ANALYTICAL
BlOCHEMlSTRY
94,
176-185 (1979)
A Rapid Method for Quantitation on Human Lymphoblastoid T. P. GILLIS, Department
of Immunofluorescence Cell Suspensions
F. H. WILSON, L. A. WILSON, AND J. J. THOMPSON of Microbiology, Louisiana State University Medical Center, 1100 Fiorida Avenue, New Orleans, Louisiana 70119 Received June 19, 1978
A quantitative fluoroimmunoassay for antibodies to, and surface antigens of, human lymphoblastoid cells (IM-1) with photon-counting spectrofluorometry is described. IM-1 cell suspensions were reacted with rabbit antiserum to human spleen vesicular membranes, were washed, and then were reacted with an excess amount of fluorochrome-conjugated (fluorescein or rhodamine) goat anti-rabbit immunoglobulin G (IgG). Under appropriate conditions, antibodies to IM- 1 cells could be detected with experimental/control fluorescence ratios ranging between 5 and 40. Moreover, detectable levels of antibody-saturated cells approached 5 x 103 cells per milliliter or a total of 1.7 x IV cells per assay. Inhibition of the fluoroimmunoassay was performed with either viable IM-1 cells or IM-1 vesicular membrane preparations and demonstrated a dose-dependent antigen inhibition. Fluorescence of sensitized cells reactive with either fluorescein- or rhodamine-labeled antiglobulins could be quantitatively distinguished in dual-labeled preparations.
Immunofluorescence is a technique with wide applicability that has been used to locate and semiquantitate antigens of various cell types (1,2). True quantitation of immunofluorescence has only recently been used for research purposes with the development of relatively sophisticated equipment that utilize either microscope attachments for microfluorometry (3) or laser-activated fluorescence for flow fluorometry (4). These quantitation methods have features that limit their application. For example, standardization procedures for microfluorometry are time consuming and may be subject to investigator bias (5,6), whereas laser-equipped flow fluorometers are cost prohibitive. Sophisticated instrumentation is not strictly required for quantitation of immunofluorescence. It has been reported (7) that fluorometric quantitation of fluorescein-conjugated antibodies attached to cell membrane antigens could be accomplished with a conventional filter fluorometer after solubilization of the cells. In addition to the solubilization 0003-2697/79/050176-10$02.00/0 Copyright All rights
0 1979 by Academic Press, Inc. of reproduction in any form reserved.
requirements, the assay took several days to complete and was relatively insensitive, requiring large numbers of cells per assay. Based on previously reported work on a quantitative fluoroimmunoassay in a bacterial system (8), we attempted fluorometric quantitation of fluorochrome-conjugated antibody on the surface of viable mammalian cells. We report here a simple, yet sensitive, fluorometric assay that detects antibodies to, and surface antigens of, human lymphoblastoid culture cells with photon-counting spectrofluorometry. METHODS
Cells. Human lymphoblastoid cells (IM- l)l in continuous culture were used for all proi Abbreviations used: IM-1, human lymphoblastoid cells; R-3, rabbit antiserum; NRS, normal rabbit serum; FITC, fluorescein isothiocyanate; TRITC, rhodamine isothiocyanate; GAR, goat antiserum; IgG, immunoglobulin G; HBSS-FCS, Hanks’ balanced salt solution with 10% fetal calf serum; DPBS, Dulbecco’s phosphate-buffered saline; FIA, fluoroimmunoassay. 176
FLUOROIMMUNOASSAY
OF LYMPHOID
cedures. Cells were grown in suspension in RPM1 1640 medium with 10% fetal calf serum (GIBCO, Grand Island, N. Y.). Seru. Rabbit antiserum (R-3) was prepared to human spleen cell vesicular membranes (9). Pooled normal rabbit serum (NRS) was used as our control for nonspecific interactions of rabbit serum with IM- 1 cells. Fluorescein isothiocyanateand rhodamine isothiocyanate-conjugated goat antisera to rabbit IgG (designated FITC-GAR IgG and TRITC-GAR IgG, respectively) were purchased from Cappel Laboratories Inc. (Cochranville, Pa.). The FITC-GAR IgG preparation contained 6.0 mg/ml antibody protein with an F/P ratio of 3.3; R/P ratio of the TRITC-GAR IgG was 5.4. Absorption of the fluorochrome conjugates was performed for 2 h at 4°C with lo6 IM-1 cells/O. 1 ml of conjugate in order to remove cross-reacting antibody to IM-1 cells. Fluoroimmunoassay . In a typical experiment, IM-1 cells were mixed with either R-3 or NRS and incubated for 45 min at 0°C to prevent “capping” of cell surface antibody aggregates. The cells were then washed twice in the cold with Hanks’ balanced salt solution with 10% fetal calf serum (HBSSFCS). FITC-GAR IgG was added to the washed cell pellets and the suspensions were further incubated at 0°C for 45 min. The cells were again washed two times in the cold with HBSS-FCS and resuspended to the appropriate cell concentration in Dulbecco’s phosphate-buffered saline (DPBS) with NaN, (0.2%) for the measurement of fluorescence. Lowered temperature (OOC) and NaN3 have been shown previously to inhibit migration of surface antibody aggregates (10). For dual-labeling experiments, 600-~1 aliquots which contained 3 x lo6 IM-1 cells in HBSS-FCS were reacted with 18 ~1 of R-3 antiserum, incubated 45 min at 0°C and then washed two times with HBSS-FCS. The cells were then suspended in 240 ~1 of either a 1: 10 dilution of FITC-GAR IgG
CELL
SUSPENSIONS
177
or 240 ~1 of a 1:4 dilution of TRITC-GAR IgG. Preliminary experiments had demonstrated that the respective amounts of fluorochrome-conjugated reagents used were sufficient to saturate the number of sensitized IM-1 cells used in these suspensions. After a 45-min incubation at 0°C the cell preparations were washed one time with HBSSFCS and one time with DPBS with NaN,. The washed cell pellets were resuspended in 3 ml of DPBS with NaN,. Unlabeled IM-1 cells were similarly washed and diluted in DPBS with NaN, to a final concentration equivalent to that of the labeled cells. Portions of these cell preparations were then mixed to give varying proportions of fluorochrome-labeled cells to unlabeled cells or fluorescein-labeled cells to rhodamine-labeled cells. Each final mixture of cells contained the same concentration of cells as the starting suspension in a volume of 500 ~1. Fluorescence data for this system were corrected by subtraction of background due to cells in buffer (7000 cps for fluorescein conditions and 1740 cps for rhodamine conditions). Inhibition of the fluoroimmunoassay was used to detect membrane antigens on intact cells and vesicular membranes. The inhibition assay was performed by the incorporation of a preincubation step of antigen with a standard amount of R-3 serum for 30 min at 37°C followed by 30 min at 0°C. After centrifugation of the mixtures (13,OOOg) for 10 min, lOO+l aliquots were removed and reacted with 6 x lo5 IM-1 target cells, as outlined above in the fluoroimmunoassay. An unabsorbed positive fluorescence control was included with buffer instead of antigen in the preincubation step. Inhibition was calculated as follows: Percentage inhibition (cps of absorbed serum samples) clx 100. (cps of unabsorbed serum samples) Fluorescence measurements. Fluores-
178
GILLIS
cence measurements were made on a MKI spectrofluorometer (Fart-and Optical Co., Inc., Valhalla, N. Y.) modified to accept a Model 1140 quantum photometer (Princeton Applied Research Corp., Princeton, N. J.) with an R 212 photomultiplier tube (Hamamatsu Corp., Middlesex, N. J.). Fluorescence measurements were expressed as photon-counting rate in counts per second at a standard deviation of 1.3%. Crossed film polarizers (incident polarizer at O”, emission polarizer at 90”, Fart-and Optical Co.) were used to minimize interference from light scatter. Polarization of excitation light alone may be sufficient to minimize scattering problems (1 l), but we have not yet studied this possibility. Slits were chosen to allow a IO-nm bandpass. Fluorescence of cell suspensions was measured in a 0.3ml quartz semimicrocuvette assembly. Excitation of fluorescence was at 485 nm for fluorescein and 535 nm for rhodamine; emission was analyzed at 525 nm for fluorescein and 580 nm for rhodamine. Slopes of linear dose-response curves for fluorescein methyl ester and rhodamine B in DPBS with NaN, were about 25,000 and 430 cps/nM, under the selective specrespectively, troscopic conditions just described. Stability was measured by the continuous recording of fluorescence signal on an SR 255B strip chart recorder (Heath Co., Benton Harbor, Mich.). Statistics. Data obtained from the duallabeling dose-response experiments were fitted by linear least-squares analysis. Variances associated with the calculated slopes and intercepts (12) were compared by the F test. When variances were not significantly different, t tests were used to determine statistical significance. RESULTS Stability of FITC-GAR IgG-Labeled IM-1 Suspensions
A suspension of IM-1 cells (final concentration of 105/ml) was reacted with R-3 and
ET AL.
