JourMi of Nrurochemistry. 1975. Vol. 24, pp. 34S351. Pcrgamon Press. Printed in Great Britain.
SERUM ‘ANTI-MYELIN ANTIBODIES : A SPECTROFLUOROMETRIC ASSAY PROCEDURE’ K. SUZUKI, M. B. BORNSTEIN and CAROLW. TIFFANY The Saul R. Korey Department of Neurology, and the Rose F. Kennedy Center for Research in Mental Retardation and Human Development, Albert Einstein College of Medicine, Bronx, NY 10461, U.S.A. (Received 12 April 1974. Accepted I 1 July 1974)
Abstract-A simple spectrofluorometric procedure has been devised to determine serum antibodies, directed to constituents of the myelin sheath. It is an adaptation of the indirect immunofluorescent technique. A suspension of highly purified bovine myelin is incubated successively with a test rabbit serum and fluoresceinisothiocyanate-conjugatedanti-rabbit gamma-globulin. Intensity of fluorescence in the final myelin suspension is determined spectrofluorometrically. Sera from rabbits with experimental allergic encephalomyelitis, induced by whole bovine spinal cord, generally gave fluorescence at least 10 times that of normal rabbit serum. Fluorescence of sera with high demyelinating activity was more intense than that of sera with equivocal demyelinating activity. The assay is specific for immunoglobulins directed to myelin constituents, organ-specific and species-independent. Rabbit anti-galactosylceramide serum with known demyelinating activity gave high fluorescence similar to that in sera of rabbits inoculated with whole spinal cord. Galactosylceramide could absorb a substantial portion of ‘anti-myelin antibodies’ of the anti-galactosylceramide serum but it did not absorb ‘anti-myelin antibodies’ of serum of rabbits with whole tissue-induced experimental allergic encephalomyelitis. This assay system may be useful for further studies of ‘antimyelin antibodies’.
I
IT HAS long been known that serum from animals with experimental allergic encephalomyelitis (EAE), induced by intradermal inoculation of whole bovine spinal cord and Freund’s adjuvant, contains circulating antibodies directed to the myelin sheath. They can be demonstrated histologically by the indirect fluorescent antibody technique (SHERWIN et al., 1961 ; APPEL & BORNSTEIN,1964). Serum from animals with EAE also possesses other biological activities. It has the capacity to cause demyelination in myelinated cultures of rat and mouse cerebellum (BORNSTEIN & APPEL, 1961). This demyelinating factor requires complement and is abolished by absorption of EAE serum with brain tissues. EAE serum also causes inhibition of bioelectric activities of cultured mouse cerebellum (BORNSTEIN & CRAIN,1965), and inhibits initiation of myelination by apparently delaying maturation of oli-
’
godendroglial cells (BORNSTEIN & RAINE,1970). This delayed oligodendroglial maturation appears to be reflected also in reduced formation of sulphatide in cultures continuously treated with EAE serum from the time of explantation (FRY et al., 1972). It is well recognized that the antigen responsible for the induction of EAE with characteristic clinical and histological features is the basic protein of the myelin sheath (KIES,1974). While sera of EAE animals inoculated with whole white matter consistently show high demyelinating activities, the demyelinating capacity of sera of EAE animals that received purified basic protein appears to be less consistent and is a subject of controversy (SEILet al., 1968; YONEZAWA et al., 1969). In 1970, DUBOIS-DALCQ et al. reported, on the other hand, that antiserum to galactosylceramide has the capacity to demyelinate CNS cultures. This finding was expanded more recently by FRYet al. (1974) who showed that antiserum to galactosylceramide not only causes demyelination but also inhibits initiation of myelination with reduced sulphatide synthesis. Furthermore, suggestive evidence exists to indicate that inhibition of bioelectric activity is caused by a factor different from the demyelinating factor. All of these findings indicate the highly complex nature of the immunological properties of the serum of animals inoculated with whole white matter, basic protein or galactosylceramide.
