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Flow cytometric detection and quantitation of immune complexes using human C1q-coated microspheres
Journal of Immunological Methods, 95 (1986) 57-61
57
Elsevier JIM 04143
Flow cytometric detection and quantitation of immune complexes using human Clq-coated microspheres Thomas M. McHugh, Daniel P. Stites *, Conrad H. Casavant and Mack J. Fulwyler Department of Laboratory Medicine, University of California at San Francisco, San Francisco, CA 94143, U.S.A. (Received 19 May 1986, accepted 22 July 1986)
A solid phase human Clq-binding fluorescent immunoassay for the measurement of immune complexes in human serum was developed. The solid phase used was 5/~m diameter polystyrene microspheres. Serum immune complexes bound to the Clq-coated microspheres were measured by flow cytometry using fluoresceinated anti-human IgG, and heat-aggregated human IgG as a standard. Patient samples were assayed and results compared to a standard fluoroimmunometric Clq-binding immune complex assay. Greater differences in circulating immune complexes were observed between the healthy control group mean and the mean of the patient values in the microsphere-flow cytometric method than were seen in the standard assay. In the microsphere-flow cytometric assay, the mean patient value was 7.5 times greater than the control mean, whereas in the standard assay the mean patient value was 2.8 times the control mean. Preliminary results suggest greater sensitivity of the microsphere-flow cytometric method over the other method. Key words: Flow cytometry; Immune complex; Immunofluorescence
Introduction
assays vary greatly in sensitivity and predictive value in various diseases. An additional complexity is that various assays, which are based on different principles, detect different types of immune complexes. In this report we describe a new flow cytometric based immune complex assay using Clq-coated microspheres and compare its performance to our standard fluoroimmunoassay (Collins et al., 1982).
Immune complexes can mediate tissue injury by their ability to activate complement after being deposited in various organs. Glomerulonephritis and vasculitis, secondary to bacterial, parasitic and viral infections, or autoimmune diseases have been associated with circulating and tissue-bound immune complexes (Theofilopoulos and Dixon, 1980). The detection and quantitation of immune complexes in serum may contribute to diagnosis and therapeutic monitoring in selected diseases. Many methods exist for the detection of immune complexes (Lambert et al., 1978) and these
Preparation of lgG aggregates
* Address correspondence to: Daniel P. Stites, M.D., Box 0100, Department of Laboratory Medicine, University of California, 3rd and Parnassus Avenues, San Francisco, CA 94143, U.S.A.
Human IgG (Gamastan, Cutter Laboratories, Berkeley, CA) was diluted to a concentration of 10 mg/ml in 0.15 M phosphate-buffered saline (PBS), pH 7.2. The preparation was aggregated by heating for 20 rain at 63°C, and was then centrifuged
Materials and methods
0022-1759/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
58 at 1500 x g for 20 min to remove large insoluble aggregates. The supernatant was adjusted to 1 m g / m l in PBS and then immediately frozen in 2 ml aliquots at - 7 0 ° C until used.
Serum samples Peripheral blood was collected in vacutainer tubes (Becton Dickinson, Rutherford, N J) without anticoagulant. Specimens were allowed to clot for 1 h at 22 ° C. Tubes were centrifuged at 400 x g for 10 min at 4°C. Serum was removed, separated into two aliquots and immediately frozen at - 7 0 ° C . Samples were allowed to thaw at 4°C prior to analysis.
Preparation of solid-phase Cl q Polystyrene microspheres of 5.0 /~m diameter (Dow Chemical Co., Indianapolis, IN) were washed in PBS several times. The microspheres were resuspended in 1 ml PBS, counted on a Coulter model Zf counter (Coulter Electronics, Hialeah, FL) and adjusted to 1 x 106 in a 12 × 75 mm glass tube. Purified human C l q (Center for Blood Research, Boston, MA) was diluted in PBS to a concentration of 50 # g / m l . 0.1 ml of C l q suspension was added to 1 × 106 microspheres and the tube was mixed and incubated for 24 h at 4oc.