FITC-GAR IgG in the standard fluoroimmunoassay procedure. Fluorescence emission was excited at 485 nm and analyzed at 525 nm. As shown in Fig. 1, buffer alone was not significantly fluorescent, but IM-1 cells showed minimal intrinsic fluorescence and/or light scatter. The addition of NRS and FITC-GAR IgG to the cells gave some fluorescent labeling. Fluctuation in the signals above noise levels probably represented cell aggregates passing through the illuminated portion of the cuvette. A l:lO,OOO dilution of the FITC-GAR IgG preparation showed evidence of fluorescence decay over the lo-min observation period; the rate of decay appeared to be slightly less than 0.5% of the initial fluorescence signal per minute. R-3 sensitized cells reacted with FITC-GAR IgG showed significantly greater fluorescence than the corresponding controls. Decay of fluorescence on these FITC-GAR IgG-labeled cells appeared similar to that of free FITC-GAR IgG and therefore could not be attributed to settling of the cell suspension. TRITCGAR IgG-labeled cells behaved similarly (not illustrated). The apparent difference in extent of “noise” at high signal levels as opposed to low signal levels is an artifact of the electronic analog processing of the digital signals by the photon-counting instrument. All signals were processed at a fixed standard deviation (1.3% in Fig. 1). As the input signal increased, this variation became larger in absolute magnitude and therefore more apparent as “noise” in the chart recording. Determination of Optimum FITC -GAR IgC
Aliquots (200 ~1) which contained 6 x lo5 IM-I cells in HBSS-FCS were reacted with either 6 ,ul of R-3 (undiluted) or NRS and various dilutions of FITC-GAR IgG (40 ~1) in the standard fluoroimmunoassay. Cells were resuspended to 1 ml prior to reading in the spectrofluorometer. As Fig. 2 demon-
FLUOROIMMUNOASSAY
OF LYMPHOID
CELL SUSPENSIONS
179
IM-1 + R-3 +FITC-GAR w
80 FITC-GAR w l:lO,fulo
60
40 IM-l+ NRS + 20 IM-1 CELLS 0
1
1
1
1
I
2
4
6
8
10
BUFFER
Time, minutes FIG. 1. Stability of fluorescence of various preparations. was analyzed at 525 nm with crossed polarizers.
strates, R-3 antibodies to IM-I cell membranes were effectively saturated with FITC-CAR IgG at a dilution of 1:lO. At the highest concentration of FITC-GAR IgG tested (15)) a slight decrease in Ruorescence was noted in the experimental reading. However, this decrease was not observed in the NRS control and resulted in a depressed experimental/control fluorescence ratio.
Excitation was at 485 nm, and emission
Titration
of R-3 Antibodies to IM-l Celi Membrane Antigens
IM-1 cells (6 x 105) in HBSS-FCS (200 ~1) were reacted with varying amounts of either R-3 serum or NRS and an excess amount of FITC-GAR (15) in the standard fluoroimmunoassay. As Fig. 3 illustrates, the IM-1 cells approached saturation with R-3
180
GILLIS ET AL. RATIO: IMMUHlNRS
14.7 9;5 , 17,7
7%-
270 -
x
240 -
5 B
210 -
2g
180 150
s
123 -
r
26,6
16.9
membranes (Fig. 4A) or intact IM-1 cells (Fig. 4B). Figure 4B also shows that the lowest concentration of cells tested (4.5 x lo5 cells/ml) gave a detectable level of inhibition in this standardized system. Sensitivity of the Fluoroimmunoassay for the Detection of ZM-1 Cells
-
W-
1
2
Relative
3
4
5
6
7
8
9
10
amount of FITC-GAR IgG
FIG. 2. Effect of FITC-GAR IgG concentration on fluorescence of cells reacted with either 6 pl of undiluted R-3 (0) or equivalent amounts of NRS (0) in the standard fluoroimmunoassay. For the abscissa, 10 units = 15 dilution of FITC-GAR IgG.
antibody at amounts of 3 ~1 or greater. Because the increase in fluorescence from 6 to 12 ~1 was minimal, we felt that in order to conserve reagents 6 ~1 per 6 x lo5 cells would suffice as a saturation end point for further experiments. At R-3 antibody concentrations of less than 3 ~1, specific fluorescence fell off rapidly as the primary antibody became limiting. This effect, shown in Fig. 3, was reflected by a decreased experimental/control fluorescence ratio for 1.5 /.Ll. Inhibition of the Fluoroimmunoassay
Inhibition of IM-1 specific immunofluorescence was tested with either intact IM-1 lymphoblastoid cells or a vesicular membrane preparation. Increasing concentrations of inhibitor were reacted with 6 ,ul of R-3 or NRS. After incubation and centrifugation, the supematant fluids were tested for residual antibody in the standard fluoroimmunoassay. Figure 4 shows that a dose-dependent inhibition of IM- 1 specific immunofluorescence was obtained by adding increasing amounts of either vesicular
Aliquots (300 ~1) of IM-1 cells containing 1.8 x lo6 cells were reacted with 18 ~1 of either R-3 or NRS and incubated at 0°C for 45 min. The cells were then washed in HBSS-FCS, and 120 ~1 of FITC-GAR IgG (1:5 dilution) was added to the washed cell pellets. Following incubation and washing, final resuspension was in 10.0 ml DPBS with 0.2% NaN,. Serial twofold dilutions in DPBS were then prepared from both the NRS-treated and R-3-treated cells. Figure 5 shows that background fluorescence (NRStreated cells) decreased linearly with dilution and approached fluorescence of buffer alone between 20 x lo3 and 40 x 103 cell/ ml. Fluorescence of R-3-treated cells similarly decreased with dilution; however, experimental/control fluorescence ratios of 2 or greater were seen with dilutions to 5.7 RATIO IMMUNE/NRS
18.6
28.3
30.4
43.8
aOi r----% 1.5
3.0 Amount
6.0 of Primary
12.0 Antibody
@I)
FIG. 3. Titration of R-3 antibodies to IM-1 cell surface antigens with constant FITC-GAR IgG (1:5). Primary antibody is either R-3 (0) or NRS (0).
