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A new assay for lytic anti-galactocerebroside (GC) antibodies employing 56rubidium release from GC-labelled liposomes
Journal of Immunological Methods, 39 (1980) 31--38 © Elsevier/North-Holland Biomedical Press
31
A NEW ASSAY FOR LYTIC ANTI-GALACTOCEREBROSIDE (GC) ANTIBODIES EMPLOYING 56RUBIDIUM RELEASE FROM GC-LABELLED LIPOSOMES
D.I. SLOVICK, T. SAIDA, R.P. LISAK and A. SCHREIBER Department of Medicine, The Middlesex Hospital, London, U.K. ; Department of Neurology and Section on Hematology-Oncology, Department of Medicine, School of Medicine, University of Pennsylvania, and University of Pennsylvania-Wistar Institute Multiple Sclerosis Research Center, Philadelphia, PA, U.SA.
(Received 31 March 1980, accepted 24 July 1980) This assay is a quick and sensitive method for determination of antibodies to galactocerebroside (GC). The use of antibody-plus-complement radioisotope release from GCbearing liposomes allows numerous samples to be handled in semi-automated fashion. By substituting alternative gangliosides or cerebrosides, the technique may be simply modified for assay of antibodies to other substances.
INTRODUCTION Biomembrane lipids, especially cerebrosides and gangliosides, have recently been under scrutiny as possible target antigens in demyelination. Complement fixation, agglutination and radioimmunoassay methods for detection of antibodies against such pure substances all suffer from uncertainty due to random micelle formation and altered stereochemical conformation resulting from the aqueous environment of such. assays. Insertion of such glycolipids into the artificial membrane of a liposome provides a closer approximation to in vivo orientation. Liposomes have been used to investigate cross-reactivity of antibodies to glycolipids (Alving and Richards, 1977), transfer of haptens on to the membranes of lymphocytes (Ozato and Henney, 1978) and complement-mediated attack via glycolipid haptens (Kinsky et al., 1969). We present here a sensitive assay for detection of lytic antibodies to galactocerebroside based on complement-mediated release of rubidium isotope from the aqueous c o m p a r t m e n t of galactocerebroside-containing liposomes. A simple filtration step is used to separate released radioisotope from residual liposomes, a considerable simplification of previously used liposome lysis systems employing release of glucose (Haxby et al., 1968), fluor/quencher combinations (Geiger and Smolarsky, 1977) or isotopes (Shin et al., 1977). This m e t h o d of assay facilitates the routine handling of large numbers of samples for a n t i b o d y determination.
32 MATERIALS
A n tisera New Zealand albino rabbits were immunized according to the schedule of Saida et al. (1979a) and absorption of sera was performed according to Niedieck (1965). Control sera included pre-immunization bleeds and bleeds from rabbits immunized with Freund's adjuvant or albumin plus adjuvant. Complement Blood was obtained from a group AB human donor and after defibrination and separation stored in aliquots at --70°C. Lyophilized guinea pig (Baltimore Biological Laboratories, Cockeysville, MD) and rabbit sera (GIBO, Grand Island, NY) were obtained commercially and reconstituted before use. Lipids Dipalmitoyl lecithin (C16:0) and dicetyl phosphate were purchased from Calbiochem (La Jolla, CA). Cholesterol and bovine brain galactocerebroside were obtained from Sigma Chemical C o m p a n y (St. Louis, MO). Chromatographic demonstration of purity of the batch of galactocerebroside used has been previously described (Saida et al., 1979b). Radioisotopes [ 3H ] Cholesterol ( [ 7-3H ] cholesterol, 12 Ci/mmole) in benzene and 86rubidium (0.5--10 Ci/g in HCI) were purchased from New England Nuclear (Boston, MA). Detection was with Aquasil scintillation cocktail (NEN) and a Beckman 8000 spectrometer. All samples were standardized for 3H quenching. Channel overlap of 3H and 86Rb was minimal. Buffer A standard veronal-buffered saline (VBS) containing 0.15 mM Ca 2÷ and 0.1 mM Mg 2÷ was used throughout. Filter Millipore cellulose filters (0.22 pm pore size) were supported on stainless steel mesh in the Millipore filter holder (Cat. No. x x l 0 025 30, Bedford, MA). Filters were pre-wetted in VBS. A vacuum was applied to allow collection of the filtrate from 1 ml in 30 sec. METHODS