This investigation was supported by research grants, 433-D-10 and 834-C-3 from the National Multiple Sclerosis Society, and NS-10885, NS-06735, NS-03356 and HD-01799 from the United States Public Health Service. Presented in part at the 5th Annual Meeting of the American Society for Neurochemistry, New Orleans, La., March l(r14. 1974, and published in an abstract form in the Transaction of the meeting (SUZUKI et al., 1974). Abbreviations used: FITC, fluoresceinisothiocyanate; EAE, experimental allergic encephalomyelitis. 345
346
K. SUZUKI, M. B. BORNSTEIN and CAROLW. TIFFANY
Biochcmical or immunological characterization of thcse biological 'factors' has been slow, due in part to thc bioassay technique by which these biological activitics a r e measured. In general elaborate facilities and specialized techniques are required for CNS culture, thc method is inherently slow, and the results obtained are at best semiquantitative. It would be desirable to devise a simpler and more quantitative procedure that can be used routinely for many test samples in order to further characterize the biological factors in EAE serum. This report describes one such attempt. The procedure appears to be sufficiently simple, quantitative and specific for the purpose for which it is designed.
ASSAY F O R "ANTI-MYELIN ANTIBODY" Tnt &It
1a~m (0.1 ml) +purlfled bovlne mydin (2 mg in 0.1 ml saline)'
I n c h t a at
3P for 60 min
I I
w a h h l c o with saline ond cemtrlfugotlon
R u * p n d myelln In 0.1 ml wllne + 0.05 ml of fluoresceinconlugaid gcai a n t i 4 I t ganmo-globulin ~rnuboteat
3P form mln w&
h l c a wlth :din. and c n M h g a t i a n
S u p m d mydln In 1 ml of O.ZM glycina buffar, pH 9.8. Intntlty of fluorrcenca d e t e n l n d wlth excltotlon rmala&~ 295 nm m d rmlulm wavalmgth 51 7 nm.
MATERIALS AND METHODS Wmk hbm contaln 0.1 ml aallna in place of the tast swum. Randomly-bred rabbits were used for induction of EAE. They received a single challenge of approximately 10 mg of FIG. I. The standard assay procedure for 'anti-myelin antibody'. lyophili7ed bovine spinal cord emulsified in complete t'rcund's adjuvant. injected intradermally in all 4 foot-pads. Animals werc hled 2 weeks after inoculation. Demyelinating activities werc assayed as described previously (BORNSTEIN once without additional saline, and the supernatant & APPLL.1961). In some experiments, stored frozen EAE decanted. The washed myelin pellets were suspended in 0.1 rabbit scra. previously prepared and assayed for demye- ml saline with momentary sonication with a probe-sonicdtor h a t i n g activities, were used. Induction of EAE was essen- at a very low energy setting (Kontes Glass Co., Vineland, tially identical in all instances. NJ). Then, 50 pl of FITC-conjugated anti-rabbit gammaFluoresceinisothiocyanate (F1TC)-conjugated IgG frac- globulin was added, and the tubes were incubated once tions prepared from either sheep or goat anti-rabbit more for 60 min at 37°C with gentle shaking. Myelin was gamma-globulin (or its subfractions) serum were purchased then washed twice with saline by vigorous suspension and from either Miles Labs, Inc., Elkhart, IN, or Cappel Labs, centrifugation as before. The blank tubes were subjected to Inc.. Downingtown, PA. Unlabelled goat anti-rabbit the same washing procedure a s sample tubes. The final myegamma-globulin and rabbit anti-human gamma-globulin lin pellets were suspended in 1.0 ml of 0 2 M-glycine-NaOH werc also products of the Miles Labs. Guinea-pig comple- buffer, p H 9.8. by sonication. Intensity of fluorescence was ment was from Grand Island Biological Co., Grand Island, determined directly on the final myelin suspension in an NY. Complete Freund's adjuvant was purchased from Difco Aminco-Bowman SPF-125 spectrofluororneter (American Labs. Detroit, MI. Galactosylceramide, lecithin and choles- Instrument Co., Silver Spring, MD) at an excitation wavetcrol were from Applied Science Labs, State College, PA. length of 295 nm and a n emission wavelength of 517 nm. Their purity was ascertained by TLC. Bovine and human Sample readings were corrected for those of the blank tubes. myelin fractions were prepared according to the method of Assays were always carried out in duplicate. NORTON& PODUSLO (I973). The staiidard assay systain. Lyophilized bovine myelin RESULTS was weighed and suspended in 150 mM-NaC1 at a conStandardization of the assay procedure. The standard centration of 20 