FLUOROIMMUNOASSAY A.
lMEMERAFBS
B.
OF LYMPHOID
CELLS
6 ‘G e -E c
25
50 Vesicular
Membraneslyll
loo
4.5
9.0 Cellslmlx
18 lo5
4. Inhibition of the standard fluoroimmunoassay by either IM-1 vesicular membrane preparation (A) or viable IM-1 lymphoblastoid cells (B). FIG.
X lo3 cells/ml. Since fluorescence was determined on only 0.3 ml of this suspension, the actual number of cells per assay would equal approximately 1.7 x 103. Beyond this dilution, values obtained could not be easily discriminated from background values, thereby establishing the lower limit of the assay for the detection of optimally sensitized IM-1 cells with this system.
181
CELL SUSPENSIONS
Double Fluorescence Assay of FITC-GAR or TRITC-GAR IgG-Labeled Cells As shown in Fig. 6, when fluoresceinlabeled cells were mixed in varying proportions with unlabeled cells and measured under spectroscopic conditions specific for fluorescein, a linear dose response was seen. If the unlabeled cells were replaced with an equal quantity of rhodamine-labeled cells, the dose response was unaffected. Correspondingly (not illustrated), when rhodamine-labeled cells were mixed in varying proportions with either unlabeled cells or fluorescein-labeled cells and measured under conditions specific for rhodamine, a linear fluorescence dose response resulted. As shown in Table 1, the slopes of the doseresponse curves for detection of either fluorescein- or rhodamine-labeled cells were not significantly different when the fluorescent cells were mixed with either unlabeled cells or cells labeled with the other fluorophore (P > 0.01 for all relevant pair com-
250
0
5. Effect of IM-1 cell dilution upon immune specific fluorescence (R-3 treated cells, 0) and background fluorescence (NRS-treated cells, 0) after reaction in the standard fluoroimmunoassay. Fluorescence at 0 cells/ml (Ix]) represents the fluorescence obtained with buffer alone in the cuvette. FIG.
20 Percentage
40 60 80 d FluoresceinLabeled Cells
100
FIG. 6. Fluorescence dose response to fluoresceinlabeled cells in the presence of rhodamine-labeled cells (0) or unlabeled cells (0). Fluorescence was excited at 485 nm and emission was analyzed at 525 nm with crossed polarizers.
182
GILLIS ET AL. TABLE STATISTICAL
ANALYSIS
1
OF FLUORESCENCE
DOSE-RESPONSE
Fluorescent emission (hE = 485 nm, AA = 525 nm)
Calculated parameter Correlation coefficient (fluorescence and % labeled cells) Slope f 1 SD (cps per % labeled cells) Coefficient of variation of the slope (%) y-Intercept f 1 SD (cps)
CURVES~
Rhodamine emission (As = 535 nm, AA = 580 nm)
Fluoresceinlabeled + unlabeled
Fluoresceinlabeled + rhodaminelabeled
Rhodaminelabeled + unlabeled
Rhodaminelabeled + fluoresceinlabeled
0.9985
0.9970
0.9974
0.9980
3,296 -’ 197
229 2 9.5
213 f 7.7
3.24 64 c 563
3.64 444 A 448
3,264 + 100 3.06 0 f 6,183
5.99 -7,800 2 12,024
(2AE = excitation wavelength; AA = wavelength at which emission was analyzed.
parisons, t test). Similarly, all y-intercepts calculated from the least-squares analysis did not vary significantly from 0 (P > 0.10 for all relevant comparisons, t test). Therefore, we concluded that the spectroscopic conditions used permitted quantitative fluoroimmunoassay of fluorescein- and rhodamine-labeled suspensions. If one arbitrarily defined an uncorrected signal that is two times more than background as being significantly different and calculated the corresponding percentage of labeled cells derived in Table 1, then the rhodamine system could be used to measure samples with 7.3% labeled cells, whereas the fluorescein system would detect as little as 2.1% labeled cells. Precision of the assays, as assessed by the coefficient of variation of the slope of the dose-response curves, was less than or equal to about 6%. Similar levels of precision were obtained with replicate determinations of single samples (not illustrated). DISCUSSION
Recent advances in fluorescence technology have made quantitation of cell surface bound fluorescent antibodies possible. However, limited availability of this tech-
nology and problems encountered in standardization procedures prompted us to test the efficacy of a quantitative fluoroimmunoassay with photon-counting spectrofluorometry. A similar approach to immune specific fluorometric quantitation of cell surface antigens with a filter fluorometer was pursued by Strom and Klein (7). Their assay involved an overnight incubation with lengthy enzymatic digestions in order to solubilize cells prior to fluorescence determinations. In contrast, with crossed film polarizers to reduce light scatter, we were able to quantitate immune specific fluorescence of IM-I cells in a matter of a few hours without solubilization. In addition, preliminary experiments not detailed here have shown that immediate solubilization of IM-1 cells prior to fluorescence determinations with 0.5% deoxycholate detergent is possible and demonstrates no effect on immune fluorescence, similar to results reported by Schreiber ef al. (13). The sensitivity of our assay is particularly noteworthy and easily comparable to other immunofluorescent techniques available. Whereas direct microscopic examination of fluorescent cells in a wet mount allows the observer to count single cells, statistics re-
FLUOROIMMUNOASSAY
OF LYMPHOID
quire that numerous cells be counted. In our laboratory, wet-mount preparations of IM-1 cells for immunofluorescence contain approximately 7.5 x lo4 cells/slide. This cell concentration yields a microscopic field that can be scored rapidly with adequate numbers of fluorescent cells. In contrast, our fluoroimmunoassay is capable of discriminating experimental/control fluorescence ratios of 2 at 5.7 X lo3 cells/ml or approximately 1.7 x lo3 cells/assay. Strom and Klein (7) reported that for some experiments significant fluorescence ratios of around 5 required between 6 x lo6 and 1 x 10’ cells/assay. Similar fluorescence ratios with our fluoroimmunoassay required the use of only 7 x lo3 cells/assay, clearly an improvement in sensitivity. The sensitivity of the FIA methods described here can also be compared to that of radiolabeling methods. The R-3 serum used in this paper has been independently characterized by complement-dependent cytotoxicity assays with 51Cr-labeled IM-1 cells (9). It was found that 2 ~1 of the serum completely lysed 6 x lo5 IM-1 cells in the presence of excess rabbit complement. When examined by fluoroimmunoassay, about 3 ~1 of this serum saturated most antigenic sites on 6 x lo5 cells (Fig. 3). Thus, the FIA was about 1.5 times less sensitive than the Cr-release assay as measured by the amount of antibody needed for maximal immunologic effect in the respective systems. This apparent difference possibly reflected formation of competent complement fixation sites on the cell surface before all available antigenic sites were saturated by antibody. At antibody/cell ratios used for maximal Cr release (equivalent to 2 pl/6 x IO5 cells in Fig. 3), the fluorescence/background ratio is still between 18.6 and 28.3 in the fluoroimmunoassay system. Therefore, both Cr release and fluoroimmunoassay could yield quantitative data at similar antibody/cell ratios. However, Cr release assays can be done with as few as 3000 cells per test (9). While fluoroimmunoassays can give sig-
CELL
SUSPENSIONS
183
nificant data with this few cells per test (about 10,000 cells/ml, specific/control fluorescence ratio about 3.0, Fig. 5), background fluorescence becomes a significant factor and would limit the ultimate sensitivity of the fluoroimmunoassay system as compared to the Cr release system. We have made no direct comparisons of the fluoroimmunoassay techniques described here to that of radioiodine-labeled antiglobulin techniques. However, many standard radioiodine antiglobulin techniques can be used to detect immunoglobulins at levels approaching 1 PM (14). Although fluorometric techniques can reach this level of sensitivity (15), the fluoroimmunoassay methods described in this paper give useful data only in ranges in excess of 100 PM. Despite this potential lOOfold sensitivity differential, fluoroimmunoassays remain useful in appropriate systems because radioiodine systems are inherently unstable due to isotope decay and present licensing and disposal problems not encountered in fluoroimmunoassays. In fact, we found that fluoroimmunoassays in our system were limited more by the “nonspecific reactivity” of the control serum with the cells, i.e., specificity of the control reagent, than by sensitivity considerations. For example, as Fig. 1 shows, fluorescence of a normal rabbit serum control after reaction with IM-1 cells and absorbed fluorescent antiglobulin was about three times that of cells alone. The same background problems would exist in any radioimmunoassay with the same normal rabbit control serum. Thus, the ratio of specific binding (immune serum) to nonspecific binding (control serum) might not be affected by the use of either fluoroimmunoassay or radioimmunoassay, although fewer total cells might be used in the radioimmunoassay system because of its increased sensitivity. Quantitative fluoroimmunoassays have other potential applications that extend their usefulness beyond that of conventional
184
GILLIS ETAL.