Liposome preparation This was performed according to H a x b y et al. (1968). Homogeneous lipid films, dried on to the lower 1.5 cm of 1.2 cm × 0.75 cm glass tubes, were composed of lecithin, cholesterol and dicetyl phosphate in the molar ratio
33 2 : 1.5 : 0.2 with 150 pg galactocerebroside/t~mole of lipid. When required, [3H]cholesterol was added as a lipid phase marker, using 2.5 #Ci/mg of lipid. The small tubes were placed inside the larger cylindrical tube of a rotary vacuum evaporator. The films were resuspended in 200 pl of VBS, containing 50--100 gCi of S6Rb. A b o u t 20 0.2 mm glass beads were added, and the tubes vortexed at r o o m temperature for 5 min. After standing for 15 min, the liposomes were diluted to approximately 2 ml with VBS. The liposomes were separated from free S6Rb by passing them through a 30 cm X 1 cm Sepharose 6B column (Pharmacia, Piscataway, NJ). Elution was with VBS. Pretreatment of the column with unlabelled liposomes (0.5 mg lipid) reduced non-specific loss of subsequently applied liposomes. The labelled liposomes were collected in 1 ml fractions; tubes comprising the peak were pooled, and the preparation was stored at 4°C until use. Stability was usually good up to 2 weeks. The batch was discarded if, after filtration, 86Rb release exceeded 10% of maximal release (see below). R u b i d i u m release assay
A dilution of liposomes was performed to give convenient numbers of cpm in a 20 pl aliquot, e.g., 50,000 cpm. 20 pl of diluted antiserum, 20 ~1 of liposome suspension and 20 pl of diluted complement were added in that order to a glass tube, covered, and incubated at 37°C for 30 min. VBS was then added to bring the volume to 1 ml, and the mixture applied to a filter. The filtrate was collected under vacuum, and a 100 pl aliquot added to the scintillation cocktail and counted. The total released counts were calculated by multiplying observed cpm by 10. 'Maximal release' was obtained b y adding 20 pl of liposome suspension, 80 pl of VBS to scintillation fluid, and counting. Controls included liposomes alone, with complement only, and with antiserum only. Results were calculated as follows: 'percent release' of S6Rb = 100 x
cpm of sample containing antiserum + complement cpm of maximal release sample
'specific percent release' of S6Rb = 100
X
cpm sample with antiserum + C -- cpm complement control maximal release cpm -- complement control cpm
Complement was used at a dilution giving a compromise o p t i m u m of high 86Rb release with antiserum, and low non-specific release w i t h o u t it. For comparative purposes, titration end points were taken at 50% of plateau rubidium release.
34 TABLE 1 A comparison of l i p o s o m e loss/disruption on filtration through various membranes. Filter Total applied counts
Total filtrate counts
% Counts lost through filter
3H
86Rb
3H
S6Rb
3H
86Rb
Nucleopore 0.44 p m
18080
16630
12910
13120
71.4
78.8
Millipore 3.0 p m 0.45 p m 0.22 p m
14 6 0 0 22110 33 788
19 320 20110 22 945
4 990 2700 850
6 990 3550 2 620
34.2 12.2 2.5
36.2 17.6 10.3
RESULTS
Efficacy of liposome filtration The efficiency of membrane filtration of labelled liposomes was assessed using a dilution of 20 pl of liposome suspension up to 1.1 ml. 0.1 ml of this was counted for S6Rb and 3H. The remaining 1 ml was applied to the filter, and 0.1 ml of filtrate was similarly counted. It was thus possible to calculate the loss of lipid ([3H]cholesterol) and aqueous (S6Rb) phase isotopic markers through the membrane. Various types and grades of filter material were compared (Table 1). 'In-depth' filters were found to be superior to 'surface'-type filter membranes. This may be attributed to deformation of liposomes through a relatively short-length pore. The optimal pore size of 'in
60 50
86Rb % Specific Releose
0
40 30
0
0
0
o
20
o o
I0
o
0 1/40 1/80 1/160 1/320 1/640 1/2560 1/10,000 1/17'80 1/5100
Dilution
of Robblt Anti Goleictocerebroside
Serum
Fig. 1. Titration of a rabbit anti-galactocerebroside antiserum, using h u m a n c o m p l e m e n t to p r o m o t e l i p o s o m e lysis. Each point represents the mean of duplicate determinations. Results with p r e - i m m u n i z a t i o n serum from the same rabbit are indicated (e).