mg/ml. The standard assay procedure for assay procedure described in Methods was adopted thc rabbit serum 'anti-myelin antibody' is shown in Fig. I. The first incubation was carried out with a mixture of 0.1 after a series of experiments to obtain the optimum ml each of the above myelin suspension and test serum in conditions. When the amount of test serum was varied a 15 ml round-bottom Corex centrifuge tube with a rubber in the assay system, there were corresponding changes stopper. Blank tubes were incubated with 0.1 ml of the mye- in the final fluorescence readings. The changes, howlin suspension and 0.1 ml of physiological saline. The tubes ever, were not quite linear against the amount of the were gently shaken for 60 min at 37°C. At the end of incuba- serum (Fig. 2). By practical considerations of obtaining tion, the myelin suspensions were shaken vigorously with a sufficiently intense fluorescence without using excesvortcx stirrer with the addition of a few ml of saline. Saline was further added to approximately 10 ml, and the tubes sive amounts of test serum, 0.1 ml was chosen for the centrifuged at 18,000 g for 10 min in a Sorval high-speed standard system. Maximum fluorescence was obtained ccntrifuge. The supernatant was decanted and the myelin with 2 mg of myelin (Fig. 3). Since increase in the pellets were washed once more with saline. The blank tubes amount of myelin caused increases in the blank reading, 2 mg of myelin was chosen for the standard assays. containing only myelin and saline were centrifuged only
Anti-myelin antibody assay
0
20
50
200
100
Amount of test serum,
347
pl/tube
FIG.2. Effect of the amount of test serum. The ‘anti-myelin antibody’ was assayed according to the standard procedure described in the text except that the volume of the test serum was varied as indicated. Appropriate volumes of saline were added to keep the total volume of all tubes constant. In this experiment, the vohme of the initial incubation with the test serum was 0.3 ml, instead of 0.2 ml, in order to permit assays with up to 0.2 ml of the test serum.
The volume of the fluorescent anti-rabbit immunoglobulin for the standard system is 50 pl, which, however, was insufficient to obtain the maximum fluorescence (Fig. 4). The choice of 50 p1 was dictated by a practical consideration of the expense and by the fact that the relative fluorescence of test samples to the blank tubes did not improve substantially because of increasing blank readings with increasing amounts of the FITCconjugated antibody. Forty-five min were sufficient for each incubation to obtain the maximum fluorescence yield. The final fluorescence was not affected substantially when the ionic strength of the glycine buffer was varied from 12.5 mM to 0.2 M,and the pH from 8.6 to 10.4. There was a slight decline in the fluorescence reading outside of this range. It should be noted, therefore, that the standard assay procedure incorporates several pragmatic compromises that must be kept in
Amount of FIR-conjugated antibody,
mind in its applications. Several attempts were also made to either dissolve the suspended myelin or to quantitatively dissociate the final antigen-antibody complex so that the fluorescence measurement could be made in a clear solution, but they were all unsatisfactory. Characterization of the assay system. The reaction does not require complement. An EAE serum sample was heated at 56°C for 25 min to inactivate endogenous complement and then assayed with or without additional exogenous guinea-pig complement. The heated serum without exogenous complement gave even slightly higher fluorescence than the unheated sample or the heated sample with exogenous complement (Table 1). The final fluorescence of EAE sera could be blocked substantially when the myelin suspension was exposed to unlabelled goat anti-rabbit gamma-globulin after the initial incubation with the test serum but before the second incubation with the FITC-conjugated goat anti-rabbit gamma-globulin (Table 2). Such blockage did not occur when the unlabelled antibody was rabbit TABLEI. EFFECTOF
COMPLEMENT ON ‘ANTI-MYELIN ANTIBODY’ ASSAY
Sample Untreated active serum Heated active serum* Heated active serum* 0.4
I
2
Amount of myelin,
mg /tube
FIG. 3. Effect of the amount of myelin. The ‘anti-myelin antibody’ was assayed according to the standard procedure described in the text except that the amount of myelin was varied as indicated.