radioimmunoassays. Cell suspensions can be directly visualized in the fluorescence microscope and have their total fluorescence measured by fluoroimmunoassay, thereby providing a convenient quantitative methodology to conduct performance evaluations of reagents to be used for fluorescence microscopy. Specifically, fluorescence parameters (16) such as desired specific fluorescence and undesired specific fluorescence could be quantitatively distinguished from background fluorescence by fluoroimmunoassay (Figs. 2 and 3) and could be related to the corresponding microscopic findings (“bright, ” “dim,” etc.). It should be noted that fluoroimmunoassay methods appear sufficiently specific (Table 1) that as few as 2.1% fluorophore-labeled cells can be measured without significant interference from unlabeled (or other fluorophorelabeled) cells. This suggests that the fluoroimmunoassay methods described here could be applied to the analysis of antibodies to, and surface antigens of, heterogenous cell mixtures. If only a fraction of cells are fluorescent, then a corrected value for fluorescence per cell could be obtained by dividing total fluorescence by the number of fluorescent cells. This value would correspond to the weighted mean value of fluorescence per cell as determined by flow microfluorometry (4). In contrast to flow microfluorometry, no information of the distribution of fluorescence in the cell population could be derived from an analysis of total fluorescence by fluoroimmunoassay. However, such fluorescence distribution information would not be required if the cells were used only as “carriers” for total antibody and/or total antigen determinations as we have illustrated in this paper. To conclude, the ultimate sensitivity of our assay, as with any immunofluorescent assay, is restricted by the primary and secondary antibody titers, the antigen concentration on the cell surface, and the amount of nonspecific fluorescence obtained with
test reagents. Therefore in systems with high background fluorescence or minimal specific fluorescence signal, the sensitivity of the assay will be reduced. In contrast to other work (16,17), the fluoroimmunoassay we described here does not require antigen purification for direct antibody detection, nor is the assay contingent upon homogeneous surface fluorescence. Rather our assay yielded total fluorescence of the cell suspension after reaction with immune-specific reagents. In addition, we showed that our fluoroimmunoassay exhibited a specific dose-dependent inhibition when either IM-1 vesicular membranes or whole IM-1 cells were used as inhibitors. This suggests that the fluoroimmunoassay system could be readily adapted to assays in which the immune sera are absorbed by other cells or cell fractions in order to determine crossreactivities of the immune sera and to identify and characterize antigenic specificities of the inhibitors. Consequently, our fluoroimmunoassay can be standardized on the basis of standard curves relating the amount of bound immunoglobulin to fluorescence with a standard lot of fluoresceinated antiglobulin, or in terms of the amount of stable antigen necessary to inhibit a previously standardized antibody system to a given degree (18,19). In summary, fluoroimmunoassay methodology proved to be: (i) quantitative, (ii) rapid; (iii) sensitive; (iv) independent of cell solubilization; (v) free from complicated verification procedures; (vi) adaptable to specific inhibition with antigen in a dosedependent fashion; and (vii) selective in double-labeling procedures. ACKNOWLEDGMENTS This investigation was supported by the following grants: National Science Foundation, BMS 74-13860; National Institutes of Health, AI-l 1907-03; Biomedical Research Support funds of the L.S.U. Schools of Dentistry and Medicine, P.H.S. SSOl-RR5704 and 5SO7-RR05376; Cancer Association of Greater New
FLUOROIMMUNOASSAY
OF LYMPHOID
Orleans, 410-17-6120; and American Heart Association-Louisiana, Inc., 410-17-6121. Thomas P. Gillis is a Graduate Student Research Fellow of the American Heart Association-Louisiana, Inc.
REFERENCES 1. Johansson, B., and Klein, E. (1970) Clin. Exp. Immunoi. 6, 421-428. 2. Galton, J., and Ivanyi, J. (1977)J. Zmmunol. Methods 17, 57-61. 3. Deelder, A. M., and Ploem, J. S. (1974) J. Immunol. Methods 4, 239-251. 4. Horan, P. D. K., and Wheeless, L. L., Jr. (1977) Science 198, 149- 157. 5. Naim, R. C. (1968) C/in. Exp. Immunol. 3, 465476. 6. Taylor, C. E. D., and Heimer, G. V. (1975) Ann. N. Y. Acad. Sci. 254, 151-156. 7. Strom, R., and Klein, E. (1969) Proc. Nat. Acad. Sci. 63, 1157-1163. 8. Gillis, T. P., and Thompson, J. J. (1978) J. Clin. Microbial. 7, 202-208. 9. Wilson, L. A., Cowdery, J. S., and Gallaspy, G. T. (1978) Transplantation 25, 73-79.
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10. Kourilsky, F. M., Silvestre, A., Neauport-Sautes, C., Loosfelt, Y., and Dausset, J. (1972) Eur. .I. Immunol. 2, 249-257. 11. Chen, R. F. (1966)Anal. Biochem. 14, 497-499. 12. Colton, T. (1974) Statistics in Medicine, pp. 189217, Little, Brown, Boston. 13. Schreiber, A. B., Hoebeke, J., Bergman, Y., Haimovich, J., and Strosberg, A. D. (1978) J. Immunol. 212, 19-23. 14. Stites, D. P., Ishizaka, K., and Fudenberg, H. H. (1972) Clin. Exp. Immunol. 10, 391-397. 15. Jameson, D. M., Weber, G., Spencer, R. D., and Mitchell, G. (1978) Rev. Sci. Instrum. 49, 510514. 16. Knapp, W., Haaijman, J. J., Schuit, H. R. E., Radl, J., Van den Berg, P., Ploem, J. S., and Hijmans, W. (1975) Ann. N. Y. Acad. Sci. 254, 94- 107. 17. Capel, P. J. A. (1975) Ann. N. Y. Acad. Sci. 254, 108-118. 18. Jongsma, P. M., Hijmans, W., and Ploem, J. (1971) Histochemie 25, 329-343. 19. Fagraeus, A., and Berquist, N. R. (1975) Ann N. Y. Acad. Sci. 254. 69-76.
