Anal. Chem. 1909, 61, 2471-2474
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LITERATURE CITED (9) Jackson, W. A.; Johnson, D. C., work in progress. (10) Naturella, M. Exptwhmtal Stalbrcs; National Bureau of Standards (1) Klssinger, P. T. J . Chem. Educ. 1983, 60, 308. kndbodc 91; NBS: WasMngton, DC. 1963; pp 6-19. (2) Johnson, D. C. Mtue 1986, 327, 451. (11) Johnson, D. C. A M I . C h h . Acte 1988, 209. 1. (3) Welch, L. E.; LecOuse, W. R.; Mead, D. A., Jr.; Hu, T.; Johnson, D. C. Anal. them. 1989, 6 7 , 555. (4) Johnson. D. C.; Pdte. T. 2. C h t o g r . Forum 1988, 7 , 37. RECEIVED for review July 1,1989. Accepted August 30,1989. (5) The Merdc Index, 10th ed.; Mer& Flahway, NJ, 1983: p 541. The majority of this work was supported by a grant from (6) Heberer, H.; Bmersohl, G. Z.Chem. 1980, 20, 361. (7) CbnQ, M.-Y.; C k n , L.-W Dong, X.-D.; Selavka. C. M.; Krull, I. S. J . Dionex Gorp., Sunnyvale, CA. The National Science FaunCtrometogr. Scl. 1987, 25, 480. dation contributed a portion of the funding through Contract (8) brew, L. A.; Johnson. D. c. J . ElectraaMi. m m . Intdackl ~ k W-m. 1989. 262, 187. CHE-8312032.
Detection of Rat Basophilic Leukemia by Cyclic Voltammetry for Monitoring Allergic Reaction Tadashi Matsunaga,* Akinori Shigematsu, and Noriyuki Nakamura Department of Biotechnology, Tokyo University of Agriculture & Technology, Koganei, Tokyo 184, Japan
Electrochemlcal detection of the rat basophlllc leukemia (RBL-1) cells has been carrled out by applying cycllc vdtammetry. The detection system consists of a basal plane pyrotytk graphite ekctrde and a porous nitroceliulose membrane fllter to trap RBL-1 cells. When the potential of the graphtte electrode was run In the range of 0-1.0 V vs SCE, RBL-1 cdls gave peak currents at 0.34 V vs SCE as well as 0.65 V vs SCE. There Is a linear relationship between the peak current at 0.34 V vs SCE and the cell numbers of RBL-1 In the range of (0.4-2.0) X 10' cells. The peak current of RBL-1 cells was attrlbuted to serotonln. When dlnltrophenylated bovlne serum albumin (DNP-BSA) as a model allergen was added to RBL-1 cells sendtlzed wlth anti-DNP IgE, the peak current decreased because of the degranulatlon of RBL-I cdls kadbg to serotonln release. On the other hand, RBL-1 cells sensltlzed wlth antCDNP IgE did not respond to egg white, pollens, house d d , and mllk.
INTRODUCTION Detection of viable cells is important in a clinical field. Various electrochemical methods have been developed for determining viable cell numbers. For example, impedance measurements of culture media have been used to determine cell numbers, although the cell numbers are measured indirectly from cell metabolite, and therefore, the results obtained sopetimes do not correlated with true cell numbers. Recently, a novel method for detecting microbial cells has been developed, based on cyclic voltammetry at a basal plane pyrolytic graphite electrode (1,2). Electron transfer between microbial cells and the graphite electrode is mediated by coenzyme A existing in the cell. Both enumeration and classification of microbial cells were possible from cyclic voltammograms by using an electrode system composed of a graphite electrode and a membrane filter retaining microbial cells. However, cyclic voltammetry of animal cells has not been reported. Therefore, cyclic voltammetry using a graphite electrode was applied to animal cells such as rat basophilic leukemia (RBG1) and mouse lymphocytes. The radioimmunosorbent test (RIST) (3-5),radioallergosorbent test (RAST) (61,and skin test (4, 7)have been used 0003-2700/89/0361-247 1$01.50/0
for sensing immediate allergic reactions. However, RIST and RAST are time-consuming and demand complicated procedures giving results independent of clinical symptoms. The skin test is dangerous because it may produce anaphylaxis in man by means of serum antibodies. A simple and safe method is still required for the detection of the immediate allergic reactions. RBL-1 cells, like normal basophils and mast cells, have an immunoglobulin E (IgE) receptor on their surface. RBL-1 cells are passively sensitized by incubating homogeneously cells with IgE. Addition of the appropriate allergen to the stimulated RBL-1 cells causes degranulation, thereby releasing histamine and serotonin (8). Therefore, RBL-1 cells can be used for the detection of immediate allergic reactions. In this paper, the allergic reaction was also monitored with the electrode system using RBL-1 cells.