35 60 50 40
86Rb % 30 Specific Rele0se
20
0 11:5o 1150
1/150
1/600 1/2000 0 1/500 111000
Dilution of Sere
Fig. 2. Titrations liposomes, using CFA (©); pooled plement controls
of various control sera in the standard lytic assay against GC-bearing human complement: rabbit, immunized with bovine serum albumin in normal rabbit sera (A); and 3 non-immunized rabbit sera (D, m, ~). Comare included. All points represent the means of duplicates.
larger volumes of VBS were used to 'wash through' the filters. In our hands, best results were obtained with 0.22 #m 'Millipore' membranes. Where possible, TF (guaranteed Triton-free) grades were used. Pre-wetting in VBS was always carried out.
Antibody plus complement-mediated liposome lysis All sera with activity against galactocerebroside gave plateaus on titration (Fig. 1). Specificity of 86Rb release was confirmed by testing the effects of control sera: pre-immunization bleeds, pooled normal rabbit sera, adjuvant only or bovine serum albumin plus adjuvant-immunized rabbit sera all gave specific S6Rb release of background level only (Fig. 2). The absorption of anti-galactocerebroside serum with galactocerebroside resulted in abolition of specific S6Rb release (Fig. 3). 5O
86Rb % Specific Rele0se
30 20 I0
o
0
i
1/30
i
i
J
J
II150 I1500 1/1500 1/5000 1/6000
Dilution of Rabbit Anfi-Oaioctocerebroside Serum Fig. 3. Lysis of GC-bearing liposomes using rabbit anti-galactocerebroside serum and human complement before ( I ) and after (o) absorption of antiserum with G C .
36 TABLE 2 A comparison of efficacy of a6Rb release from GC-bearing liposomes of guinea pig, human and rabbit complement; rabbit anti- GC serum ( 1/20) was used throughout. Complement source
Dilution
Test mixture
% of 86Rb max. release
Calculated % 85Rb specific release
Guinea pig complement
1/4 1/4 1/8 1/8 1/12 1/12 1/16 1/16
liposomes liposomes liposomes liposomes liposomes liposomes liposomes liposomes
26.4 54.6 16.7 59.1 15.4 61.4 14.7 59.5
38
1/4 1/4 1/8 1/8 1/12 1/12 1/16 1/16
liposomes liposomes liposomes liposomes liposomes liposomes liposomes liposomes
21.6 45.6 18.4 46.5 15.9 41.2 14.7 59.5
30
1/4 1/4 1/8 1/8 1/12 1/12 1/16 1/16
liposomes liposomes liposomes liposomes liposomes liposomes liposomes liposomes
20.1 56.6 21.0 72.9 16.5 66.6 15.2 63.2
46
Rabbit complement
Human complement
+ antiserum + antiserum + antiserum + antiserum + antiserum + antiserum + antiserum + antiserum + antiserum + antiserum + antiserum + antiserum + antiserum
51 54 53
34 30 32
66 60 57
Efficacy of different complement sources Considerable differences were found between the abilities of human, rabbit and guinea pig sera to cause rubidium release from liposomes at the same dilution and with the same rabbit anti-galactocerebroside serum. Freshly frozen human serum was slightly more effective than lyophilized guinea pig serum, and considerably more effective than rabbit serum ( T a b l e 2).
Demonstration of cross-reactivity causing rubidium release The relative degree of cross-reactivity of sera directed against mono- and digalactosyldiglyceride, and a serum raised to 'mixed-gangliosides' was t e s t e d . T h e r e s u l t s a r e s h o w n in F i g . 4. T h e d e c r e a s i n g o r d e r o f c r o s s - r e a c t i o n o f a n t i - m o n o > a n t i - d i g a l a c t o d i g l y c e r i d e > ' m i x e d - g a n g l i o s i d e s ' as s h o w n b y 8 6 R b - r e l e a s e c o n f i r m s t h e f i n d i n g s o f A l v i n g e t al. ( 1 9 7 4 ) .