Flow cytometric Clq-binding immune complex assay (FACS assay) Assays were carried out in 12 x 75 mm glass tubes. Clq-coated microspheres were washed three times in 0.15 M PBS + 0.5% bovine serum albumin + 0.1% Tween 20, pH 6.4 (PBS-BSA-Tw). Serum samples were heat-inactivated for 30 rain at 56°C. The heat-aggregated IgG was diluted in PBS to concentrations of 10, 25, 50, 100 and 200 /~g/ml. 0.5 ml of zero standard, IgG standards, and sera diluted 1:11, were pipetted into tubes. 1 x 105 Clq-coated microspheres were added to each tube and incubated for 2 h at 22°C. Each tube was washed three times with PBS-BSA-Tw. The final microsphere pellet was resuspended in a 1 : 60 dilution of fluorescein-isothiocyanate (FITC)-conjugated goat F(alY)2 anti-human IgG (Tago, Burlingame, CA) and incubated for 1 h at 22°C. Tubes were washed as above, the pellet was resuspended in PBS and held on ice in the dark
until FACS analysis. BSA (l%)-coated microspheres without C l q were used with each serum specimen as a blank.
FA CS analysis Microspheres were analyzed on a FACS analyzer (Becton Dickinson Immunocytometry Systems, Mountain View, CA) interfaced to a Hewlett-Packard model 9816 computer and printer. Fluorescence excitation and emission wavelengths were selected using optical filters. The excitation filter was a 22 nm bandpass dichroic filter centered at 485 nm. A short-pass dichroic mirror at 505 nm was used to reflect fluorescence emission from FITC. A minimum of 10 000 microspheres were analyzed per tube. Data was acquired and processed using Consort 30 software (Becton Dickinson). Fluorescence profiles were analyzed from singlet microspheres and aggregates of microspheres were eliminated from the analysis by electronic gating using the electronic impedence sizing (Coulter volume) parameter on the FACS analyzer. Fluorescence emission was logarithmically amplified and displayed as a fluorescence profile histogram. The peak modal channel (PMC) of the fluorescence peak was used as the assay measurement unit. PMC was plotted versus/~g/ml of aggregated IgG and results expressed as /~g equivalents of aggregated I g G / m l .
Standard Cl q-binding immune complex assay The fluorescent immune complex assay used was as described by Collins et al. (1982). Briefly, diluted human C l q was bound to polymeric StiQ (IDT, Santa Clara, CA) samplers. Heat-aggregated IgG was used as the standard and serum samples were used at 1:11. Clq-coated and BSA-coated samplers were incubated with samples for 2 h at 22°C. Samplers were washed and FITC-conjugated goat F(ab')2 anti-human IgG (Kallestad Laboratories, Austin, TX) was added and incubated for 45 min at 22°C. Samplers were washed as before and the fluorescence read in a FIAX fluorometer (IDT, Santa Clara, CA). BSA-coated samplers were used as the blank. Fluorescence was reported in fluorescent signal units and was plotted versus /xg/ml aggregated IgG. Results were expressed as #g equivalents of aggregated I g G / m l .
59 TABLE I
Results
NORMAL VALUES FOR IMMUNE COMPLEXES IN SERUM
Fig. 1 shows fluorescence histograms of the heat-aggregated I g G s t a n d a r d curve. P M C is indicated i n each histogram. Sensitivity of the assay is best from 0/~g equivalents to approximately 4 0 / t g equivalents I g G / m l . T a b l e I lists i m m u n e complex assay results from the healthy volunteers. Previous work has shown that sera from healthy individuals can contain up to 25 /~g equivalents I g G / m l w h e n assayed b y our s t a n d a r d i m m u n e complex assay. Values in n o r m a l sera are generally lower in the F A C S assay. T a b l e II lists i m m u n e complex results o n selected p a t i e n t sera d e m o n s t r a t e d to have high or b o r d e r l i n e i m m u n e complex results when tested b y our s t a n d a r d assay. T h e range of n o r m a l values i n the F A C S assay is from 0 to 11/~g e q u i v a l e n t s / ml ( m e a n +_ 2 s t a n d a r d deviations). As c a n be seen, all p a t i e n t sera h a d significantly elevated i m m u n e complexes i n the F A C S assay. I n general, the p a t i e n t values were well above the n o r m a l range i n the F A C S assay.