pl/tube
FIG.4. Effect of the amount of FITC-conjugated anti-rabbit gamma-globulin. The ‘anti-myelin antibody’ was assayed according to the standard procedure described in the text except that the volume of the FITC-conjugated antibody for the second incubation was varied as indicated.
Exogenous complement
-
+
Fluorescence 1310 1650 1440
* Active EAE rabbit serum was heated at 56°C for 25 min to inactivate the endogenous complement. The heat-inactivated serum was assayed for the ‘anti-myelin antibody’ according to the standard procedure with or without additional 0.05 ml of guinea-pig complement during the initial incubation. Saline, 0.05 ml, was added to other samples instead of the complement.
K. SUZUKI. M. 9. BORNSTEINand CAROLW . TI1 FANY
34x
TABLE 2. BLOCKINGOF ‘ANTI-MYELIN’
FLUORESCENCE
T A B L4.~ MYLLIN OF
DIFFERENT
sri.riEs FOR ‘ANTI-MYELIN
ANTIBODY’ ASSAY
Test serum
Blocking agent
Fluorescence
..
1400
Test serum EAE rabbit EAE rabbit EAE rahbit Normal rabbit Normal rabbit Normal rabbit
~
Anti-rabbit gamma-globulin Anti -hu ma n gamma-globulin -
Anti-rabbit gamma-globulin An ti-human gamma-globulin
500 1410 24
Normal rabbit Normal rabbit EAE rabbit I EAE rabbit 1 EAE rabbit 2 EAE rabbit 2
64
anti-human gamma-globulin. This finding indicated that the fluorescence measured by the standard system is specifically due to rabbit gamma-globulin coupled on to myelin during the initial incubation. When FITC-conjugated antibodies specific toward different rabbit immunoglobulin classes were used for the second incubation, EAE sera gave much higher final fluorescence readings than normal control sera in all instances, suggesting that all immunoglobulin classes are involved in the reaction (Table 3). For practical purposes, however, FITC-conjugated anti-rabbit gamma-globulin or anti-rabbit IgC appears to be the choice because of the high fluorescence yields. The inyclin fraction purified from human cerebral whitc matter could replace bovine myelin in the standard system for assaying the serum ‘anti-myelin antibody’ in rabbits with EAE induced by whole bovine spinal cord (Table 4). This result suggested that the assay procedure measures antibodies specific to components of the myelin sheath but not those specific to
Bovine Human Bovine Human Bovine Human
48 475 575 475
25
.
510
EAE was induced in rabbits with bovine spinal cord and complete Freunds adjuvant. ‘Anti-myelin antibody’ was assayed with the use of both bovine and human myelin. otherwise according to the standard assay procedure described in the text.
