Anal. Chem. 1990, 62, 2103-2106
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Homogeneous Chemiluminescent Immunoassay Based on Complement-Mediated Hemolysis of Red Blood Cells Yoshiro Tatsu* and Susumu Yoshikawa Government Industrial Research Institute, Osaka, Midorigaoka, Ikeda, Osaka 563, J a p a n
A novel method was developed for the homogeneous chemiluminescent Immunoassay uslng complement-medlated hemolysis of sheep red Mood cells. The chemllumlnescent reaction of lumlnol and H,Op was catalyzed by hemoglobins leaked from hemolyzed sheep red blood cells. The chemlumlnescence was measured by counting photons. When uslng nonhemolyzed cells, chemllumlnescence was virtually not observed. Only hemoglobin released from the cells was chemllumlnescent, so that the extent of hemolysis could be measured wlthout separating hemolyzed and nonhemolyzed cells. The Immunoassay was done for the lmmunoagents: complement, hemolysin (anti-sheep red blood cell antibody), and anti-human albumin antibody. I n the assay of complement or anti-human albumin antibody, sheep red blood cells used were bound wlth hemolysln or human albumin, respectively. The cell was hemolyzed by the action of antlgen-antibody binding and subsequent activation of complement. The extent of hemolysis depended on the concentration of the antibody or complement. The calibration curves were obtained by chemllumlnometrlc measurements on the added diluted antibody or complement. The sensitivity was 0.047 CH50 unWmL for complement and below 1.0 pg/mL of anti-human albumin antibody.
INTRODUCT1ON In recent years, a large number of immunoassay techniques using chemiluminescence have been developed (I), since chemiluminescent measurements are the most sensitive methods using nonradioactive probes. From a practical point of view, homogeneous measurement, i.e. without separating binding and nonbinding immunoagents, would be favorable for simplifying assay procedures. There have been, however, few reported homogeneous measurements using chemiluminescence (2). Here we report a novel type of homogeneous chemiluminescent immunoassay which uses complementmediated hemolysis of red blood cells. The complement contains a series of proteases which are initially activated by the formation of antigen-antibody complexes and finally construct channels of ca. 100 8, on the cell membrane which lead to the lysis of the cell (3). Complement-mediated hemolysis has been used for immunochemical measurements, for example, determining the complement level (4),plaque forming assay (5), and the complement fixation test (6). Hemoglobin in red blood cells is a useful chromophore for measurement of hemolysis by spectrometry. The use of spectrometry, however, has the disadvantage that the separation of cells is necessary because the presence of nonhemolyzed cells interferes with the measurement. Homogeneous measurements using complementmediated lytic action were also developed by the liposome technique: the marker solution was loaded within the lipid bilayer containing antigen. Glucose (7a), enzymes (7b), dyes (7c), or electroactive species (7d) were used as markers. Only the marker leaked from the liposome by complement-mediated lysis was detected. These methods have a serious defect in 0003-2700/90/0362-2 103$02.50/0
that it is impossible to avoid the leakage of the marker during preparation and storage of liposome (8). The use of a biological membrane for these measurements was reported by Humphries and McConnell (9). They used mammalian erythrocyte ghost loaded with a spin label, tempocholine chloride. D'Orazio and Rechnitz used an electrochemically active marker, trimethylphenylammonium chloride, within sheep erythrocyte ghost and detected it by an ion-selective electrode (10). The present report describes the use of intact red blood cells, with hemoglobin present, which can be used as the catalyst for the chemiluminescent reaction of luminol with peroxides, such as hydrogen peroxide and sodium percarbonate. Hemoglobin is known as an effective catalyst (11) and its concentration within the cell is 0.3 kg/L (12). Hemoglobin released from the cells by complement-mediated hemolysis could in the presence of hydrogen peroxide catalyze the chemiluminescent reaction, which can be detected photometrically. Chemiluminescence was hardly observed for nonhemolyzed cells and hence homogeneous measurements involving both hemolyzed and nonhemolyzed cells are possible. This method was applied to the assays of complement, hemolysin, and anti-human albumin antibody.