Serum number
FACS PMC a
/xg eq. b
Standard assay, FSU c
/zg eq.
1 2 3 4 5 6 7 8 9 10
80 65 115 130 109 125 58 62 73 110
4 3 8 9 7 9 2 2 3 7
18 16 26 11 12 19 13 7 8 14
15 13 23 7 8 16 9 3 4 11
.,Y SD
88 38
5 3
14 6
11 6
a Peak modal channel from FACS immune complex assay. b #g equivalents of IgG/ml calculated from standard curve. c Fluorescence signal units from standard fluoroimmunoassay.
Discussion The l a b o r a t o r y detection of i m m u n e complexes is complicated b y the variability i n b i n d i n g of the complexes to the selected substrate. This variabil-
psc:146k P.c-1681 i
PMC:38
[~
170
0
170
0
85
85
255
0
85
170
255
0
85
170
255
i
PMC/~179
255
PMC:194
0
85
A
170
255
k
0
PMC:189
85
_/% 170
255
Fig. 1. Fluorescence profile histograms of standard curve in FACS immune complex assay. A: 0 ttg/ml; B: 10/~g/ml; C: 25 ~g/mi; D: 50 /~g/ml; E: 100 /~g/ml; F: 200/xg/ml. 10000 microspheres were assayed at each concentration. Horizontal axis represents particle fluorescence on a logarithmic scale expressed as FACS channel number. Vertical axis is relative number of events.
60 TABLE II IMMUNE COMPLEX VALUES IN PATIENT SERA Serum number 1 2 3
4
5 6 7 8 9 10 Normal range (£+2 SD)
Disease category
FACS (/,g eq.)
Standard assay (/~g eq.)
Rheumatoid arthritis Rheumatoid arthritis Rheumatoid arthritis with streptococcal glomerulonephritis Systemic lupus
45 36
38 23
50
41
erythrematosus Infectious mononucleosis Leukemia Leukemia AIDS AIDS AIDS
30
28
21 37 33 35 40 45
26 40 34 29 26 30
0-11
0-23
ity prohibits comparison of patient values between methodologies and raises questions as to the significance of a single assay result (Jones et al., 1982; Nydegger et al., 1983). In addition, current assays do not offer detection of various immunoglobulin classes involved in the immune complex or the antigen present in the complex. Microspheres have previously been used as a substrate for immunological based assays (Capel, 1974; Jolley et al., 1984). However, these applications did not use flow cytometry to detect signals generated from the microspheres. Recently, Bonnefoy et al. (1986) demonstrated the use of fluorescent microspheres and flow cytometry in detecting Fc receptors on human leukocytes. In their application microspheres were used as the fluorescent label to amplify the signal detected by flow cytometry. Horan (1977) first described the potential of using a flow cytometric solid-phase immunofluorescent assay with microspheres as the solid phase based upon the original idea by Fulwyler. We have applied the use of microspheres and flow cytometry to the detection of immune complexes. Our initial studies are encouraging in the apparent increase in sensitivity of this
assay over our standard method. While we have demonstrated the detection of IgG containing immune complexes in our Clq assay, it would be possible and desirable to detect other immunoglobulins or antigens present (Valentijn et al., 1984). A variation of our assay could be the use of microspheres of different sizes, each size coated with a different substrate for detection of immune complexes. In theory, one size microsphere preparation could be coated with Clq or monoclonai antibody to Clq and another size preparation coated with monoclonal antibody to C3d. Once the microspheres have been coated, aliquots of microspheres are mixed together and patient sera added, followed by a fluoresceinated anti-immunoglobulin reagent. Since the FACS analyzer can accurately detect particles based on size, it would be possible to select a known microsphere population and determine the fluorescence without interference from other size microspheres. This approach would allow for the simultaneous detection of immune complexes using two distinct capture techniques. Reckel et al. (1984) have demonstrated the potential for using a monoclonal anti-Clq based ELISA assay to detect immune complexes and monoclonal anti-C3d would mimic the Raji cell assay (Jones et al., 1982). Recent unpublished data from our laboratory has demonstrated the potential for using multiple sized microspheres as substrates for various antigens in a simultaneous flow cytometric detection system for infectious disease serology. The use of microspheres as solid supports in immunological based assays with flow cytometric analysis should allow for the development of assays with high sensitivity, and the potential exists for simultaneous performance of multiple assays using microspheres of different sizes. Furthermore, laboratories using flow cytometry for other determinations can now employ this sophisticated instrument to a variety of serologic assays using the coated microsphere technique we have described.