37
15 86Rb Specific Releose
I0
5
0
I/9
L
I
1/30
1/150
I
J
I
1/300 1/600 1/1500
Dilution of Antiserum
Fig. 4. Titration of rabbit antisera to monogalaetosyl diglyeeride (o), digalactosyl diglyceride (©) and mixed gangliosides (A).
DISCUSSION
The use of liposomes allows an approximation to in vivo orientation of glycolipids inserted into their membranes. Such preparations are appropriate for studying antibodies capable of causing complement mediated lysis, and measurement of the release of liposome-entrapped 86Rb forms a convenient assay for such antibody activity. However, till now, no simple method has been available for separation of released isotope from whole unlysed liposomes; the m e t h o d of Shin et al. (1977) requires separate chromatographic separation for each sample. We report here that simple filtration under controlled conditions provides good separation, enabling large numbers of assays to be run simultaneously and harvested quickly. Reproducibility between samples has been shown to be well within 10%. With the composition of liposomes employed here, the slow rate of deterioration of a new batch of liposomes allows it to be used for approximately 2 weeks before background 86Rb release becomes t o o high. It is necessary to emphasize that other types of liposomes are much more fragile, exhibit a short half-life and may n o t tolerate filtration. The use of double labelling, i.e., of lipid and aqueous parts of liposomes, although n o t essential, gives a useful check on the initial preparation of liposomes, and on the subsequent filtration step. It is important to use a determined dilution of liposomes and then titrate the complement to achieve a range of 86Rb release, from low background to high antibody mediated release with a k n o w n positive serum. The different activities of various sources of complement m a y reflect differing sensitivities to an anti-complementary c o m p o n e n t in rabbit antisera. Alternatively, it m a y reflect an optimal activity on the part of human complement components in penetrating an artificial membrane of the characteristics used here, containing C16:0 chain length lecithin. The relative sus-
38 ceptibilities of liposomes to complement-mediated attack are discussed by Shin et al. (1977). The observations of Alving et al. (1974) on the relative degree of crossreaction seen with antisera against related glycolipids to galactocerebroside were confirmed. This p h e n o m e n o n can apparently be varied by altering the lecithin chain length of the 'carrier' liposome employed. This may n o t be simply a question of increased hapten 'exposure' to antibody but may reflect altered ability of fixed complement to bring about liposomal lysis, as discussed above. A further possibility is of different degrees of alternativepathway activation. The ease with which liposome may be prepared allows rapid incorporation of a wide range of glycolipid haptens. The assay described will allow further investigation of lyric antibodies from h u m a n and experimental animal diseases to be undertaken simply and on a large scale. ACKNOWLEDGEMENTS D.I.S. acknowledges the encouragement and assistance shown on his visit to University of Pennsylvania, especially by Ms. M. Manning and Drs. A. Levinson, D. Pleasure, D. Silberberg and B. Zweiman. The work was supported by the Ethel ReiUy F u n d , Middlesex Hospital Medical School, L o n d o n , USPHS NS-11034 and National Multiple Sclerosis Society (U.S.) RG 894-B-2. REFERENCES Alving, C.R. and R.L. Richards, 1977, Immunochemistry 14, 383. Alving, C.R., J.W. Fowble and K.C. Joseph, 1974, Immunochemistry 11,475. Geiger, B. and M. Smolarsky, 1977, J. Immunol. Methods 17, 7. Haxby, J.A., C.B. Kinsky and S.C. Kinsky, 1968, Proc. Natl. Acad. Sci. U.S.A. 61,300. Kinsky, S.C., J.A. Haxby, D.A. Zopf, C.R. Alving and C.B. Kinsky, 1969, Biochemistry 8, 4149. Niedieck, B., 1965, Prog. Allergy 18, 253. Ozato, K. and C.S. Henney, 1978, J. Immunol. 121, 2405. Saida, K., T. Saida, M.J. Brown and D.H. Silberberg, 1979a, Am. J. Pathol. 95, 99. Saida, T., K. Saida, S. Dorfman, D.H. Silberberg, A.S. Sumner, M.C. Manning, R.P. Lisak and M.J. Brown, 1979b, Science 204, 1103. Shin, M.L., W.A. Paznekas, A.S. Abramovitz and M.M. Mayer, 1977, J. Immunol. 119, 1358.