EXPERIMENTAL SECTION Apparatus. AU chemiluminescentmeasurements were carried out by the photon counting method. Figure 1 is a schematic diagram of the experimental setup. The sample tube was placed in the temperature-controlled sample box and the chemiluminescent reaction was initiated by injection of the reagents using a microsyringe. The light was transmitted from the reaction to a photomultiplier tube, Hamamatsu R-2757 at 2.0 kV, through optical fibers. Pulses of photoelectrons were appropriately discriminated by output voltage and counted by a signal processor, Hamamatsu C2550. Reagents. Luminol was purchased from Wako Pure Chemicals and used without further purification. All chemicals used were of reagent grade. Rabbit antiserum to sheep red blood cells (hemolysin)was purchased from Cordis. Lyophilized guinea pig complement was obtained from Handai-Biken. Rabbit anti-human albumin antiserum from Seikagaku-Kogyo was 2.9 mg/mL of the antibody determined by immunoprecipitation. Sheep red blood cells or the hemolysin-sensitizedcells for measurement of complement activity were purchased from Cappel or Ishizu-seiyaku, which are preserved in Alserver solution (30 mM citrate buffered saline containing 0.114 M glucose, pH 6.1). The modification of the cells by human albumin was done with chromium chloride (13). The cells were washed with saline several times and suspended at 1 x lo9 cells/mL. Four times recrystallized human albumin was obtained from Nakarai tesque and dissolved in saline at 10 mg/mL. Human albumin solution (1 mL) was added to 5 mL of the cell suspension. Freshly prepared chromium chloride solution (CrC13.6H20,0.5 mM, 1.0 mL) was then mixed and incubated at 37 "C for 1 h. The cells were washed several times with saline. Assay Procedures. The used buffers were veronal buffered saline of pH 7.4 containing 0.1% gelatin (abbreviated as GVB) or GVB containing 0.5 mM CaC12and 0.15 mM MgCl, (GVB2+). Sheep red blood cells were washed with GVB containing 0.01 M EDTA and then several times with GVB2+. The cells were suspended in GVB2+at a concentration of 1 X log cells/mL. Hemolysin was heated at 56 "C for 30 min to remove any complement 0 1990 American Chemical Society
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Figure 1. The instrument for chemiluminescent measurements: (a) microsyringe;(b) sample tube; (c) temperatureGontrolled water; (d) dark box; (e) optical fiber; (f) photomultiplier: (9) signal processor; (h) high voltage; (i) computer.
activity and was diluted with GVB2+ solution. Lyophilized complement was reconstituted as directed (with Green's solution containing sodium acetate and boric acid) and was incubated with sheep red blood cells at 4 "C for 30 min to remove any anti-sheep antibodies. Complement solution reconstituted was frozen at -70 "C and the solution thawed was used within 1 day of thawing below 4 O C so that there was almost no loss of activity. The complement activity was within the ordinal level around 200 CH50 units/mL, measured by Mayer's method (4),where CH50 is a unit of activity of the complements and 1 CH50 unit of complement can hemolyze 50% of 5 X lo8 sensitized red blood cells in 7.5 mL of total solution. The hemolysin assay was performed as follows. Red blood ells were mixed with an equivolume of diluted hemolysin at 0 "C. Five microliters of the mixture was incubated at 37 "C for 10 min and cooled at 4 "C for 30 min. GVB2+(20 pL) and 500-fold diluted complement solution (13 pL), containing 0.35 CH50 units/mL, was added and incubated at 37 "C for 1 h. The total sample volume at this point was 38 pL. After the hemolytic reaction, 37 pL of GVB was added and the solution was kept at 4 "C until the chemiluminescence was measured. The measurement was done as soon as possible although the hemolysis by complement at 4 "C was not seen to continue during a 24-h period. The complement assay was done in the same way except that sheep red blood cells were the hemolysin-sensitized ones. The sensitized cells (5 x 108cells/mL, 0.4 mL) were mixed with diluted complement (2.6 mL) at 0 "C. Thirty-eight microliters of the mixture was incubated at 37 "C for 1 h, followed by the same procedure as the hemolysin assay. The assay of anti-human albumin antibody was also done in a similar way. Albumin-modified cells (2.5 X lo8 cells/mL, 10 pL) were mixed with diluted serum containing anti-human albumin antibody (10 pL) and incubated at 37 "C for 15 min. After the incubation, complement solution (0.67 CH50 unit/mL, 17.5 pL) was added and incubated at 37 "C for another 60 min. After hemolytic reaction, 37.5 pL of GVB was added and the solution was kept at 4 "C until chemiluminescence measurements were performed. In order to measure chemiluminescence,50 pL of each of 1mM luminol solution and 5 mM HzOzsolution were added. The photon counting was carried out over a period of 205 s with a gate time of 50 ms. The final sample volume was 175 pL.
