Corresponding author. Tel.: +7-383-2-22-26-74; fax: +7-3832-22-70-28. E-mail address: [email protected] (S.V. Sennikov).
qualitative and quantitative estimation of cytokine production. At a number of diseases of the immune system the quantification of cytokines in blood serum and conditioned medium from stimulated blood cells is essential to determine the immunopathogenetic stage of development of a disease, to choose the proper immunotherapy and to estimate the efficacy of specific immunocorrection (Bienvenu et al., 1993; Cortes and Kurzrock, 1997; Naoumov and Rossol, 1997; Papanicolaou et al., 1998; Sennikov et al., 2001, 2002; Steffen and Ebersole, 1996). Consequently, the development of new methods of estimation of cytokine content in physiologic media and in conditioned medium from cell cultures is
0022-1759/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0022-1759(03)00007-3
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important not only for fundamental research, but also for medical practice. We used electrochemiluminescence method (ECL) developed by IGEN (USA) for quantitative definition of interleukin (IL)-2, IL-4, IL-10 and interferon-g (IFN-g) in human blood serum and in conditioned medium from mitogen-stimulated mononuclear blood cells. The method is based on a reaction of electronic transfer in which molecule precursor tripropylamine (TPA) and ruthenium tris-bipiridine chelate (Ru(bpy32 +) or TAG) are being activated, losing one electron on a surface of an electrode in the process. Therefore, both TPA and ruthenium get the ability to emit a photon of specific length of a wave 620 nm at a moment of restoration of Ru3 + up to Ru2 + (Blackburn et al., 1991; Yang et al., 1994). Ruthenium property to form N-hydroxysuccinymidether (NHS) of ruthenium(II) tris-bipiridine chelate or TAG-NHS Ester allows to use a reaction of electronic transfer with the subsequent photon emission for a quantitative estimation of TAG-labeled molecules in a solution. Pairs of antibodies with different epitopes of binding are labeled either by TAG or biotin. TAG- and biotin-labeled antibodies are added to an assay (1– 4 Ag/ml) that is incubated for 90– 120 min at room temperature and with regular stirring in order to form a complex of labeled antibodies with cytokine molecules. Then paramagnetic streptavidin-coated beads are added to an assay and the following incubation lasts for 10– 30 min to form bead – complex conjugate. Bead –complex conjugate enters luminometer chamber. The mobile magnet pulls in the conjugate to an electrode on a surface of which a reaction of electrochemiluminescence occurs. These reactions result in emission of photon in a wavelength of 620 nm registered by luminometer. The received data are processed by computer. The available quantitative methods for estimation of cytokines in biological and culture media have certain lacks, which concern such parameters as accuracy of definition, sensitivity of a method, use of a radioactive label, complexity of fulfillment of a technique, time for fulfillment of a technique, limited range of determined concentration of cytokines in serum and conditioned medium (Bienvenu et al., 1993). As opposed to the majority of systems widespread for today, such as ELISA, RIA and others, ECL
method of quantitative definition of cytokines has a number of advantages by these parameters. ECL method allows to carry out the analysis of tests with the minimum expenses of time. It does not require a change in temperature of assays. All reactions proceed at room temperature. The method is ecological since a used label is not radioactive. The large range of determined cytokines concentration in the same experiment allows activity with different samples without dissolution. The high sensitivity of a method allows to reveal in assays protein concentrations of less than 1 pg/ml. A method gives an opportunity of working with molecules from 1000 Da and above, as well as with oligonucleotides and nucleotides (e.g. cAMP) with high resolution (Yu and Bruno, 1996).