BlOCHEMlSTRY
94,
176-185 (1979)
A Rapid Method for Quantitation on Human Lymphoblastoid T. P. GILLIS, Department
of Immunofluorescence Cell Suspensions
F. H. WILSON, L. A. WILSON, AND J. J. THOMPSON of Microbiology, Louisiana State University Medical Center, 1100 Fiorida Avenue, New Orleans, Louisiana 70119 Received June 19, 1978
A quantitative fluoroimmunoassay for antibodies to, and surface antigens of, human lymphoblastoid cells (IM-1) with photon-counting spectrofluorometry is described. IM-1 cell suspensions were reacted with rabbit antiserum to human spleen vesicular membranes, were washed, and then were reacted with an excess amount of fluorochrome-conjugated (fluorescein or rhodamine) goat anti-rabbit immunoglobulin G (IgG). Under appropriate conditions, antibodies to IM- 1 cells could be detected with experimental/control fluorescence ratios ranging between 5 and 40. Moreover, detectable levels of antibody-saturated cells approached 5 x 103 cells per milliliter or a total of 1.7 x IV cells per assay. Inhibition of the fluoroimmunoassay was performed with either viable IM-1 cells or IM-1 vesicular membrane preparations and demonstrated a dose-dependent antigen inhibition. Fluorescence of sensitized cells reactive with either fluorescein- or rhodamine-labeled antiglobulins could be quantitatively distinguished in dual-labeled preparations.
Immunofluorescence is a technique with wide applicability that has been used to locate and semiquantitate antigens of various cell types (1,2). True quantitation of immunofluorescence has only recently been used for research purposes with the development of relatively sophisticated equipment that utilize either microscope attachments for microfluorometry (3) or laser-activated fluorescence for flow fluorometry (4). These quantitation methods have features that limit their application. For example, standardization procedures for microfluorometry are time consuming and may be subject to investigator bias (5,6), whereas laser-equipped flow fluorometers are cost prohibitive. Sophisticated instrumentation is not strictly required for quantitation of immunofluorescence. It has been reported (7) that fluorometric quantitation of fluorescein-conjugated antibodies attached to cell membrane antigens could be accomplished with a conventional filter fluorometer after solubilization of the cells. In addition to the solubilization 0003-2697/79/050176-10$02.00/0 Copyright All rights
0 1979 by Academic Press, Inc. of reproduction in any form reserved.
requirements, the assay took several days to complete and was relatively insensitive, requiring large numbers of cells per assay. Based on previously reported work on a quantitative fluoroimmunoassay in a bacterial system (8), we attempted fluorometric quantitation of fluorochrome-conjugated antibody on the surface of viable mammalian cells. We report here a simple, yet sensitive, fluorometric assay that detects antibodies to, and surface antigens of, human lymphoblastoid culture cells with photon-counting spectrofluorometry. METHODS
Cells. Human lymphoblastoid cells (IM- l)l in continuous culture were used for all proi Abbreviations used: IM-1, human lymphoblastoid cells; R-3, rabbit antiserum; NRS, normal rabbit serum; FITC, fluorescein isothiocyanate; TRITC, rhodamine isothiocyanate; GAR, goat antiserum; IgG, immunoglobulin G; HBSS-FCS, Hanks’ balanced salt solution with 10% fetal calf serum; DPBS, Dulbecco’s phosphate-buffered saline; FIA, fluoroimmunoassay. 176
FLUOROIMMUNOASSAY
OF LYMPHOID
cedures. Cells were grown in suspension in RPM1 1640 medium with 10% fetal calf serum (GIBCO, Grand Island, N. Y.). Seru. Rabbit antiserum (R-3) was prepared to human spleen cell vesicular membranes (9). Pooled normal rabbit serum (NRS) was used as our control for nonspecific interactions of rabbit serum with IM- 1 cells. Fluorescein isothiocyanateand rhodamine isothiocyanate-conjugated goat antisera to rabbit IgG (designated FITC-GAR IgG and TRITC-GAR IgG, respectively) were purchased from Cappel Laboratories Inc. (Cochranville, Pa.). The FITC-GAR IgG preparation contained 6.0 mg/ml antibody protein with an F/P ratio of 3.3; R/P ratio of the TRITC-GAR IgG was 5.4. Absorption of the fluorochrome conjugates was performed for 2 h at 4°C with lo6 IM-1 cells/O. 1 ml of conjugate in order to remove cross-reacting antibody to IM-1 cells. Fluoroimmunoassay . In a typical experiment, IM-1 cells were mixed with either R-3 or NRS and incubated for 45 min at 0°C to prevent “capping” of cell surface antibody aggregates. The cells were then washed twice in the cold with Hanks’ balanced salt solution with 10% fetal calf serum (HBSSFCS). FITC-GAR IgG was added to the washed cell pellets and the suspensions were further incubated at 0°C for 45 min. The cells were again washed two times in the cold with HBSS-FCS and resuspended to the appropriate cell concentration in Dulbecco’s phosphate-buffered saline (DPBS) with NaN, (0.2%) for the measurement of fluorescence. Lowered temperature (OOC) and NaN3 have been shown previously to inhibit migration of surface antibody aggregates (10). For dual-labeling experiments, 600-~1 aliquots which contained 3 x lo6 IM-1 cells in HBSS-FCS were reacted with 18 ~1 of R-3 antiserum, incubated 45 min at 0°C and then washed two times with HBSS-FCS. The cells were then suspended in 240 ~1 of either a 1: 10 dilution of FITC-GAR IgG
CELL
SUSPENSIONS
177
or 240 ~1 of a 1:4 dilution of TRITC-GAR IgG. Preliminary experiments had demonstrated that the respective amounts of fluorochrome-conjugated reagents used were sufficient to saturate the number of sensitized IM-1 cells used in these suspensions. After a 45-min incubation at 0°C the cell preparations were washed one time with HBSSFCS and one time with DPBS with NaN,. The washed cell pellets were resuspended in 3 ml of DPBS with NaN,. Unlabeled IM-1 cells were similarly washed and diluted in DPBS with NaN, to a final concentration equivalent to that of the labeled cells. Portions of these cell preparations were then mixed to give varying proportions of fluorochrome-labeled cells to unlabeled cells or fluorescein-labeled cells to rhodamine-labeled cells. Each final mixture of cells contained the same concentration of cells as the starting suspension in a volume of 500 ~1. Fluorescence data for this system were corrected by subtraction of background due to cells in buffer (7000 cps for fluorescein conditions and 1740 cps for rhodamine conditions). Inhibition of the fluoroimmunoassay was used to detect membrane antigens on intact cells and vesicular membranes. The inhibition assay was performed by the incorporation of a preincubation step of antigen with a standard amount of R-3 serum for 30 min at 37°C followed by 30 min at 0°C. After centrifugation of the mixtures (13,OOOg) for 10 min, lOO+l aliquots were removed and reacted with 6 x lo5 IM-1 target cells, as outlined above in the fluoroimmunoassay. An unabsorbed positive fluorescence control was included with buffer instead of antigen in the preincubation step. Inhibition was calculated as follows: Percentage inhibition (cps of absorbed serum samples) clx 100. (cps of unabsorbed serum samples) Fluorescence measurements. Fluores-
178
GILLIS
cence measurements were made on a MKI spectrofluorometer (Fart-and Optical Co., Inc., Valhalla, N. Y.) modified to accept a Model 1140 quantum photometer (Princeton Applied Research Corp., Princeton, N. J.) with an R 212 photomultiplier tube (Hamamatsu Corp., Middlesex, N. J.). Fluorescence measurements were expressed as photon-counting rate in counts per second at a standard deviation of 1.3%. Crossed film polarizers (incident polarizer at O”, emission polarizer at 90”, Fart-and Optical Co.) were used to minimize interference from light scatter. Polarization of excitation light alone may be sufficient to minimize scattering problems (1 l), but we have not yet studied this possibility. Slits were chosen to allow a IO-nm bandpass. Fluorescence of cell suspensions was measured in a 0.3ml quartz semimicrocuvette assembly. Excitation of fluorescence was at 485 nm for fluorescein and 535 nm for rhodamine; emission was analyzed at 525 nm for fluorescein and 580 nm for rhodamine. Slopes of linear dose-response curves for fluorescein methyl ester and rhodamine B in DPBS with NaN, were about 25,000 and 430 cps/nM, under the selective specrespectively, troscopic conditions just described. Stability was measured by the continuous recording of fluorescence signal on an SR 255B strip chart recorder (Heath Co., Benton Harbor, Mich.). Statistics. Data obtained from the duallabeling dose-response experiments were fitted by linear least-squares analysis. Variances associated with the calculated slopes and intercepts (12) were compared by the F test. When variances were not significantly different, t tests were used to determine statistical significance. RESULTS Stability of FITC-GAR IgG-Labeled IM-1 Suspensions
A suspension of IM-1 cells (final concentration of 105/ml) was reacted with R-3 and
ET AL.