RESULTS AND DISCUSSION Preliminary chemiluminescent measurements were performed with hemoglobins in order to test the sensitivity and response of the instrument. Fifty microliters of 1mM luminol was added to 75 pL of hemoglobin solution followed by 50 pL of 5 mM HzO,. The detection limit of this instrument using optical fibers was less than 0.1 pg of hemoglobin (Figure 2). Several authors reported the detection limit of hemoglobin as being in the nanogram range using the luminol reaction a t pH 12 (11). Although the luminol reaction was more efficient in alkaline solution, neutral and isotonic solutions were preferable in the following experiment since intact cells were used. With large amounts of hemoglobin, the intensity approached a maximum. The volume of each sheep red blood
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Figwe 2. Dependence of chemiluminescence of lumind on the amount of hemoglobin. Fifty microliters of 1 mM luminol and 50 pL of 5 mM H,O, were added to 75 pL of twlcecrystailized bovine hemoglobin. All reagents were dissolved In GVB.
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Figure 3. Time course of chemiluminescent intensity during the reaction of 50 pL of 1 mM luminol and 50 pL of 5 mM H,O, with 75 pL of a suspension of sheep red blood cells suspension: (a) 100% lysed cells; (b) 0 % lysed cells
cell was 32 pm3, which encapsulated 1.0 X lo-" g of hemoglobin. In the following experiments 2.5 x lo6 cells were suspended in 175 pL of solution. The total amount of hemoglobin is calculated as ca. 25 pg, which is within the measurable range using the chemiluminescent technique (Figure 2). Thus, 0.1-25 p g of hemoglobin (Le. 0.4 to 100% of the total amount used) could be determined by the equipment. In order to develop a method without separation procedures, it is essential that the chemiluminescent reaction does not take place in nonhemolyzed cells so that only hemoglobins from hemolyzed cells catalyze the reaction. Figure 3 shows a time course of the chemiluminescent intensity for both hemolyzed and nonhemolyzed cells. It is clear that sheep red blood cells without hemolysis showed almost no chemiluminescence. The total count in 205 s was 8.11 X lo3,whereas for hemolyzed cells it was 9.70 X lo6. With this equipment the average dark count in 205 s was (6.00 f 0.10) X lo3. Nonspecific chemiluminescence of ca. 2000 counts was observed in nonhemolyzed cells, which could be attributed to leaked hemoglobins or any enzymes present. Complement-mediated hemolysis has been accounted for by the one-hit theory (14). Complement is activated by the existence of one molecule of IgM or two adjacent molecules of IgG binding on the cell membrane. The concentration of proteins in red blood cells is 0.3 kg/L (12), 99% of which is hemoglobin. When one molecule of IgM binds onto a red blood cell, the cell releases IO8molecules of hemoglobin. The immunological signal, i.e. the formation of an antigen-antibody complex, is amplified by the hemolysis as large amounts of hemoglobin are released. This immunochemical amplification enables the sensitive detection of a specific immunoreaction. The above observations suggest that no chemiluminescence takes place within the nonhemolyzed cells. From his experiment with liposome, Frimer reported that H,O, can cross the
ANALYTICAL CHEMISTRY, VOL. 62, NO. 19, OCTOBER 1, 1990 1000000
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Figure 4. Dependence of chemiluminescence on the concentration of H202. Seventy-five microliters of GVB containing 5 pL of red blood cell suspenslon was added to 50 pL of 1 mM luminol and Injected with 50 pL of H202. Circles are for hemolyzed cells, triangles for nonhemoiyzed cells, and squares for the buffer without the cell.
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Flgure 6. Chemiiuminometric response curve to dilution of hemolysin (rabbit antisheep red blood cell antibody). Maximum count in the gate time of 50 ms is plotted to the dilution factor. Trlangtes show the blank without antibodies.
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/ rnM Figure 5. Dependence of chemiluminescence on the concentration of luminol. Seventy-five microlkers of GVB containing 5 pL of red blood cell suspension was added to 50 pL of luminol and injected with 50 pL of 5 mM H,02. Circles are for hemolyzed cells, triangles for nonhemolyzed cells, and squares for the buffer without the cell. lipid bilayer (15).However, the probability of luminol, a large anionic molecule, crossing the membrane is very small. The chemiluminescence of luminol has the same fluorescence spectrum as 3-aminophthalicacid, which was the final product of the luminol reaction (16). In GVB the fluorescent maximum of 3-aminophthalic acid was observed at 420 nm. Hemoglobin is very concentrated within the cells and its absorption maximum was observed at 410 nm. The fluorescence of aminopbthalic acid was remarkably quenched in the solution containing the concentrated hemoglobins (data not shown). Even if luminol was able to permeate through the membrane into the cell, the chemiluminescence it would cause could not be detected because of this overlap in the spectra. Thus only hemoglobin leaked from the cell gave chemiluminescence, making homogeneous measurements feasible. The chemiluminescence was observed immediately after addition of H20z. It reached at maximum level 1 min later and then decreased with first-order kinetics. An approximately linear relationship existed between the total count and the maximum count in a 50-ms period. In order to simplify the acquisition of data, the measure of chemiluminescent intensity was taken to be the maximum count. The optimum conditions for chemiluminescent measurements were determined as follows. Figures 4 and 5 show the dependence of the chemiluminescence on the concentration of H202and luminol, respectively. In each experiment, the light intensity of the buffer solution was almost the dark level. When nonhemolyzed cells were used, the background level increased with high concentrations of Hz02,but no change
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Figure 7. Chemiluminometric response curve to the dilution of complement of guinea pig. Maximum count in the gate time of 50 ms is plotted to the dilution factor. Triangles show the blank without complement.