2. Materials and methods 2.1. Equipment Quantitative protein detection was made on ORIGENR Analyser provided by IGEN. 2.2. Antibodies Polyclonal and monoclonal antibodies (Ab) were purchased at R&D System (UK). Monoclonal Ab MAB202 and polyclonal Ab AB-202-NA were used against recombinant human (rh) IL-2; monoclonal MAB204 and polyclonal AB-204-NA were used against rhIL-4; monoclonal MAB217 and polyclonal AB-217-NA were used against rhIL-10; monoclonal Ab MAB284 and polyclonal AB-284-NA were used against rhIFN-g. All lyophilized Ab was reconstituted at 1 mg/ml in PBS with 0.1% BSA. Labeled Ab were dissolved to 1 or 2 Ag/ml in PBS with 1.5% Tween 20 for detection in serum, and in PBS with 1% Tween 20 + 1% BSA for detection in conditioned medium, directly before use. 2.3. Cytokines Recombinant cytokines IL-2, IL-4 and IL-10 purchased at R&D System were used for calibration curve (CC). IFN-g was purchased at Thomae-Biberach/Riss (Germany). All recombinant proteins were
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dissolved in PBS, pH 7.4, with 0.05% NaN3 and BSA 0.1% to 2 Ag/ml concentration; aliquots were stored at 70 jC before use.
CO2. Cells were stimulated by 10 Ag/ml ConA (Sigma, USA). For construction of calibration curve we used the same medium.
2.4. Gelfiltration
2.9. Antibody biotinylation procedure
Gelfiltration was performed at PD-10 Column Sephadex G-25M (Pharmacia, Sweden).
ORIGENR Biotin-LC-Sulfo-NHS Ester or sulfosuccinimidyl-6-(biotinamido)hecsanoate was donated by IGEN. Polyclonal Ab was dissolved in PBS pH 7.8 (amine-free) at 1 mg/ml. Biotin was dissolved in sterile distilled water at 2 mg/ml. An optimum molar ratio of biotin – antibody 20:1 was used. The mixture of biotin – antibodies was incubated for 60 min at room temperature. The reaction was stopped by addition of 20-Al 2 M glycine. The mixture of labeled Ab was cleared from nonbinding label on PD-10 Column (Pharmacia). Biotinylated Ab was stored at + 4 jC before use.
2.5. Streptavidin-coated beads DynabeadsR M-280 streptavidin-coated beads were received at IGEN. Polystyrene beads, which have a magnetic basis, diameter 2.8 Am, are covered with streptavidin, and covalently connected with a bead surface. Beads in 10 mg/ml concentration are dissolved in PBS pH 7.4 with 0.1% BSA and 0.02% NaN3 and stored at 4– 8 jC. Before use the beads were dissolved at 1 mg/ml in PBS with 1.5% Tween 20 or in PBS with 1% Tween 20 + 1% BSA for detection in serum or conditioned medium, respectively. 2.6. Buffers Buffers were donated by IGEN. The cleaning buffer or ORIGENR Cell Cleaner contains KOH and Triton X-100. The analytical buffer or ORIGENR Assay Buffer is a phosphate buffer containing tripropylamine (TPA). The buffers are stored at room temperature. 2.7. Serum For calibration we used the normal human serum purchased at Rockland (UK). For analysis we used serum of healthy volunteers, prepared by standard protocol. 2.8. Conditioned medium Peripheral blood mononuclear cells (PBMC) were isolated by standard method on ficoll-urografin gradient. The mononuclear cells were incubated in RPMI-1640 medium supplemented with 10% heat inactivated FCS (ICN), 2 mM/l L-glutamin, 100 mg/ l ampicillin and 50 mg/l gentamicin at 1 106 cells/ml in 24-well plates (1 ml/well) for 48 h at 37 jC and 5%
2.10. Antibody TAG labeling procedure ORIGENR TAG-NHS Ester or N-hydroxysuccinimide ether (NHS) of a ruthenium(II) tris-bioyridine chelate was donated by IGEN. Optimum molar ratio for TAG/Ab binding with maximum labeling of Ab was 5:1. ORIGENR TAGNHS Ester (75 Ag) was dissolved in 50-Al DMSO and 1.5 Ag/Al was obtained. Ab mixture with TAG label is incubated for 60 min at room temperature at constant shaking. The reaction is stopped by addition of 20-Al 2 M glycine. To remove nonreacting label, the solution was subjected to gelfiltration on PD-10 Column (Pharmacia). 2.11. Statistics The Wilcoxon pair test was used to compare results from unstimulated cells with that from stimulated cells.