31
A NEW ASSAY FOR LYTIC ANTI-GALACTOCEREBROSIDE (GC) ANTIBODIES EMPLOYING 56RUBIDIUM RELEASE FROM GC-LABELLED LIPOSOMES
D.I. SLOVICK, T. SAIDA, R.P. LISAK and A. SCHREIBER Department of Medicine, The Middlesex Hospital, London, U.K. ; Department of Neurology and Section on Hematology-Oncology, Department of Medicine, School of Medicine, University of Pennsylvania, and University of Pennsylvania-Wistar Institute Multiple Sclerosis Research Center, Philadelphia, PA, U.SA.
(Received 31 March 1980, accepted 24 July 1980) This assay is a quick and sensitive method for determination of antibodies to galactocerebroside (GC). The use of antibody-plus-complement radioisotope release from GCbearing liposomes allows numerous samples to be handled in semi-automated fashion. By substituting alternative gangliosides or cerebrosides, the technique may be simply modified for assay of antibodies to other substances.
INTRODUCTION Biomembrane lipids, especially cerebrosides and gangliosides, have recently been under scrutiny as possible target antigens in demyelination. Complement fixation, agglutination and radioimmunoassay methods for detection of antibodies against such pure substances all suffer from uncertainty due to random micelle formation and altered stereochemical conformation resulting from the aqueous environment of such. assays. Insertion of such glycolipids into the artificial membrane of a liposome provides a closer approximation to in vivo orientation. Liposomes have been used to investigate cross-reactivity of antibodies to glycolipids (Alving and Richards, 1977), transfer of haptens on to the membranes of lymphocytes (Ozato and Henney, 1978) and complement-mediated attack via glycolipid haptens (Kinsky et al., 1969). We present here a sensitive assay for detection of lytic antibodies to galactocerebroside based on complement-mediated release of rubidium isotope from the aqueous c o m p a r t m e n t of galactocerebroside-containing liposomes. A simple filtration step is used to separate released radioisotope from residual liposomes, a considerable simplification of previously used liposome lysis systems employing release of glucose (Haxby et al., 1968), fluor/quencher combinations (Geiger and Smolarsky, 1977) or isotopes (Shin et al., 1977). This m e t h o d of assay facilitates the routine handling of large numbers of samples for a n t i b o d y determination.
32 MATERIALS
A n tisera New Zealand albino rabbits were immunized according to the schedule of Saida et al. (1979a) and absorption of sera was performed according to Niedieck (1965). Control sera included pre-immunization bleeds and bleeds from rabbits immunized with Freund's adjuvant or albumin plus adjuvant. Complement Blood was obtained from a group AB human donor and after defibrination and separation stored in aliquots at --70°C. Lyophilized guinea pig (Baltimore Biological Laboratories, Cockeysville, MD) and rabbit sera (GIBO, Grand Island, NY) were obtained commercially and reconstituted before use. Lipids Dipalmitoyl lecithin (C16:0) and dicetyl phosphate were purchased from Calbiochem (La Jolla, CA). Cholesterol and bovine brain galactocerebroside were obtained from Sigma Chemical C o m p a n y (St. Louis, MO). Chromatographic demonstration of purity of the batch of galactocerebroside used has been previously described (Saida et al., 1979b). Radioisotopes [ 3H ] Cholesterol ( [ 7-3H ] cholesterol, 12 Ci/mmole) in benzene and 86rubidium (0.5--10 Ci/g in HCI) were purchased from New England Nuclear (Boston, MA). Detection was with Aquasil scintillation cocktail (NEN) and a Beckman 8000 spectrometer. All samples were standardized for 3H quenching. Channel overlap of 3H and 86Rb was minimal. Buffer A standard veronal-buffered saline (VBS) containing 0.15 mM Ca 2÷ and 0.1 mM Mg 2÷ was used throughout. Filter Millipore cellulose filters (0.22 pm pore size) were supported on stainless steel mesh in the Millipore filter holder (Cat. No. x x l 0 025 30, Bedford, MA). Filters were pre-wetted in VBS. A vacuum was applied to allow collection of the filtrate from 1 ml in 30 sec. METHODS