was observed with high concentrations of luminol. This increase may be caused by a small amount of hemoglobin or enzymes. It is necessary to use a lower concentration of H20z for homogeneous measurement in order to obtain a low background chemiluminescence. The hemolyzed cells gave much stronger chemiluminescence which increased with increasing concentration of H202. The chemiluminescence was less dependent on the concentration of luminol than on that of H202. Luminol is a well-known compound but the mechanism of the chemiluminescent reaction is not yet clear (17). The difference in the concentration dependence between luminol and HzOzcould reflect the reaction mechanism. To get the optimum range, the following experiments were done using 1 mM luminol and 5 mM H202. Figure 6 shows the chemiluminescentresponse of sheep red blood cells with different dilutions of hemolysin. As dilution increased chemiluminescence decreased. Since the hemolysin was heated to deactivate complement components, this response curve depends only on the concentration of anti-sheep red blood cell antibody in hemolysin solution. An 8000-fold dilution of hemolysin could be detected in this experiment. Since normal rabbit serum contains an immunoglobulin concentration of ca. 1 mg/mL, the detection limit is estimated as in the sub-pg/mL range. Figure 7 shows the response of sensitized sheep red blood cells to varying dilutions of complement. As the complement was diluted the chemiluminescence decreased. The sensitivity was comparable to absorption methods ( 4 ) as a 3615-fold dilution of complement, which had 0.047 CH50 unit/mL, was detected. The reproducibility in Figure 6 was less than that in Figure 7 . This is attributable to the difference in the
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/ ug/ml Figure 8. Chemiluminometry of rabbit anti-human albumin antibody using human albumin modlfied red blood cells. Maximum count in the gate time of 50 ms is plotted to the concentration of the antibody. Circles represent the modiRed cells. Triangles represent the cells which were modified with a fifth of albumin used for the circles. Squares represent nonmodified cells. [Ab]
number of steps in the assays. The former needs the extra steps for the formation of the immunecomplex prior to the complement activation. By the modification of red blood cells by antigens, this method can be developed to wide application to other analytes. Circled plots in Figure 8 show the response of sheep red blood cells modified with human albumin to varying the concentration of anti-human albumin antibody. The antibodies could bind on the cell membrane via a specific antigen-antibody reaction and activate the complement, in a similar manner to hemolysin, and finally hemolyze the cells. As the level of antibody increased, the chemiluminescence increased. Saturation of the intensity was observed above 4 pg/mL of the antibody. This saturation probably means that the modified human albumin is entirely bound with antibody. The triangles show the results of experiments where the cells were modified with only one-fifth of the human albumin used for the results depicted in circles. The chemiluminescence was weak but a gradual increase was observed. The cells without modification gave no chemiluminescence and had no dependence on the antibodies (squares). Therefore the solutions contained no anti-sheep red blood cell antibody. The dependence of the chemiluminescence on the modification of human albumin clearly indicates that the concentration of anti-human albumin antibody could be specifically assayed by this method. The sensitivity was below 1 pg/mL of anti-human albumin antibody. The sensitivity and calibration curve of this method were the same as those for the absorption method, which was done on a larger scale (1.5 mL at hemolysis reaction). The same ratio of reagents was used and measurements of supernatant of the cell suspension a t 541 nm were performed (data not shown). Although the sensitivity of chemiluminescent measurements is higher than that of absorption measurement in general, the sensitivity of this method is governed by the hemolysin reaction itself. The extent of hemolysis is governed
by the concentration of membrane bound antigen, antibody, complement, and red blood cells. A s m d amount of red blood cells is hemolyzed by a small amount of complement and antibody. Thus, optimizing the conditions of the hemolysis reaction and the reaction size would improve the sensitivity. The advantage of the present chemiluminescent technique lies in the capacity to perform small scale (175 pL) and homogeneous measurements, which are impossible with the absorption method. Although the sensitivity was not superior to other sensitive methods such as radioimmunoassay, the advantages of this method are ease and speed of measurements. The homogeneous immunoassay can be performed by using chemiluminescent measurements with no other specific chemicals than luminol and H202. In this study, the immunoassays were performed for proteins related to immunochemistry. For the other proteins or nonprotein substances such as carbohydrates and drugs, this homogeneous method would be applicable by a complement fixation technique or by competitive reaction between analyte and antigen-modified cells. The modification methods of the cells in the hemagglutination test are good examples of the application to the other analytes.