EXPERIMENTAL SECTION Materials. Sodium 2,4dinitrobenzenesulfonate was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo,Japan). Bovine serum albumin (BSA), 5-hydroxytryptamine hydrochloride (serotonin),and compound 48/80, condensation product of Nmethyl-p-methoxyphenethylamine with formaldehyde were obtained from Sigma Chemical Co. (St. Louis, MO). Monoclonal m o w anti-DNP IgE (9),purified from the ascitic fluid of BALB/c X C5,BL-F1,mice bearing the SPE-7 hybridoma, was purchased from Seikagaku Kogyo Co., Ltd. (Osaka, Japan). Other reagents were commercially available analyticalreagents or laboratmy grade materials and were used as received. Distilled-deionized water was used in all procedures. Preparation of Dinitrophenylated Bovine Serum Albumin (DNP-BSA). A DNP-BSA conjugate (13.8 mol of DNP/mol of BSA) was prepared by using sodium dinitrobenzenesulfonate and BSA as previously described by Eisen et al. (10). The DNP-BSA was employed as a model allergen. Preparation of Allergen Extracts. Immediate allergic reaction was performed with allergens (egg white, yolk, common ragweed pollen, evening primrose pollen, house dust, cow's milk, and DNP-BSA)and mouse anti-DNP IgE. These allergens except DNP-BSA were sonicated in the phosphate buffered saline (PBS, 1.5 mM KH2P04,7.3 mM Na,,HP04, 137 mM NaC1,2.7 mM KC1, pH 7.4) for 45 min and incubated at 4 O C for 12 h. Then allergens were centrifuged at 4 "C and 3000g for 30 min and the supernatants were obtained. Then, these extracts were passed through the sterilized membrane fiiter (pore size, 0.45 pm) and dialyzed for 3 days against 5 L of PBS. The protein concentration of allergen extracts was determined by the Lowry method (11). 0 1989 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 61, NO. 22, NOVEMBER 15, 1989
-
4 2
4-
c
W L L
0 3
Fl(~we1.sohemetrcdiagamoftheelectrodesystemforthedetecbbn of the allergic r e " : 1, W b n generator; 2, potentlostat; 3, X-Y recorder; 4, reference electrode (saturated calomel electrode);5, counter electrode (platinum wire); 0, working electrode (basal plane pyrolytlc graphite), 7, RBL-1 cell; 8, membrane filter; 9, holder.