Liposome preparation This was performed according to H a x b y et al. (1968). Homogeneous lipid films, dried on to the lower 1.5 cm of 1.2 cm × 0.75 cm glass tubes, were composed of lecithin, cholesterol and dicetyl phosphate in the molar ratio
33 2 : 1.5 : 0.2 with 150 pg galactocerebroside/t~mole of lipid. When required, [3H]cholesterol was added as a lipid phase marker, using 2.5 #Ci/mg of lipid. The small tubes were placed inside the larger cylindrical tube of a rotary vacuum evaporator. The films were resuspended in 200 pl of VBS, containing 50--100 gCi of S6Rb. A b o u t 20 0.2 mm glass beads were added, and the tubes vortexed at r o o m temperature for 5 min. After standing for 15 min, the liposomes were diluted to approximately 2 ml with VBS. The liposomes were separated from free S6Rb by passing them through a 30 cm X 1 cm Sepharose 6B column (Pharmacia, Piscataway, NJ). Elution was with VBS. Pretreatment of the column with unlabelled liposomes (0.5 mg lipid) reduced non-specific loss of subsequently applied liposomes. The labelled liposomes were collected in 1 ml fractions; tubes comprising the peak were pooled, and the preparation was stored at 4°C until use. Stability was usually good up to 2 weeks. The batch was discarded if, after filtration, 86Rb release exceeded 10% of maximal release (see below). R u b i d i u m release assay
A dilution of liposomes was performed to give convenient numbers of cpm in a 20 pl aliquot, e.g., 50,000 cpm. 20 pl of diluted antiserum, 20 ~1 of liposome suspension and 20 pl of diluted complement were added in that order to a glass tube, covered, and incubated at 37°C for 30 min. VBS was then added to bring the volume to 1 ml, and the mixture applied to a filter. The filtrate was collected under vacuum, and a 100 pl aliquot added to the scintillation cocktail and counted. The total released counts were calculated by multiplying observed cpm by 10. 'Maximal release' was obtained b y adding 20 pl of liposome suspension, 80 pl of VBS to scintillation fluid, and counting. Controls included liposomes alone, with complement only, and with antiserum only. Results were calculated as follows: 'percent release' of S6Rb = 100 x
cpm of sample containing antiserum + complement cpm of maximal release sample
'specific percent release' of S6Rb = 100
X
cpm sample with antiserum + C -- cpm complement control maximal release cpm -- complement control cpm
Complement was used at a dilution giving a compromise o p t i m u m of high 86Rb release with antiserum, and low non-specific release w i t h o u t it. For comparative purposes, titration end points were taken at 50% of plateau rubidium release.
34 TABLE 1 A comparison of l i p o s o m e loss/disruption on filtration through various membranes. Filter Total applied counts
Total filtrate counts
% Counts lost through filter
3H
86Rb
3H
S6Rb
3H
86Rb
Nucleopore 0.44 p m
18080
16630
12910
13120
71.4
78.8
Millipore 3.0 p m 0.45 p m 0.22 p m
14 6 0 0 22110 33 788
19 320 20110 22 945
4 990 2700 850
6 990 3550 2 620
34.2 12.2 2.5
36.2 17.6 10.3
RESULTS
Efficacy of liposome filtration The efficiency of membrane filtration of labelled liposomes was assessed using a dilution of 20 pl of liposome suspension up to 1.1 ml. 0.1 ml of this was counted for S6Rb and 3H. The remaining 1 ml was applied to the filter, and 0.1 ml of filtrate was similarly counted. It was thus possible to calculate the loss of lipid ([3H]cholesterol) and aqueous (S6Rb) phase isotopic markers through the membrane. Various types and grades of filter material were compared (Table 1). 'In-depth' filters were found to be superior to 'surface'-type filter membranes. This may be attributed to deformation of liposomes through a relatively short-length pore. The optimal pore size of 'in
60 50
86Rb % Specific Releose
0
40 30
0
0
0
o
20
o o
I0
o
0 1/40 1/80 1/160 1/320 1/640 1/2560 1/10,000 1/17'80 1/5100
Dilution
of Robblt Anti Goleictocerebroside
Serum
Fig. 1. Titration of a rabbit anti-galactocerebroside antiserum, using h u m a n c o m p l e m e n t to p r o m o t e l i p o s o m e lysis. Each point represents the mean of duplicate determinations. Results with p r e - i m m u n i z a t i o n serum from the same rabbit are indicated (e).