Acknowledgement The authors thank Charlene Anderson for her excellent preparation of the manuscript.
61
References Bonnefoy, J.H., J. Banchereau, J.P. Aubry and J. Wijdenes, 1986, J. Immunol. Methods 88, 25. Capel, P.J.A., 1974, J. Immunol. Methods 5, 165. Collins, M.M., C.H. Casavant and D.P. Stites, 1982, ft. Clin. Microbiol. 15, 456. Horan, P.K. and L.L. Wheeless, 1977, Science 198, 149. Jolley, M.E., C.H.J. Wang, S.J. Ekenberg, M.S. Zuelke and D.M. Kelso, 1984, J. Immunol. Methods 67, 21. Jones, D.B., N.J. Goulding, C.R. Casey and P.J. GaUagher, 1982, J. Immunol. Methods 53, 201.
Lambert, P.H., F.J. Dixon, R.H. Zubler, V. Agnell, et al., 1978, J. Ciin. Lab. Immunol. 1, 1. Nydegger, U.E., A. Corvetta, P.J. Spaeth and M. Spycher, 1983, Clin. Immuno]. Immunopathol. 26, 56. Reckel, R.P., J. Harris, E. Botsko, R. Wellerson and S. Varga, 1984, Diagn. ImmunoL 2, 228. Theofilopoulos, A.N. and F.J. Dixon, 1980, Hosp. Pract. May, 107. Valentijn, R.M., L.A. Van Es and M.R. Daha, 1984, J. Clin. Lab. ImmunoI. 14, 81.
57
Elsevier JIM 04143
Flow cytometric detection and quantitation of immune complexes using human Clq-coated microspheres Thomas M. McHugh, Daniel P. Stites *, Conrad H. Casavant and Mack J. Fulwyler Department of Laboratory Medicine, University of California at San Francisco, San Francisco, CA 94143, U.S.A. (Received 19 May 1986, accepted 22 July 1986)
A solid phase human Clq-binding fluorescent immunoassay for the measurement of immune complexes in human serum was developed. The solid phase used was 5/~m diameter polystyrene microspheres. Serum immune complexes bound to the Clq-coated microspheres were measured by flow cytometry using fluoresceinated anti-human IgG, and heat-aggregated human IgG as a standard. Patient samples were assayed and results compared to a standard fluoroimmunometric Clq-binding immune complex assay. Greater differences in circulating immune complexes were observed between the healthy control group mean and the mean of the patient values in the microsphere-flow cytometric method than were seen in the standard assay. In the microsphere-flow cytometric assay, the mean patient value was 7.5 times greater than the control mean, whereas in the standard assay the mean patient value was 2.8 times the control mean. Preliminary results suggest greater sensitivity of the microsphere-flow cytometric method over the other method. Key words: Flow cytometry; Immune complex; Immunofluorescence
Introduction
assays vary greatly in sensitivity and predictive value in various diseases. An additional complexity is that various assays, which are based on different principles, detect different types of immune complexes. In this report we describe a new flow cytometric based immune complex assay using Clq-coated microspheres and compare its performance to our standard fluoroimmunoassay (Collins et al., 1982).
Immune complexes can mediate tissue injury by their ability to activate complement after being deposited in various organs. Glomerulonephritis and vasculitis, secondary to bacterial, parasitic and viral infections, or autoimmune diseases have been associated with circulating and tissue-bound immune complexes (Theofilopoulos and Dixon, 1980). The detection and quantitation of immune complexes in serum may contribute to diagnosis and therapeutic monitoring in selected diseases. Many methods exist for the detection of immune complexes (Lambert et al., 1978) and these
Preparation of lgG aggregates
* Address correspondence to: Daniel P. Stites, M.D., Box 0100, Department of Laboratory Medicine, University of California, 3rd and Parnassus Avenues, San Francisco, CA 94143, U.S.A.