Preparation of RBL-1 Cells. RBL-1 cells were cultured in Eagle's minimum essential medium with Earle's balanced salt solution supplemented with 10% fetal bovine serum and antibiotics in the atmosphere of 5 % COz at 37 OC. Cell viabilities were determined by using the trypan blue exclusion method. Cells were used for the experiments when viabilities exceeded 90%. Apparatus. The electrode system for the detection of the allergic reaction is depicted in Figure 1. The electrode system consisted of a basal plane pyrolytic graphite electrode (surface area, 0.19 cm2; Union Carbide Corp., New York), a counter electrode (platinum wire), and a membrane filter for retaining RBL-1 cells. The reference electrode was the saturated calomel electrode (SCE). Cyclic voltammograms were obtained by using a potentiostat (Model HA301, Hokuto Denko, Tokyo, Japan), a function generator (Model HBlO4, Hokuto Denko, Tokyo, Japan), and an X-Y recorder (Model F35, Riken Denshi, Tokyo, Japan). After each run, the graphite electrode was polished with emery paper. Procedure for the Detection of Immediate Allergic Reaction. The cultured RBL-1 cells were centrifuged at 100g for 1 min and washed twice with cold (4"C) Tris-A buffer (25 mM Tris-base, 120 mM NaCl, 5 mM KCI, and 0.3 mg.mL-' BSA, pH 7.6). The cells were suspended to a density of 1.0 X 106cebmL-l in 1mL Tris-ACM buffer (Tris-A buffer containing 0.6 mM CaClz and 1.0 mM MgC12). Then monoclonal mouse anti-DNP IgE and allergen extracts were added to the RBL-1 cell suspension for a final concentration of 3 pg-mL-' and 5 Mg of protein-mL-', respectively. After the cells were incubated for 2 h at 37 O C in 5% COD 0.1-0.2 mL of cell suspension was dropped on the membrane filter (pore size, 0.45pm). Immediately, the cells were fiied on the membrane filter by fitration using an aspirator. The cells on the membrane fiter were attached to the b a d plane pyrolytic graphite. Cyclic voltammetry was run in the range of 0-1.0 V vs SCE in 10 mL of PBS (pH 7.0). The degranulation of cells was confirmed by microscopy (12). Sonication of RBGl Cells. RBL-1 cells (1.0 X 106cells) were suspended in 1 mL of PBS (pH 7.0) and then disrupted by the ultrasonic disrupter (Model UR-SOOP, TOMY Seiko Co., Ltd., Tokyo, Japan) operated for 5 min at 0 "C over 5 times. The sonicated cells were centrifuged at 4 "C and lOOg, and the exudate of sonicated cells was obtained. Collected cells were rwuspended in 1mL of PBS and cell suspension (0.1 mL) was dropped on the membrane filter and used for measurement as described above. Preparation of Basic Granule. RBL-1 cells (1.0 x 105cells) were washed with 0.32 M sucrose solution containing 40 pM EDTA.2Na and 1 mM Tris-HC1 buffer (pH 7.4) and sonicated in sucrose solution. The sonicated solution was centrifuged at 6OOg for 10 min and the supernatant containing the exudate from the cells was collected. The supernatant was centrifuged at 7O00g for 10 min, and basic granules were obtained as precipitate (13). RESULTS AND DISCUSSION Cycric Voltammetry of RBL-1 Cells. Figure 2 shows the cyclic voltammograms of RBGl cells and mouse lymphocytes.
0
0,5
Potential
1,G
( V v s . SCE)
Flgure 2. Cyclic voltammograms of (A) RBL-1 cells (1.2 X lo5 cells) and (B) mouse lymphocytes (8.0 X lo6 cells) on the membrane filter. Scan rate was 10 mV.s-'. The measurements were performed In 10 mL of PBS (pH 7.0).
0
1.0
2,o
Cell nwnkrs ( X 105 cells) Flgwe 3. Relationship between peak current at 0.34 V vs SCE and RBL-1 cell numbers. Scan rate was 10 mV.s-'. The measurements were performed in 10 mL of PBS (pH 7.0).
Anodic waves appeared at 0.34 and 0.68 V vs SCE for RBL-1 cells and 0.65 V vs SCE for mouse lymphocytes. The peak current at 0.34 V vs SCE was 0.76 pA for 1.2 X 1oBR B L l cells. Upon scan reversal, no corresponding reduction peak was obtained. This shows that the electrode reactions of RBL-1 cells and lymphocytes were irreversible. The differential-pulse voltammograms of RBL-1 cells have also been obtained. The differential-pulse voltammograms were obtained by using a polarograph (Fuso Seisakusho, Model 312) and an X-Y recorder. The peak current appeared at the same potential as that of the cyclic vo1ta"ograms. The sensitivity was not improved by the differential-pulse voltammograms. Figure 3 shows the relationship between the peak currents at 0.34 V vs SCE and the cells numbers of RBL-1 on the membrane filter. The peak current increased linearly with increasing the cell numbers up to 2.0 X lo5 cells. The minimum detectable cell numbers were 0.4 x lo6 cells. These results show that cell numbers of RBL-1 in the range of (0.4-2.0) X lo5 cells can be determined from the peak current of cyclic voltammetry. Mechanism of Electrochemical Reaction in RBL-1 Cells. Recently, it was found that an electron transfer between cells and the graphite electrode is mediated by coenzyme A (CoA) present in the cell wall for various microorganisms. The peak currents of the cyclic voltammograms of yeasts, Gram-positive bacteria, and Gram-negative bacteria were observed at 0.65-0.74 V vs SCE which were attributed to the oxidation of CoA present in the cells (1,2,14). The peak currents of lymphocyte, macrophage, ascites tumor, and erythrocyte were obtained around 0.65 V vs SCE (unpublished
ANALYTICAL CHEMISTRY, VOL. 61, NO. 22, NOVEMBER 15, 1989
i.