35 60 50 40
86Rb % 30 Specific Rele0se
20
0 11:5o 1150
1/150
1/600 1/2000 0 1/500 111000
Dilution of Sere
Fig. 2. Titrations liposomes, using CFA (©); pooled plement controls
of various control sera in the standard lytic assay against GC-bearing human complement: rabbit, immunized with bovine serum albumin in normal rabbit sera (A); and 3 non-immunized rabbit sera (D, m, ~). Comare included. All points represent the means of duplicates.
larger volumes of VBS were used to 'wash through' the filters. In our hands, best results were obtained with 0.22 #m 'Millipore' membranes. Where possible, TF (guaranteed Triton-free) grades were used. Pre-wetting in VBS was always carried out.
Antibody plus complement-mediated liposome lysis All sera with activity against galactocerebroside gave plateaus on titration (Fig. 1). Specificity of 86Rb release was confirmed by testing the effects of control sera: pre-immunization bleeds, pooled normal rabbit sera, adjuvant only or bovine serum albumin plus adjuvant-immunized rabbit sera all gave specific S6Rb release of background level only (Fig. 2). The absorption of anti-galactocerebroside serum with galactocerebroside resulted in abolition of specific S6Rb release (Fig. 3). 5O
86Rb % Specific Rele0se
30 20 I0
o
0
i
1/30
i
i
J
J
II150 I1500 1/1500 1/5000 1/6000
Dilution of Rabbit Anfi-Oaioctocerebroside Serum Fig. 3. Lysis of GC-bearing liposomes using rabbit anti-galactocerebroside serum and human complement before ( I ) and after (o) absorption of antiserum with G C .
36 TABLE 2 A comparison of efficacy of a6Rb release from GC-bearing liposomes of guinea pig, human and rabbit complement; rabbit anti- GC serum ( 1/20) was used throughout. Complement source
Dilution
Test mixture
% of 86Rb max. release
Calculated % 85Rb specific release
Guinea pig complement
1/4 1/4 1/8 1/8 1/12 1/12 1/16 1/16
liposomes liposomes liposomes liposomes liposomes liposomes liposomes liposomes
26.4 54.6 16.7 59.1 15.4 61.4 14.7 59.5
38
1/4 1/4 1/8 1/8 1/12 1/12 1/16 1/16
liposomes liposomes liposomes liposomes liposomes liposomes liposomes liposomes
21.6 45.6 18.4 46.5 15.9 41.2 14.7 59.5
30
1/4 1/4 1/8 1/8 1/12 1/12 1/16 1/16
liposomes liposomes liposomes liposomes liposomes liposomes liposomes liposomes
20.1 56.6 21.0 72.9 16.5 66.6 15.2 63.2
46
Rabbit complement
Human complement
+ antiserum + antiserum + antiserum + antiserum + antiserum + antiserum + antiserum + antiserum + antiserum + antiserum + antiserum + antiserum + antiserum
51 54 53
34 30 32
66 60 57
Efficacy of different complement sources Considerable differences were found between the abilities of human, rabbit and guinea pig sera to cause rubidium release from liposomes at the same dilution and with the same rabbit anti-galactocerebroside serum. Freshly frozen human serum was slightly more effective than lyophilized guinea pig serum, and considerably more effective than rabbit serum ( T a b l e 2).
Demonstration of cross-reactivity causing rubidium release The relative degree of cross-reactivity of sera directed against mono- and digalactosyldiglyceride, and a serum raised to 'mixed-gangliosides' was t e s t e d . T h e r e s u l t s a r e s h o w n in F i g . 4. T h e d e c r e a s i n g o r d e r o f c r o s s - r e a c t i o n o f a n t i - m o n o > a n t i - d i g a l a c t o d i g l y c e r i d e > ' m i x e d - g a n g l i o s i d e s ' as s h o w n b y 8 6 R b - r e l e a s e c o n f i r m s t h e f i n d i n g s o f A l v i n g e t al. ( 1 9 7 4 ) .