Human IgG (Gamastan, Cutter Laboratories, Berkeley, CA) was diluted to a concentration of 10 mg/ml in 0.15 M phosphate-buffered saline (PBS), pH 7.2. The preparation was aggregated by heating for 20 rain at 63°C, and was then centrifuged
Materials and methods
0022-1759/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
58 at 1500 x g for 20 min to remove large insoluble aggregates. The supernatant was adjusted to 1 m g / m l in PBS and then immediately frozen in 2 ml aliquots at - 7 0 ° C until used.
Serum samples Peripheral blood was collected in vacutainer tubes (Becton Dickinson, Rutherford, N J) without anticoagulant. Specimens were allowed to clot for 1 h at 22 ° C. Tubes were centrifuged at 400 x g for 10 min at 4°C. Serum was removed, separated into two aliquots and immediately frozen at - 7 0 ° C . Samples were allowed to thaw at 4°C prior to analysis.
Preparation of solid-phase Cl q Polystyrene microspheres of 5.0 /~m diameter (Dow Chemical Co., Indianapolis, IN) were washed in PBS several times. The microspheres were resuspended in 1 ml PBS, counted on a Coulter model Zf counter (Coulter Electronics, Hialeah, FL) and adjusted to 1 x 106 in a 12 × 75 mm glass tube. Purified human C l q (Center for Blood Research, Boston, MA) was diluted in PBS to a concentration of 50 # g / m l . 0.1 ml of C l q suspension was added to 1 × 106 microspheres and the tube was mixed and incubated for 24 h at 4oc.
Flow cytometric Clq-binding immune complex assay (FACS assay) Assays were carried out in 12 x 75 mm glass tubes. Clq-coated microspheres were washed three times in 0.15 M PBS + 0.5% bovine serum albumin + 0.1% Tween 20, pH 6.4 (PBS-BSA-Tw). Serum samples were heat-inactivated for 30 rain at 56°C. The heat-aggregated IgG was diluted in PBS to concentrations of 10, 25, 50, 100 and 200 /~g/ml. 0.5 ml of zero standard, IgG standards, and sera diluted 1:11, were pipetted into tubes. 1 x 105 Clq-coated microspheres were added to each tube and incubated for 2 h at 22°C. Each tube was washed three times with PBS-BSA-Tw. The final microsphere pellet was resuspended in a 1 : 60 dilution of fluorescein-isothiocyanate (FITC)-conjugated goat F(alY)2 anti-human IgG (Tago, Burlingame, CA) and incubated for 1 h at 22°C. Tubes were washed as above, the pellet was resuspended in PBS and held on ice in the dark
until FACS analysis. BSA (l%)-coated microspheres without C l q were used with each serum specimen as a blank.
FA CS analysis Microspheres were analyzed on a FACS analyzer (Becton Dickinson Immunocytometry Systems, Mountain View, CA) interfaced to a Hewlett-Packard model 9816 computer and printer. Fluorescence excitation and emission wavelengths were selected using optical filters. The excitation filter was a 22 nm bandpass dichroic filter centered at 485 nm. A short-pass dichroic mirror at 505 nm was used to reflect fluorescence emission from FITC. A minimum of 10 000 microspheres were analyzed per tube. Data was acquired and processed using Consort 30 software (Becton Dickinson). Fluorescence profiles were analyzed from singlet microspheres and aggregates of microspheres were eliminated from the analysis by electronic gating using the electronic impedence sizing (Coulter volume) parameter on the FACS analyzer. Fluorescence emission was logarithmically amplified and displayed as a fluorescence profile histogram. The peak modal channel (PMC) of the fluorescence peak was used as the assay measurement unit. PMC was plotted versus/~g/ml of aggregated IgG and results expressed as /~g equivalents of aggregated I g G / m l .