LITERATURE CITED (1) Methods Enzymol. 1986, 133, section 2. (2) (a) Ikariyama, Y.; Kunoh, H.; Aizawa, M. Blochem. Hophys. Res. Commun. 1985, 728 (2), 987-992. (b) Messeri, G.; et ai. clfn. Chem. 1984, 30 (5), 653-657. (c) Campbell, A. K.; Patel, A. Biochem. J. 1983, 276, 185-194. (d) Kohen, F.; Pauagli, M.;Kim, J. B.; Linder, H. R.; Boguslaski, R. C., FEBS Lett. 1979, 704 (I), 201-205. (3) TanumJensen, J.; Bhakdi-lehnen, B.; Bjerrum, 0. J.; Speth, V. Scan. J. Immunol. 1978, 7 , 45-56. (4) Kabat, E. A,; Mayer, M. M. Experimental Immunochemistry;Charles C Thomas: Springfield, IL, 1961; Chaptdr 4. (5) Jerne, N. K.; Nordin, A. A. Science 1963, 740, 405. (6) Wasserman, E.; Levine, L. J . Immunol. 1981, 8 7 , 290. (7) (a) Kinsky. S. C.; Haxby, J. A.; Zopf, D. A,; Alving. C. R.; Kinsky, C. B. Biochemistry 1989, 8 (lo), 4149. (b) Haga, M.; Itagaki, H.; Sugawara, S.; Okano, T. Biochem. Biophys. Res. Commun. 1@80, 95 (I), 187-192. (c) Urneda, M.; Ishimori, Y.; Yoshikawa, K.; Takada, M.; Yasuda, T. J. Immunol. Methods 1988, 95, 15-21. (d) Shlba, K.; Umezawa, Y.; Watanabe, T.; Ogawa, S.; Fujiwara, S. Anal. Chem. 1980, 52(11),1610-1613. (8) Vistnes, A. I.Nonisotopic Immunoassay; Ngo, T. T., Ed.; Plenum: New York, 1988; pp 361-368. (9) Humphries. G. K.; McConnell, H. M. Roc. Narl. Acad. Sei. U . S . A . 1974, 77 (5), 1691-1694. (IO) D’Orazio, Paul; Rechnitz, G. A. Anal. Chem. 1977, 49 (13). 2083-2086. (11) (a) Neufeld, H. A.; Conkiin, C. J.; Towner, R. D., Anal. Hochem. 1885, 72, 303-309. (b) Olsson, T.; Bergstroem. K.; Thore, A. Bblumin. Chemllumin . (Int . Symp Anal. Appl Blolumi Chemilumi .), 2nd. 1981, 659-666. (c) Kaiabina, L. V. Vestn. Kiev. Politskh. Insr., Khim. Mashinostr. Tekhnoi. 1981, 78, 54-55. Chem. Abstr. 1982, 96, 1 3 9 3 3 ~ . (12) Rajeshwar Rao, G.; Narasinhalu, P. R.; Krishna Reddy, V.; Ahmed Hussian, M. Indian Vet. J. 1982, 39, 429-432. (13) Gold, E. R.;Fundenberg, H. H. J. Immund. 1967, 99 (5), 859-866. (14) Kabat, E. A. Structural Concept in Immunology and Immunochemktry; Hog Rinehart Winston, New York, 1968; Chapter 11. (15) Frimer, Aryeh, A.; Forman, Arthur; Borg, Donald C. I s r . J . Chem. 1983. 23, 442-445. (16) White, E. H.; Bursey, M. M. J. Am. Chem. SOC.1964, 8 6 , 941-942. (17) Gundermann. K.-D.; McCapra, F. Chemiluminescence in Organic Chemistry; Springer: Berlin, 1987; p 77.
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RECEIVED for review January 17, 1990. Revised manuscript received June 18, 1990. Accepted June 29, 1990.