FITC-GAR IgG in the standard fluoroimmunoassay procedure. Fluorescence emission was excited at 485 nm and analyzed at 525 nm. As shown in Fig. 1, buffer alone was not significantly fluorescent, but IM-1 cells showed minimal intrinsic fluorescence and/or light scatter. The addition of NRS and FITC-GAR IgG to the cells gave some fluorescent labeling. Fluctuation in the signals above noise levels probably represented cell aggregates passing through the illuminated portion of the cuvette. A l:lO,OOO dilution of the FITC-GAR IgG preparation showed evidence of fluorescence decay over the lo-min observation period; the rate of decay appeared to be slightly less than 0.5% of the initial fluorescence signal per minute. R-3 sensitized cells reacted with FITC-GAR IgG showed significantly greater fluorescence than the corresponding controls. Decay of fluorescence on these FITC-GAR IgG-labeled cells appeared similar to that of free FITC-GAR IgG and therefore could not be attributed to settling of the cell suspension. TRITCGAR IgG-labeled cells behaved similarly (not illustrated). The apparent difference in extent of “noise” at high signal levels as opposed to low signal levels is an artifact of the electronic analog processing of the digital signals by the photon-counting instrument. All signals were processed at a fixed standard deviation (1.3% in Fig. 1). As the input signal increased, this variation became larger in absolute magnitude and therefore more apparent as “noise” in the chart recording. Determination of Optimum FITC -GAR IgC
Aliquots (200 ~1) which contained 6 x lo5 IM-I cells in HBSS-FCS were reacted with either 6 ,ul of R-3 (undiluted) or NRS and various dilutions of FITC-GAR IgG (40 ~1) in the standard fluoroimmunoassay. Cells were resuspended to 1 ml prior to reading in the spectrofluorometer. As Fig. 2 demon-
FLUOROIMMUNOASSAY
OF LYMPHOID
CELL SUSPENSIONS
179
IM-1 + R-3 +FITC-GAR w
80 FITC-GAR w l:lO,fulo
60
40 IM-l+ NRS + 20 IM-1 CELLS 0
1
1
1
1
I
2
4
6
8
10
BUFFER
Time, minutes FIG. 1. Stability of fluorescence of various preparations. was analyzed at 525 nm with crossed polarizers.
strates, R-3 antibodies to IM-I cell membranes were effectively saturated with FITC-CAR IgG at a dilution of 1:lO. At the highest concentration of FITC-GAR IgG tested (15)) a slight decrease in Ruorescence was noted in the experimental reading. However, this decrease was not observed in the NRS control and resulted in a depressed experimental/control fluorescence ratio.
Excitation was at 485 nm, and emission
Titration
of R-3 Antibodies to IM-l Celi Membrane Antigens
IM-1 cells (6 x 105) in HBSS-FCS (200 ~1) were reacted with varying amounts of either R-3 serum or NRS and an excess amount of FITC-GAR (15) in the standard fluoroimmunoassay. As Fig. 3 illustrates, the IM-1 cells approached saturation with R-3
180
GILLIS ET AL. RATIO: IMMUHlNRS
14.7 9;5 , 17,7
7%-
270 -
x
240 -
5 B
210 -
2g
180 150
s
123 -
r
26,6
16.9
membranes (Fig. 4A) or intact IM-1 cells (Fig. 4B). Figure 4B also shows that the lowest concentration of cells tested (4.5 x lo5 cells/ml) gave a detectable level of inhibition in this standardized system. Sensitivity of the Fluoroimmunoassay for the Detection of ZM-1 Cells
-
W-
1
2
Relative
3
4
5
6
7
8
9
10
amount of FITC-GAR IgG
FIG. 2. Effect of FITC-GAR IgG concentration on fluorescence of cells reacted with either 6 pl of undiluted R-3 (0) or equivalent amounts of NRS (0) in the standard fluoroimmunoassay. For the abscissa, 10 units = 15 dilution of FITC-GAR IgG.
antibody at amounts of 3 ~1 or greater. Because the increase in fluorescence from 6 to 12 ~1 was minimal, we felt that in order to conserve reagents 6 ~1 per 6 x lo5 cells would suffice as a saturation end point for further experiments. At R-3 antibody concentrations of less than 3 ~1, specific fluorescence fell off rapidly as the primary antibody became limiting. This effect, shown in Fig. 3, was reflected by a decreased experimental/control fluorescence ratio for 1.5 /.Ll. Inhibition of the Fluoroimmunoassay
Inhibition of IM-1 specific immunofluorescence was tested with either intact IM-1 lymphoblastoid cells or a vesicular membrane preparation. Increasing concentrations of inhibitor were reacted with 6 ,ul of R-3 or NRS. After incubation and centrifugation, the supematant fluids were tested for residual antibody in the standard fluoroimmunoassay. Figure 4 shows that a dose-dependent inhibition of IM- 1 specific immunofluorescence was obtained by adding increasing amounts of either vesicular
Aliquots (300 ~1) of IM-1 cells containing 1.8 x lo6 cells were reacted with 18 ~1 of either R-3 or NRS and incubated at 0°C for 45 min. The cells were then washed in HBSS-FCS, and 120 ~1 of FITC-GAR IgG (1:5 dilution) was added to the washed cell pellets. Following incubation and washing, final resuspension was in 10.0 ml DPBS with 0.2% NaN,. Serial twofold dilutions in DPBS were then prepared from both the NRS-treated and R-3-treated cells. Figure 5 shows that background fluorescence (NRStreated cells) decreased linearly with dilution and approached fluorescence of buffer alone between 20 x lo3 and 40 x 103 cell/ ml. Fluorescence of R-3-treated cells similarly decreased with dilution; however, experimental/control fluorescence ratios of 2 or greater were seen with dilutions to 5.7 RATIO IMMUNE/NRS
18.6
28.3
30.4
43.8
aOi r----% 1.5
3.0 Amount
6.0 of Primary
12.0 Antibody
@I)
FIG. 3. Titration of R-3 antibodies to IM-1 cell surface antigens with constant FITC-GAR IgG (1:5). Primary antibody is either R-3 (0) or NRS (0).