Standard Cl q-binding immune complex assay The fluorescent immune complex assay used was as described by Collins et al. (1982). Briefly, diluted human C l q was bound to polymeric StiQ (IDT, Santa Clara, CA) samplers. Heat-aggregated IgG was used as the standard and serum samples were used at 1:11. Clq-coated and BSA-coated samplers were incubated with samples for 2 h at 22°C. Samplers were washed and FITC-conjugated goat F(ab')2 anti-human IgG (Kallestad Laboratories, Austin, TX) was added and incubated for 45 min at 22°C. Samplers were washed as before and the fluorescence read in a FIAX fluorometer (IDT, Santa Clara, CA). BSA-coated samplers were used as the blank. Fluorescence was reported in fluorescent signal units and was plotted versus /xg/ml aggregated IgG. Results were expressed as #g equivalents of aggregated I g G / m l .
59 TABLE I
Results
NORMAL VALUES FOR IMMUNE COMPLEXES IN SERUM
Fig. 1 shows fluorescence histograms of the heat-aggregated I g G s t a n d a r d curve. P M C is indicated i n each histogram. Sensitivity of the assay is best from 0/~g equivalents to approximately 4 0 / t g equivalents I g G / m l . T a b l e I lists i m m u n e complex assay results from the healthy volunteers. Previous work has shown that sera from healthy individuals can contain up to 25 /~g equivalents I g G / m l w h e n assayed b y our s t a n d a r d i m m u n e complex assay. Values in n o r m a l sera are generally lower in the F A C S assay. T a b l e II lists i m m u n e complex results o n selected p a t i e n t sera d e m o n s t r a t e d to have high or b o r d e r l i n e i m m u n e complex results when tested b y our s t a n d a r d assay. T h e range of n o r m a l values i n the F A C S assay is from 0 to 11/~g e q u i v a l e n t s / ml ( m e a n +_ 2 s t a n d a r d deviations). As c a n be seen, all p a t i e n t sera h a d significantly elevated i m m u n e complexes i n the F A C S assay. I n general, the p a t i e n t values were well above the n o r m a l range i n the F A C S assay.
Serum number
FACS PMC a
/xg eq. b
Standard assay, FSU c
/zg eq.
1 2 3 4 5 6 7 8 9 10
80 65 115 130 109 125 58 62 73 110
4 3 8 9 7 9 2 2 3 7
18 16 26 11 12 19 13 7 8 14
15 13 23 7 8 16 9 3 4 11
.,Y SD
88 38
5 3
14 6
11 6
a Peak modal channel from FACS immune complex assay. b #g equivalents of IgG/ml calculated from standard curve. c Fluorescence signal units from standard fluoroimmunoassay.
Discussion The l a b o r a t o r y detection of i m m u n e complexes is complicated b y the variability i n b i n d i n g of the complexes to the selected substrate. This variabil-
psc:146k P.c-1681 i
PMC:38
[~
170
0
170
0
85
85
255
0
85
170
255
0
85
170
255
i
PMC/~179
255
PMC:194
0
85
A
170
255
k
0
PMC:189
85
_/% 170
255
Fig. 1. Fluorescence profile histograms of standard curve in FACS immune complex assay. A: 0 ttg/ml; B: 10/~g/ml; C: 25 ~g/mi; D: 50 /~g/ml; E: 100 /~g/ml; F: 200/xg/ml. 10000 microspheres were assayed at each concentration. Horizontal axis represents particle fluorescence on a logarithmic scale expressed as FACS channel number. Vertical axis is relative number of events.
60 TABLE II IMMUNE COMPLEX VALUES IN PATIENT SERA Serum number 1 2 3
4
5 6 7 8 9 10 Normal range (£+2 SD)
Disease category
FACS (/,g eq.)
Standard assay (/~g eq.)