37
15 86Rb Specific Releose
I0
5
0
I/9
L
I
1/30
1/150
I
J
I
1/300 1/600 1/1500
Dilution of Antiserum
Fig. 4. Titration of rabbit antisera to monogalaetosyl diglyeeride (o), digalactosyl diglyceride (©) and mixed gangliosides (A).
DISCUSSION
The use of liposomes allows an approximation to in vivo orientation of glycolipids inserted into their membranes. Such preparations are appropriate for studying antibodies capable of causing complement mediated lysis, and measurement of the release of liposome-entrapped 86Rb forms a convenient assay for such antibody activity. However, till now, no simple method has been available for separation of released isotope from whole unlysed liposomes; the m e t h o d of Shin et al. (1977) requires separate chromatographic separation for each sample. We report here that simple filtration under controlled conditions provides good separation, enabling large numbers of assays to be run simultaneously and harvested quickly. Reproducibility between samples has been shown to be well within 10%. With the composition of liposomes employed here, the slow rate of deterioration of a new batch of liposomes allows it to be used for approximately 2 weeks before background 86Rb release becomes t o o high. It is necessary to emphasize that other types of liposomes are much more fragile, exhibit a short half-life and may n o t tolerate filtration. The use of double labelling, i.e., of lipid and aqueous parts of liposomes, although n o t essential, gives a useful check on the initial preparation of liposomes, and on the subsequent filtration step. It is important to use a determined dilution of liposomes and then titrate the complement to achieve a range of 86Rb release, from low background to high antibody mediated release with a k n o w n positive serum. The different activities of various sources of complement m a y reflect differing sensitivities to an anti-complementary c o m p o n e n t in rabbit antisera. Alternatively, it m a y reflect an optimal activity on the part of human complement components in penetrating an artificial membrane of the characteristics used here, containing C16:0 chain length lecithin. The relative sus-
38 ceptibilities of liposomes to complement-mediated attack are discussed by Shin et al. (1977). The observations of Alving et al. (1974) on the relative degree of crossreaction seen with antisera against related glycolipids to galactocerebroside were confirmed. This p h e n o m e n o n can apparently be varied by altering the lecithin chain length of the 'carrier' liposome employed. This may n o t be simply a question of increased hapten 'exposure' to antibody but may reflect altered ability of fixed complement to bring about liposomal lysis, as discussed above. A further possibility is of different degrees of alternativepathway activation. The ease with which liposome may be prepared allows rapid incorporation of a wide range of glycolipid haptens. The assay described will allow further investigation of lyric antibodies from h u m a n and experimental animal diseases to be undertaken simply and on a large scale. ACKNOWLEDGEMENTS D.I.S. acknowledges the encouragement and assistance shown on his visit to University of Pennsylvania, especially by Ms. M. Manning and Drs. A. Levinson, D. Pleasure, D. Silberberg and B. Zweiman. The work was supported by the Ethel ReiUy F u n d , Middlesex Hospital Medical School, L o n d o n , USPHS NS-11034 and National Multiple Sclerosis Society (U.S.) RG 894-B-2. REFERENCES Alving, C.R. and R.L. Richards, 1977, Immunochemistry 14, 383. Alving, C.R., J.W. Fowble and K.C. Joseph, 1974, Immunochemistry 11,475. Geiger, B. and M. Smolarsky, 1977, J. Immunol. Methods 17, 7. Haxby, J.A., C.B. Kinsky and S.C. Kinsky, 1968, Proc. Natl. Acad. Sci. U.S.A. 61,300. Kinsky, S.C., J.A. Haxby, D.A. Zopf, C.R. Alving and C.B. Kinsky, 1969, Biochemistry 8, 4149. Niedieck, B., 1965, Prog. Allergy 18, 253. Ozato, K. and C.S. Henney, 1978, J. Immunol. 121, 2405. Saida, K., T. Saida, M.J. Brown and D.H. Silberberg, 1979a, Am. J. Pathol. 95, 99. Saida, T., K. Saida, S. Dorfman, D.H. Silberberg, A.S. Sumner, M.C. Manning, R.P. Lisak and M.J. Brown, 1979b, Science 204, 1103. Shin, M.L., W.A. Paznekas, A.S. Abramovitz and M.M. Mayer, 1977, J. Immunol. 119, 1358.