2473
0
basophil I C leukemio c e l l
IU
baslc granule
IgE
release o f basic granule
Figure 5. Schematic diagram for the detection of immediate allergic
0
0
0
I
0
60
30 incubation time
reaction.
(
min
5.0
)
Figure 4. Time-course of (0)peak current and (0)serotonin content in RBL-1 cells when RBL-1 cells (1.4 X lo5 cells) were degranulated wlth compound 48/80 in 10 mL of Tris-ACM buffer (pH 7.0). Scan rate
was
10
mv-s-‘.
data, Matsunaga et al.). As described above, RBL-1 cells showed the peak currents at 0.34V and 0.65 V w SCE. These results suggest that the peak current of animal cells including RBL-1 cells obtained around 0.65 V vs SCE results from the electrochemical oxidation of CoA. However, the peak current at 0.34 V vs SCE obtained from RBL-1 cells seems to be related with other compounds. RBL-1 cells have cytoplasmic granules containing mediators of anaphylaxis such as histamine, serotonin, eosinophil chemotactic factor, and neutrophil chemotactic factor. The peak current of serotonin was obtained at 0.33 V vs SCE at pH 7.0 when BPG electrode was employed. Previous papers have reported that the peak potential appeared at 0.29-0.34 V vs SCE around pH 7.0 at pyrolytic graphite electrodes (15)and carbon fiber microelectrodes (16). The peak potential of the RBL-1 cell was similar to those of serotonin in our experiment and the previous papers. On the other hand, the peak current was not obtained from other mediators of anaphylaxis such as histamine. Moreover, no corresponding reduction peak was obtained in serotonin, too. The electrode reaction of serotonin was also irreversible. The oxidation peak currents of RBL-1 cells and serotonin increased linearly with the square root of the scan rate as expected for a totally irreversible reaction. The slope of peak potentials vs pH lime was 50-55 mV in the range of pH 6.0-8.0 for RBL-1 cells and serotonin. These experimental results support that the generation of peak current results from electrochemical oxidation of serotonin. Then, serotonin was eluted by sonicating RBL-1 cells in the buffer solution. As a result, the peak current from R B L l cells decreased to almost zero after 30 min of sonication. The amount of serotonin in the exudate of sonicated cells after elution was consistent with that of the RBL-1 cells before elution. Therefore, the serotonin concentration in the exudate solution of sonicated cells was determined by high-performance liquid chromatography (HPLC, Shimadzu Co. Model LC-6A system) on Shim-pack CLC-ODS (150 mm X 6.0 mm diameter). As a result, 0.90 pM of serotonin was detected in the exudate of sonicated cells (1 mL). Next, granules in the RBL-1 cells were expelled by degranulation. As a result, the serotonin content of R B L l cells decreased. Figure 4 shows the timecourse of the peak current and the serotonin content of RBL-1 cells when RBL-1 cells were degranulated with compound 48/80. It is employed as the inducer of degranulation. After degranulation, cells were washed and centrifuged for electrochemical measurement. Then, the serotonin content of cells and the amount of serotonin released were determined by high-performance liquid chromatography (HPLC). The serotonin content of degranulated RBL-1 cells decreased gradually because serotonin eluted. The peak current at 0.34 V v9 SCE also decreased with
< a Y
2.5 al a U L
0 0
0.5 Potential
( V vs
1.0
SCE)
m e 8. Cyclic voltammograms of (A) sensitized RBL-1 cells (2.0 X lo5 cells) and (9) degranulated RBL-1 cells (2.0 X lo5 cells). Scan rate was 10 mV-s-‘. The measurements were performed in 10 m~ of PBS (pH 7.0).