3. Results We recommend to optimize the conditions of interaction Ab, cytokine, streptavidin and TPA for each Ab pair. The optimization of conditions is carried out on such parameters as antibody concentration
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ratio, incubation duration, the number of streptavidin beads added in assay and TPA amount. In the present work the selection of antibody ratio was done by the following scheme: TAG/biotin ratio—1:1, 1:2, 2:1, 2:2 Ag/ml at a range of standard dilutions of recombinant cytokine from 0 to 10 000 pg/ml. Fig. 1 shows the selection of the antibody ratio given on IL-4 as an example. We tested 30, 90 and 120 min of antibody and assay incubation to find the optimum time. One hundred and twenty minutes is usually enough for the first incubation of antibodies with assay, and 30 min for the second incubation of immune complex with streptavidin beads. This protocol is efficient while working with assay of donors blood serum, but the procedure should be corrected while making measurements in conditioned medium. In this case it is better to make biotin – streptavidin reaction prior to incubation of streptavidin beads with assays, thus preventing binding of free biotin of conditioned medium. The protocol for conditioned medium: 20 – 30-min incubation of antibodies and streptavidin beads mixture, and 90 – 120-min incubation with assay. Fig. 2 illustrates the selection of the protocol for definition IL-4 in conditioned mediums. According to ORIGENR manufacturer’s protocol, effective kinetics of formation of immune complexes can be reached with 30 Ag of beads for a test.
Fig. 1. Selection of the antibody ratio for detection of IL-4 in human serum. Ratio of labeled antibody (TAG/biotin) were (n) 1—1:1; (o) 2—1:2; (z) 3—2:1; (D) 4—2:2. rhIL-4 were diluted in Rockland serum in concentrations 0, 10, 100, 1000 and 10 000 pg/ ml. Optimal CC were obtained for ratio 1:1.
Fig. 2. Selection of the optimal protocol for detection of IL-4 in conditioned medium. Ratio of labeled antibody (TAG/biotin) 1:1. rhIL-4 were diluted in culture medium in concentrations 0, 10, 100, 1000 and 10 000 pg/ml. Protocol 1: 25 Al biotinylated antibody (1 Ag/ml) + 25 Al streptavidin-coated beads (1 mg/ml)—incubation of 30 min. Addition of 25 Al sample + 25 Al ruthenylated antibody (1 Ag/ml)—incubation of 120 min. Dilution with 200 Al of assay buffer. Protocol 2: 25 Al sample + 25 Al ruthenylated antibody (1 Ag/ ml) + 25 Al biotinylated antibody (1 Ag/ml)—incubation of 120 min. Addition of 25 Al streptavidin-coated beads (1 mg/ml)—incubation of 30 min. Dilution with 200 Al of assay buffer. Optimal CC were obtained for protocol 2.
Streptavidin has high affinity to biotin (Kd = 10 15 M) and is characterized by a low degree of nonspecific binding. One milligram of DynabeadsRM280 binds at least 300 pmol of free biotin or 5– 10 Ag of biotinylated antibodies. The selection of optimum quantity of beads for the most effective kinetics of formation of immune complex can be performed by dilutions in 20, 30, 40 and 60 Ag for an assay. In our experiments the amount of streptavidin beads in reaction did not effect such parameters as sensitivity and range of determined concentration (data not shown). While preparing standard dilutions, it is necessary to take into account what matrix is to be used. In case the quantification of cytokines is made in blood serum of donors, it is necessary to prepare standard dilutions of recombinant cytokines in normal human serum. It will help to lower ‘‘matrix effects’’ as much as possible. It is better, but not necessary, to use the serum previously cleared from admixture of determined protein. Such approach will raise ECL sensitivity (Collins, 1988).