FLUOROIMMUNOASSAY A.
lMEMERAFBS
B.
OF LYMPHOID
CELLS
6 ‘G e -E c
25
50 Vesicular
Membraneslyll
loo
4.5
9.0 Cellslmlx
18 lo5
4. Inhibition of the standard fluoroimmunoassay by either IM-1 vesicular membrane preparation (A) or viable IM-1 lymphoblastoid cells (B). FIG.
X lo3 cells/ml. Since fluorescence was determined on only 0.3 ml of this suspension, the actual number of cells per assay would equal approximately 1.7 x 103. Beyond this dilution, values obtained could not be easily discriminated from background values, thereby establishing the lower limit of the assay for the detection of optimally sensitized IM-1 cells with this system.
181
CELL SUSPENSIONS
Double Fluorescence Assay of FITC-GAR or TRITC-GAR IgG-Labeled Cells As shown in Fig. 6, when fluoresceinlabeled cells were mixed in varying proportions with unlabeled cells and measured under spectroscopic conditions specific for fluorescein, a linear dose response was seen. If the unlabeled cells were replaced with an equal quantity of rhodamine-labeled cells, the dose response was unaffected. Correspondingly (not illustrated), when rhodamine-labeled cells were mixed in varying proportions with either unlabeled cells or fluorescein-labeled cells and measured under conditions specific for rhodamine, a linear fluorescence dose response resulted. As shown in Table 1, the slopes of the doseresponse curves for detection of either fluorescein- or rhodamine-labeled cells were not significantly different when the fluorescent cells were mixed with either unlabeled cells or cells labeled with the other fluorophore (P > 0.01 for all relevant pair com-
250
0
5. Effect of IM-1 cell dilution upon immune specific fluorescence (R-3 treated cells, 0) and background fluorescence (NRS-treated cells, 0) after reaction in the standard fluoroimmunoassay. Fluorescence at 0 cells/ml (Ix]) represents the fluorescence obtained with buffer alone in the cuvette. FIG.
20 Percentage
40 60 80 d FluoresceinLabeled Cells
100
FIG. 6. Fluorescence dose response to fluoresceinlabeled cells in the presence of rhodamine-labeled cells (0) or unlabeled cells (0). Fluorescence was excited at 485 nm and emission was analyzed at 525 nm with crossed polarizers.
182
GILLIS ET AL. TABLE STATISTICAL
ANALYSIS
1
OF FLUORESCENCE
DOSE-RESPONSE
Fluorescent emission (hE = 485 nm, AA = 525 nm)
Calculated parameter Correlation coefficient (fluorescence and % labeled cells) Slope f 1 SD (cps per % labeled cells) Coefficient of variation of the slope (%) y-Intercept f 1 SD (cps)
CURVES~
Rhodamine emission (As = 535 nm, AA = 580 nm)
Fluoresceinlabeled + unlabeled
Fluoresceinlabeled + rhodaminelabeled
Rhodaminelabeled + unlabeled
Rhodaminelabeled + fluoresceinlabeled
0.9985
0.9970
0.9974
0.9980
3,296 -’ 197
229 2 9.5
213 f 7.7
3.24 64 c 563
3.64 444 A 448
3,264 + 100 3.06 0 f 6,183
5.99 -7,800 2 12,024
(2AE = excitation wavelength; AA = wavelength at which emission was analyzed.
parisons, t test). Similarly, all y-intercepts calculated from the least-squares analysis did not vary significantly from 0 (P > 0.10 for all relevant comparisons, t test). Therefore, we concluded that the spectroscopic conditions used permitted quantitative fluoroimmunoassay of fluorescein- and rhodamine-labeled suspensions. If one arbitrarily defined an uncorrected signal that is two times more than background as being significantly different and calculated the corresponding percentage of labeled cells derived in Table 1, then the rhodamine system could be used to measure samples with 7.3% labeled cells, whereas the fluorescein system would detect as little as 2.1% labeled cells. Precision of the assays, as assessed by the coefficient of variation of the slope of the dose-response curves, was less than or equal to about 6%. Similar levels of precision were obtained with replicate determinations of single samples (not illustrated). DISCUSSION
Recent advances in fluorescence technology have made quantitation of cell surface bound fluorescent antibodies possible. However, limited availability of this tech-
nology and problems encountered in standardization procedures prompted us to test the efficacy of a quantitative fluoroimmunoassay with photon-counting spectrofluorometry. A similar approach to immune specific fluorometric quantitation of cell surface antigens with a filter fluorometer was pursued by Strom and Klein (7). Their assay involved an overnight incubation with lengthy enzymatic digestions in order to solubilize cells prior to fluorescence determinations. In contrast, with crossed film polarizers to reduce light scatter, we were able to quantitate immune specific fluorescence of IM-I cells in a matter of a few hours without solubilization. In addition, preliminary experiments not detailed here have shown that immediate solubilization of IM-1 cells prior to fluorescence determinations with 0.5% deoxycholate detergent is possible and demonstrates no effect on immune fluorescence, similar to results reported by Schreiber ef al. (13). The sensitivity of our assay is particularly noteworthy and easily comparable to other immunofluorescent techniques available. Whereas direct microscopic examination of fluorescent cells in a wet mount allows the observer to count single cells, statistics re-
FLUOROIMMUNOASSAY
OF LYMPHOID
quire that numerous cells be counted. In our laboratory, wet-mount preparations of IM-1 cells for immunofluorescence contain approximately 7.5 x lo4 cells/slide. This cell concentration yields a microscopic field that can be scored rapidly with adequate numbers of fluorescent cells. In contrast, our fluoroimmunoassay is capable of discriminating experimental/control fluorescence ratios of 2 at 5.7 X lo3 cells/ml or approximately 1.7 x lo3 cells/assay. Strom and Klein (7) reported that for some experiments significant fluorescence ratios of around 5 required between 6 x lo6 and 1 x 10’ cells/assay. Similar fluorescence ratios with our fluoroimmunoassay required the use of only 7 x lo3 cells/assay, clearly an improvement in sensitivity. The sensitivity of the FIA methods described here can also be compared to that of radiolabeling methods. The R-3 serum used in this paper has been independently characterized by complement-dependent cytotoxicity assays with 51Cr-labeled IM-1 cells (9). It was found that 2 ~1 of the serum completely lysed 6 x lo5 IM-1 cells in the presence of excess rabbit complement. When examined by fluoroimmunoassay, about 3 ~1 of this serum saturated most antigenic sites on 6 x lo5 cells (Fig. 3). Thus, the FIA was about 1.5 times less sensitive than the Cr-release assay as measured by the amount of antibody needed for maximal immunologic effect in the respective systems. This apparent difference possibly reflected formation of competent complement fixation sites on the cell surface before all available antigenic sites were saturated by antibody. At antibody/cell ratios used for maximal Cr release (equivalent to 2 pl/6 x IO5 cells in Fig. 3), the fluorescence/background ratio is still between 18.6 and 28.3 in the fluoroimmunoassay system. Therefore, both Cr release and fluoroimmunoassay could yield quantitative data at similar antibody/cell ratios. However, Cr release assays can be done with as few as 3000 cells per test (9). While fluoroimmunoassays can give sig-
CELL
SUSPENSIONS
183
nificant data with this few cells per test (about 10,000 cells/ml, specific/control fluorescence ratio about 3.0, Fig. 5), background fluorescence becomes a significant factor and would limit the ultimate sensitivity of the fluoroimmunoassay system as compared to the Cr release system. We have made no direct comparisons of the fluoroimmunoassay techniques described here to that of radioiodine-labeled antiglobulin techniques. However, many standard radioiodine antiglobulin techniques can be used to detect immunoglobulins at levels approaching 1 PM (14). Although fluorometric techniques can reach this level of sensitivity (15), the fluoroimmunoassay methods described in this paper give useful data only in ranges in excess of 100 PM. Despite this potential lOOfold sensitivity differential, fluoroimmunoassays remain useful in appropriate systems because radioiodine systems are inherently unstable due to isotope decay and present licensing and disposal problems not encountered in fluoroimmunoassays. In fact, we found that fluoroimmunoassays in our system were limited more by the “nonspecific reactivity” of the control serum with the cells, i.e., specificity of the control reagent, than by sensitivity considerations. For example, as Fig. 1 shows, fluorescence of a normal rabbit serum control after reaction with IM-1 cells and absorbed fluorescent antiglobulin was about three times that of cells alone. The same background problems would exist in any radioimmunoassay with the same normal rabbit control serum. Thus, the ratio of specific binding (immune serum) to nonspecific binding (control serum) might not be affected by the use of either fluoroimmunoassay or radioimmunoassay, although fewer total cells might be used in the radioimmunoassay system because of its increased sensitivity. Quantitative fluoroimmunoassays have other potential applications that extend their usefulness beyond that of conventional
184
GILLIS ETAL.