Rheumatoid arthritis Rheumatoid arthritis Rheumatoid arthritis with streptococcal glomerulonephritis Systemic lupus
45 36
38 23
50
41
erythrematosus Infectious mononucleosis Leukemia Leukemia AIDS AIDS AIDS
30
28
21 37 33 35 40 45
26 40 34 29 26 30
0-11
0-23
ity prohibits comparison of patient values between methodologies and raises questions as to the significance of a single assay result (Jones et al., 1982; Nydegger et al., 1983). In addition, current assays do not offer detection of various immunoglobulin classes involved in the immune complex or the antigen present in the complex. Microspheres have previously been used as a substrate for immunological based assays (Capel, 1974; Jolley et al., 1984). However, these applications did not use flow cytometry to detect signals generated from the microspheres. Recently, Bonnefoy et al. (1986) demonstrated the use of fluorescent microspheres and flow cytometry in detecting Fc receptors on human leukocytes. In their application microspheres were used as the fluorescent label to amplify the signal detected by flow cytometry. Horan (1977) first described the potential of using a flow cytometric solid-phase immunofluorescent assay with microspheres as the solid phase based upon the original idea by Fulwyler. We have applied the use of microspheres and flow cytometry to the detection of immune complexes. Our initial studies are encouraging in the apparent increase in sensitivity of this
assay over our standard method. While we have demonstrated the detection of IgG containing immune complexes in our Clq assay, it would be possible and desirable to detect other immunoglobulins or antigens present (Valentijn et al., 1984). A variation of our assay could be the use of microspheres of different sizes, each size coated with a different substrate for detection of immune complexes. In theory, one size microsphere preparation could be coated with Clq or monoclonai antibody to Clq and another size preparation coated with monoclonal antibody to C3d. Once the microspheres have been coated, aliquots of microspheres are mixed together and patient sera added, followed by a fluoresceinated anti-immunoglobulin reagent. Since the FACS analyzer can accurately detect particles based on size, it would be possible to select a known microsphere population and determine the fluorescence without interference from other size microspheres. This approach would allow for the simultaneous detection of immune complexes using two distinct capture techniques. Reckel et al. (1984) have demonstrated the potential for using a monoclonal anti-Clq based ELISA assay to detect immune complexes and monoclonal anti-C3d would mimic the Raji cell assay (Jones et al., 1982). Recent unpublished data from our laboratory has demonstrated the potential for using multiple sized microspheres as substrates for various antigens in a simultaneous flow cytometric detection system for infectious disease serology. The use of microspheres as solid supports in immunological based assays with flow cytometric analysis should allow for the development of assays with high sensitivity, and the potential exists for simultaneous performance of multiple assays using microspheres of different sizes. Furthermore, laboratories using flow cytometry for other determinations can now employ this sophisticated instrument to a variety of serologic assays using the coated microsphere technique we have described.
Acknowledgement The authors thank Charlene Anderson for her excellent preparation of the manuscript.
61
References Bonnefoy, J.H., J. Banchereau, J.P. Aubry and J. Wijdenes, 1986, J. Immunol. Methods 88, 25. Capel, P.J.A., 1974, J. Immunol. Methods 5, 165. Collins, M.M., C.H. Casavant and D.P. Stites, 1982, ft. Clin. Microbiol. 15, 456. Horan, P.K. and L.L. Wheeless, 1977, Science 198, 149. Jolley, M.E., C.H.J. Wang, S.J. Ekenberg, M.S. Zuelke and D.M. Kelso, 1984, J. Immunol. Methods 67, 21. Jones, D.B., N.J. Goulding, C.R. Casey and P.J. GaUagher, 1982, J. Immunol. Methods 53, 201.
Lambert, P.H., F.J. Dixon, R.H. Zubler, V. Agnell, et al., 1978, J. Ciin. Lab. Immunol. 1, 1. Nydegger, U.E., A. Corvetta, P.J. Spaeth and M. Spycher, 1983, Clin. Immuno]. Immunopathol. 26, 56. Reckel, R.P., J. Harris, E. Botsko, R. Wellerson and S. Varga, 1984, Diagn. ImmunoL 2, 228. Theofilopoulos, A.N. and F.J. Dixon, 1980, Hosp. Pract. May, 107. Valentijn, R.M., L.A. Van Es and M.R. Daha, 1984, J. Clin. Lab. ImmunoI. 14, 81.