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Working with samples of conditioned medium, we prepared the standard in identical culture medium with identical contents of FCS, antibiotics, etc. Calibration curve (CC) built for rhIL-2 defined in blood serum is shown in Fig. 3. The ratio of TAG/ biotin Ab against rhIL-2, chosen by us at optimization of conditions, was 2:2 Ag/ml. The range of standard rhIL-2 dilutions for CC was 0 – 3000 pg/ml. The sensitivity of the method enabled to detect up to 1 pg/ml IL-2 in samples of blood serum of donors. CC appropriate to standard dilution in culture medium is shown in Fig. 3. For this CC by us the same range of the standards 0– 3000 pg/ml was used. Sensitivity to IL-2 in culture medium was higher (0.9 pg/ml). The reaction condition:
25 Al sample + 25 Al ruthenylated antibody (2 Ag/ ml)—incubation of 20 min. 25 Al streptavidin-coated beads (1 mg/ml) + 25 Al biotinylated antibody (2 Ag/ml)—incubation of 20 min. Mixing of both cocktails and incubation of 90 min. Dilution with 150 Al of assay buffer. Read assay.
Fig. 4 shows an example of CC for IL-4 built on standard rhIL-4 dilutions in human serum. The most
Fig. 3. Calibration curves for hIL-2. rhIL-2 were diluted in Rockland serum (n) and culture medium (o) in concentrations 0, 12, 37, 111, 333, 1000 and 3000 pg/ml. The measurement of ECL signal for each point of the calibration curve was measured in doublet. The calibration curves were plotted on average values of two measurements.
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Fig. 4. Calibration curves for hIL-4. rhIL-4 were diluted in Rockland serum (n) and culture medium (o) in concentrations 0, 12, 37, 111, 333, 1000, 3000 and 9000 pg/ml. The measurement of ECL signal for each point of the calibration curve was measured in doublet. The calibration curves were plotted on average values of two measurements.
optimum ratio of labeled antibodies to IL-4 was 1:1 Ag/ml. The sensitivity for IL-4 defined in a serum was 2 pg/ml. The range of standard dilutions used for CC was 0– 9000 pg/ml. As well as in the case with IL-2, at measurement of IL-4 in conditioned medium (Fig. 4), the sensitivity was higher and was equal to 1.8 pg/ml.
Fig. 5. Calibration curves for hIL-10. rhIL-10 were diluted in Rockland serum (n) and culture medium (o) in concentrations 0, 12, 37, 111, 333, 1000, 3000 and 9000 pg/ml. The measurement of ECL signal for each point of the calibration curve was measured in doublet. The calibration curves were plotted on average values of two measurements.
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in culture medium. In both cases, the dilution range was 0 –9000 pg/ml; the sensitivity of the method at working with conditioned medium assays was 2 pg/ ml. The reaction condition:
25 Al sample + 25 Al ruthenylated antibody (1 Ag/ ml) + 25 Al biotinylated antibody (2 Ag/ml)— incubation of 120 min. Addition of 25 Al streptavidin-coated beads (1 mg/ ml)—incubation of 30 min. Dilution with 200 Al of assay buffer. Read assay. Fig. 6. Calibration curves for hIFN-g. rhIFN-g were diluted in Rockland serum (n) and culture medium (o) in concentrations 0, 12, 37, 111, 333, 1000, 3000 and 9000 pg/ml. The measurement of ECL signal for each point of the calibration curve was measured in doublet. The calibration curves were plotted on average values of two measurements.
The reaction condition:
The measurement of IFN-g concentrations in human serum has shown that for this cytokine, ECL method also has a high sensitivity (1 pg/ml) at 2:1 Ag/ ml Ru/biotin Ab ratio. The dilution range for CC shown in Fig. 6 was 0 – 9000 pg/ml. CC received in culture medium was also shown in Fig. 6. The sensitivity for assays of conditioned medium was 1 pg/ml. The reaction condition:
25 Al sample + 25 Al ruthenylated antibody (1 Ag/ ml) + 25 Al biotinylated antibody (1 Ag/ml)— incubation of 120 min. Addition of 25 Al streptavidin-coated beads (1 mg/ ml)—incubation of 30 min. Dilution with 200 Al of assay buffer. Read assay. Fig. 5 shows CC for IL-10 defined in blood serum. The minimum IL-10 concentration, detected by ECL method, was 1.23 pg/ml. Such sensitivity was achieved at 1:2 Ag/ml ratio of antibodies labeled by ruthenium and biotin, respectively. Fig. 5 shows CC for IL-10 built on standard dissolved
25 Al sample + 25 Al ruthenylated antibody (2 Ag/ ml) + 25 Al biotinylated antibody (1 Ag/ml)— incubation of 90 min. Addition of 25 Al streptavidin-coated beads (1 mg/ ml)—incubation of 30 min. Dilution with 200 Al of assay buffer. Read assay. The results of the cytokine level measurement in the serum and conditioned medium from 10 donors were shown in Table 1. The definition of IL-10 concentration in conditioned mediums was not carried out.