radioimmunoassays. Cell suspensions can be directly visualized in the fluorescence microscope and have their total fluorescence measured by fluoroimmunoassay, thereby providing a convenient quantitative methodology to conduct performance evaluations of reagents to be used for fluorescence microscopy. Specifically, fluorescence parameters (16) such as desired specific fluorescence and undesired specific fluorescence could be quantitatively distinguished from background fluorescence by fluoroimmunoassay (Figs. 2 and 3) and could be related to the corresponding microscopic findings (“bright, ” “dim,” etc.). It should be noted that fluoroimmunoassay methods appear sufficiently specific (Table 1) that as few as 2.1% fluorophore-labeled cells can be measured without significant interference from unlabeled (or other fluorophorelabeled) cells. This suggests that the fluoroimmunoassay methods described here could be applied to the analysis of antibodies to, and surface antigens of, heterogenous cell mixtures. If only a fraction of cells are fluorescent, then a corrected value for fluorescence per cell could be obtained by dividing total fluorescence by the number of fluorescent cells. This value would correspond to the weighted mean value of fluorescence per cell as determined by flow microfluorometry (4). In contrast to flow microfluorometry, no information of the distribution of fluorescence in the cell population could be derived from an analysis of total fluorescence by fluoroimmunoassay. However, such fluorescence distribution information would not be required if the cells were used only as “carriers” for total antibody and/or total antigen determinations as we have illustrated in this paper. To conclude, the ultimate sensitivity of our assay, as with any immunofluorescent assay, is restricted by the primary and secondary antibody titers, the antigen concentration on the cell surface, and the amount of nonspecific fluorescence obtained with
test reagents. Therefore in systems with high background fluorescence or minimal specific fluorescence signal, the sensitivity of the assay will be reduced. In contrast to other work (16,17), the fluoroimmunoassay we described here does not require antigen purification for direct antibody detection, nor is the assay contingent upon homogeneous surface fluorescence. Rather our assay yielded total fluorescence of the cell suspension after reaction with immune-specific reagents. In addition, we showed that our fluoroimmunoassay exhibited a specific dose-dependent inhibition when either IM-1 vesicular membranes or whole IM-1 cells were used as inhibitors. This suggests that the fluoroimmunoassay system could be readily adapted to assays in which the immune sera are absorbed by other cells or cell fractions in order to determine crossreactivities of the immune sera and to identify and characterize antigenic specificities of the inhibitors. Consequently, our fluoroimmunoassay can be standardized on the basis of standard curves relating the amount of bound immunoglobulin to fluorescence with a standard lot of fluoresceinated antiglobulin, or in terms of the amount of stable antigen necessary to inhibit a previously standardized antibody system to a given degree (18,19). In summary, fluoroimmunoassay methodology proved to be: (i) quantitative, (ii) rapid; (iii) sensitive; (iv) independent of cell solubilization; (v) free from complicated verification procedures; (vi) adaptable to specific inhibition with antigen in a dosedependent fashion; and (vii) selective in double-labeling procedures. ACKNOWLEDGMENTS This investigation was supported by the following grants: National Science Foundation, BMS 74-13860; National Institutes of Health, AI-l 1907-03; Biomedical Research Support funds of the L.S.U. Schools of Dentistry and Medicine, P.H.S. SSOl-RR5704 and 5SO7-RR05376; Cancer Association of Greater New
FLUOROIMMUNOASSAY
OF LYMPHOID
Orleans, 410-17-6120; and American Heart Association-Louisiana, Inc., 410-17-6121. Thomas P. Gillis is a Graduate Student Research Fellow of the American Heart Association-Louisiana, Inc.
REFERENCES 1. Johansson, B., and Klein, E. (1970) Clin. Exp. Immunoi. 6, 421-428. 2. Galton, J., and Ivanyi, J. (1977)J. Zmmunol. Methods 17, 57-61. 3. Deelder, A. M., and Ploem, J. S. (1974) J. Immunol. Methods 4, 239-251. 4. Horan, P. D. K., and Wheeless, L. L., Jr. (1977) Science 198, 149- 157. 5. Naim, R. C. (1968) C/in. Exp. Immunol. 3, 465476. 6. Taylor, C. E. D., and Heimer, G. V. (1975) Ann. N. Y. Acad. Sci. 254, 151-156. 7. Strom, R., and Klein, E. (1969) Proc. Nat. Acad. Sci. 63, 1157-1163. 8. Gillis, T. P., and Thompson, J. J. (1978) J. Clin. Microbial. 7, 202-208. 9. Wilson, L. A., Cowdery, J. S., and Gallaspy, G. T. (1978) Transplantation 25, 73-79.
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10. Kourilsky, F. M., Silvestre, A., Neauport-Sautes, C., Loosfelt, Y., and Dausset, J. (1972) Eur. .I. Immunol. 2, 249-257. 11. Chen, R. F. (1966)Anal. Biochem. 14, 497-499. 12. Colton, T. (1974) Statistics in Medicine, pp. 189217, Little, Brown, Boston. 13. Schreiber, A. B., Hoebeke, J., Bergman, Y., Haimovich, J., and Strosberg, A. D. (1978) J. Immunol. 212, 19-23. 14. Stites, D. P., Ishizaka, K., and Fudenberg, H. H. (1972) Clin. Exp. Immunol. 10, 391-397. 15. Jameson, D. M., Weber, G., Spencer, R. D., and Mitchell, G. (1978) Rev. Sci. Instrum. 49, 510514. 16. Knapp, W., Haaijman, J. J., Schuit, H. R. E., Radl, J., Van den Berg, P., Ploem, J. S., and Hijmans, W. (1975) Ann. N. Y. Acad. Sci. 254, 94- 107. 17. Capel, P. J. A. (1975) Ann. N. Y. Acad. Sci. 254, 108-118. 18. Jongsma, P. M., Hijmans, W., and Ploem, J. (1971) Histochemie 25, 329-343. 19. Fagraeus, A., and Berquist, N. R. (1975) Ann N. Y. Acad. Sci. 254. 69-76.