decreasing the serotonin content of the cells. Decrease of the serotonin content of RBL-1 cells agreed with the amount of serotonin released. Detection of Immediate Allergic Reaction by Using Cyclic Voltammetry of RBL-1 Cells. Allergic reactions are included in immunological responses. IgE antibodies, which are secreted by activated B lymphocytes, are responsible for allergic reactions. The Fc region of IgE molecules binds to specific receptor proteins on the surface of mast cells in tissues and basophilic leucocytes in the blood. The bound IgE molecules in turn serve as receptors for allergens. Allergens cross-link those membrane-bound IgE antibodies with complementary allergen-bindingsites, thereby triggering the cells to secrete histamine and serotonin. Histamine and serotonin cause dilation and increased permeability of blood vessels and are largely responsible for the clinical manifestations of such allergic reactions as pollenosis, asthma, and hives. R B L l cells, like mast cells and basophilic leucocytes, have IgE receptor on their surface and show the same response to allergens (17). Figure 5 shows the principle for the detection of immediate allergic reaction. When allergen was reacted with RBL-1 cells sensitized with antiallergen IgE, RBL-1 cells were degranulated to release serotonin (8,12). As a result, the serotonin content of the cell decreased, leading to the decrease of the peak current of cyclic vo1ta”ograms. RBL-1 cells were reacted with mouse anti-DNP IgE. Then, when allergen, DNP-BSA was added to R B L l cells sensitized with anti-DNF’ IgE, RBL-1 cells were degranulated. Figure 6 shows the cyclic voltammograms of passively sensitized RBL-1 cells with IgE and degranulated RBL-1 cells. Anodic waves of the sensitized RBL-1 cells appeared at the same potential as that of the normal RBL-1 cells. The peak current was also similar to that of the normal RBL-1 cells. However, the peak current decreased when the sensitized RBL-1 cells were degranulated
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ANALYTICAL CHEMISTRY, VOL. 61, NO. 22, NOVEMBER 15, 1989
Table 11. Peak Currents of RBL-1Cells When Various Allergens Were Added with Anti-DNP IgE"
Y 0
a
0
Serotonin content
( n m o ~ / l ~ s cI eS )~
extract of allergen (3 pgmL-')
peak current, pA
none egg white yolkb pollen evening primrose common ragweed house dust milk DNP-BSA
1.00 1.02 0.72 0.94 0.96 0.90 1.00 0.70
"RBL-1 cells were incubated in Tris-ACM solution for 2 h and 1.6 X lo5 cells of RBL-1 were employed for experiments. *The
Figure 7. Relationship between peak current at 0.34 V vs SCE and serotonin content in RBL-1 cells. Scan rate was 10 mV-s-'. The measurements were performed in PBS (pH 7.0).
yolk was similarly decreased peak currents of RBL-1 cells without IgE.
Table I. Peak Currents of RBL-1Cells When Anti-DNP IgE and DNP Derivatives as a Model Allergen Were Added"
ever, stimulation by yolk decreased the peak current of R B L l cells without IgE. Therefore, no specific current decrease by yolk was discriminated from a specific current decrease by DNP-BSA using RBL-1 cells not sensitized with IgE as a reference. The peak currents were reproducible with a relative error of 5% when RBL-1 cells prepared at the same time were used for the experiments. These results suggested that IgEspecific degranulation of immediate allergic reaction can be detected from the peak current of cyclic voltammetry. The novel concept deacribed here for detecting intermediate allergic reaction lays the groundwork for the development of methods detecting specific allergens for a person. Further developmental studies in our laboratory are now directed toward detection of allergic reaction using human IgE in the serum.
addition none DNP-BSA (3 pgmL-') IgE (5 FgmL-') IgE (5 pgmL-') DNP (1 pgmL-') IgE (5 pgmL-') + DNP-BSA (3 pgmL-')
+
peak current, pA 1.26
1.20 1.34
1.30 0.96
" RBL-1 cells were incubated in Tris-ACM solution for 2 h and 2.0 X l @ cells of RBL-1 were employed for experiments.