Table 1 Cytokine levels in serum and conditioned medium from PBMSa Fluids and treatment
Serum Medium conditioned by unstimulated PBMC Medium conditioned by ConA-stimulated PBMC
Mean F standard error (pg/ml) IL-2
IL-4
IL-10
IFN-g
13.6 F 4.01 33.1 F 8.42 113.0 F 32.79*
24.8 F 5.74 379.3 F 102.18 1208.5 F 211.85*
41.1 F 6.76 – –
12.9 F 2.33 234.3 F 73.29 1155.2 F 420.14*
a Supernatants were harvested at 48 h from 106 PBMCs. Data from 10 donors are shown. * The cytokine level was significantly higher as compared to unstimulated cells ( p < 0.01).
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4. Discussion Growing clinical interest to this parameter causes the improvement of methods of quantitative definition of cytokine contents in blood serum and conditioned medium from cell culture. The estimation of cytokine content in physiologic media and conditioned medium from cell cultures is important not only for fundamental research, but also for clinical use in making diagnosis and for immunocorrective therapy of various diseases of the immune and hematopoietic systems. The main benefits of this method are the following: accuracy of detection, sensitivity, simplicity of fulfillment of a technique, nonuse of radioactive label, reproducibility of measurements and big range of determined concentrations of cytokines in serum and conditioned medium. As of opposed to the majority of systems widespread today, such as ELISA, RIA and others, ECL method of quantitative definition of cytokines has a number of advantages by these parameters. The washing procedure is not required while using this method. The reaction is made at room temperature and only a small volume of assay is needed for analysis. The main parameters of ECL method of definition of IL-2, IL-4, IL-10 and IFN-g content in blood serum and conditioned medium from cell culture are shown in comparison with similar parameters shown by the firms that manufactured the commercial sets for quantification of cytokine contents. Large range of concentrations defined by ECL method allows definition of the cytokine concentration within the limits of CC in almost any studied assay. ECL method of definition of the content of cytokines in blood serum and conditioned medium from cell culture provides good reproducibility of the results at correct storage of standards and reagents. The sensitivity of ECL method is higher or comparable to sensitivity of submitted sets. The kit of some companies for ultra-exact measurements up to hundreds of femtograms (fg) has a small range of standard dilutions in CC that limits the area of their application. The use of chemiluminescence label provides ecological purity of the method fulfillment. The duration of incubation of assays with labeled antibodies makes 2 – 2.5 h, which together with the absence of washing procedures considerably reduces the time of the method fulfillment.
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The short time of incubation depends on the kinetics of antigen –antibody reaction passing and is provided in ECL method by immunomagnetic method of antigen capture by means of mobile magnet microbeads. Immunomagnetic method (in comparison with other test systems) provides more free and effective antigen binding in various complex biological liquids. Magnetic beads provide delivery of immune complexes directly on a surface of electrode (anode) where the reaction of electrochemiluminescence at the TPA presence takes place. The obtained results on the use of ECL method for definition of the level of various cytokines in blood serum and conditioned medium allow to conclude that the parameters of the method are comparable to the main parameters shown for commercial kits and even surpassing them by some parameters. These parameters are the following: simplicity of method fulfillment, time of technique, absence of washing procedures, small volume of assay, large range of determined concentration of cytokines in serum and conditioned medium, and reproducibility of the method. ECL can be used for both scientific and clinical studies.
Acknowledgements We are very grateful to IGEN for the equipment and consultation on ECL method.
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