with DNP-BSA. Addition of DNP-BSA to sensitized RBL-1 cells with IgE brought about degranulation of cells. Figure 7 shows the relationship between the peak current and the serotonin content of the degranulated RBL-1 cells when RBL-1 cells sensitized with anti-DNP IgE were degranulated with DNP-BSA by IgE-mediated reaction. The peak current obtained from the degranulated cells decreased with decreasing the serotonin content of the cells. This indicates that the peak current decrease was attributed to decrease of serotonin present in the RBL-1 cells after degranulation. Serotonin release by immediate allergic reaction can be detected by measuring the peak current of cyclic voltammograms. Then, the relationship between the degranulation of R B L l cells and the peak current was examined. RBL-1 cells were incubated with mouse anti-DNP IgE and DNP-BSA for 2 h. After the incubation, the peak current decreased from 0.63 pA/106 cells to 0.48 pA/l@ cells. On the other hand, the peak current did not decrease when DNP-BSA, IgE, or DNP and IgE were added to RBL-1 cells (Table I). These results suggested that cross-linking of receptor-bound IgE molecules by DNP-BSA decreased the peak current, while the binding of IgE with the receptor or the binding of DNP with receptor-bound IgE molecule did not decrease the peak current. Degranulation of RBL-1 cells occurred by cross-linking of sensitized cells with DNP-BSA. Table I1 shows the peak currents of RBL-1 cells when they were stimulated with various allergen extracts and mouse anti-DNP IgE. Various allergen extracts and mouse anti-DNP IgE did not show a peak current when cyclic voltammograms were directly obtained from these samples. Stimulation of RBL-1 cells by egg white, pollens (evening primrose and common ragweed), house dust, and cow's milk did not show the decrease of peak current. Only DNP-BSA and yolk decreased the peak current. How-
LITERATURE CITED Matsunaga, T.; Namba, Y. Anal. C h m . 1984, 56, 798-801. Matsumga, T.; Namba, Y. Anal. c%n. Acta 1984, 759, 87-94. Merrett, J.; Merett, T. 0. C k . AWgy 1087, 77, 409-416. Green-Graif, Y.; Ewan, P. W. C&. A k g y 1087, 17, 431-438. Walsh, 8. J.; EIHott, C.; Baker, R. S.; Bamett, D.;B v k y , R. W.; Htll, D. J.; Howden, M. E. H. Int. Arch. A&gy Appl. Immund. 1987, 84, 228-232. Kelly, K. A.; Lang. G. M.; Bundeson, P. 0.; HdforbStrevens, V.; Bottcher. I.; Sehon, A. H. J . Immunol. AWtwcb 1980, 39, 317-333. Derborg. S.; Einarsson, R.; Longbottom, J. L. Mndbook of Expcvimental lmmunoiogu; Weir, D. M., Ed.; Blackwell Scientific Publicatlons: London, 1973; Chapter 10. Ishizaka, T.; IsMzaka, K. Prog. A&gy 1984, 34, 188-235. Eshhar, 2.; Ofarim, M.; Waks, T. J. I-. 1980, 124, 775-780. Little. J. R.; Eisen, H. N. hMhcd.s kr Immundogy and Immurochemlsfry, Vol. 7 ; Williams, C. A., Chase, M. W., Eds.; Academic Press: New York, 1967; DD 128-133. Lowry, 0. H.; Ro~brough,N. J.; Farr, A. L.; Randall, R. J. J. Biol. chem.1951, 793. 285-275. Weili, B. J.; Renoux. M. L. Ce//Immunol. 1982. 68, 220-233. Saeki, K.; Endo, E.; Yamasaki. H. Jpn. J . phamcd. 1972. 22. 27-32. Matsunaga, T.; Nakajima, T. Appl. Envkon. MicrobioIl. 1985, 50, 238-242. Ponchon, J. L.; Cespuglio, R.; Gonon, F.; Joovet, M.;Pujol, J. F. A M I . Chem. 1979, 57, 1483-1486. Wrona, M. 2.; Dryhllrst, G. J . Org. Chem. 1087, 52, 2817-2625. Mdeculsr Biology of the CeW; Albert, B., Bray, D., Lewis, J., Raff, M., Roberts, K., Watson, J. D., Eds.; Garland Publishing, Inc.: New York, 1983; pp 967-969.
RECEIVED for review May 31, 1988. Revised manuscript received June 6,1989. Accepted August 18, 1989. This work was partially supported by Grant-in-Aid for Special Project Research No. 63108002 from the Ministry of Education, Science and Culture.