bovine species. Absorption experiments were carried out with purified bovine myelin. spinal cord of several mammalian species. and several bovinc organs. Absorption of active EAE serum with either purified inyelin or lyophilized spinal cord tissues prior to the assay procedure resulted in substantial decreases in thc final fluorescence readings while absorption with bovine non-neural tissues generally showed minor reductions in the final fluorescence (Table 5). These 2 experiments supportcd the organ-specific and speciesindependent nature of the ‘anti-myelin antibodies’ as measured by this procedure. The significance of the moderate absorption by bovine splecn is not clear at present. ‘Atiti-my/iti crritihodies’ in rahhit iriiwunc. s m m . Sera of EAE rabbits with known high demyelinating activities consistently gave high values in our assay system while normal control rabbit sera always gave very low values. Generally the intensity of fluorescence of normal serum was less than 10 pcr ccnt of sera with known high demyelinating activities. In order to assess the potential usefulness of the procedure in correlating the fluorescence intensity dnd the demyelinating activity. 2 EAE serum samples with known strong demyelinating
T A B L3.~ IMMUNOGLOBULIN CLASS1.S OF ‘ANTI-MYtLIN
Anti-gamma globulin Anti-gamma globulin Anti-IgC (heavy and light chain) Anti-IgC (hcavy and light chain) Anti-IgC (heavy chain) Anti-IgC (heavy chain) Anti-IgA An ti- IgA Anti-IgM Anti-lgM
Fluorescence
5
After the initial incubation with test sera, the myelin suspensions were treated with unlabelled goat IgG fraction specific to rabbit or human gamma-globulin prior to the incubation with the FITC-conjugated immunoglobulins. This intermediate incubation was carried out with a mixture of 0.1 ml myelin suspension in saline and 0.05 ml unlabelled immunoglobulin at 37°C for 60 min.
FITC-antibody
Myelin source
ANTIBOI>Y’
Test serum
Fluorescence
EA E/Normal
EAE Normal EAE Normal EAE Normal EAE Normal EAE Normal
1310 I05
12.5
878 XI 673 99
I05 0 298 57
10.8 6.X -
5.2
FITC-conjugated goat IgG, specific to different classes of rabbit immunoglobulins, were used for the assay of ‘anti-myelin antibody’ in EAE and normal rabbit sera.
Anti-myelin antibody assay TABLE 5. AILSORPTION OF ‘ANTI-MYELIN ANTIBODY’ BY
TISSUES
349
TABLE 6. CORRELATION OF THE DEMYELINATING FACTOR A N D THE ‘ANTI-MYELIN ANTIBODY’
Pretreatment
Fluorescence
% Change
2100 560 540 720 680 690 630 I780 1360 I990 2000
-
None Bovine myelin Bovine spinal cord Rabbit spinal cord Rat spinal cord Mouse spinal cord Guinea-pig spinal cord Bovine kidney Bovine spleen Bovine heart Bovine testis
Demyelinating activity
Serum
- 73 - 14 - 66 - 68 - 67 - 70 - 15 - 35 -5
Fluorescence
++ ++
EAE rabbit EAE rabbit EAE rabbit EAE rabbit Control rabbit Control rabbit
840 1430 348 568 59 68
k It
~~~
Four EAE and 2 control rabbit sera of known demyeh a t i n g activity were assayed blindly for the ‘anti-myelin antibody’ according to the standard procedure
-5
Ten mg of lyophilized tissues were incubated with 0.4 ml of the test serum at 37’C for 60 min with gentle shaking. The tissues were removed by centrifugation, and the supernatants were assayed for the ‘anti-myelin antibody’ according to the standard procedure as described in the text. activity. 2 EAE sera with equivocal demyelinating activity, a n d 2 normal rabbit sera were subjected to the assay procedure blindly. The results showed an apparent positive correlation between the demyelinating activities and the relative intensity of fluorescence by this procedure (Table 6). In view of the positive demyelinating activity of antiga1actosvlcer;~inidcserum. 2 samples of such scra wcrc subjected to the standard assay procedure. Both samples showed high fluorescence comparable to that obtained with sera with high demyelinating capacity from rabbits inoculated with whole spinal cord (Table 7). The fluorescence of both the EAE and anti-galactosylceramide sera could be absorbed by pretreatment with bovine myelin (Table 8). However, the difference in the nature of the fluorescence derived from the 2 dif-
ferent immune sera became apparent when the sera were pretreated with galactosylceramide adsorbed on cellulose powder. Galactosylceramide could absorb most of the ‘anti-myelin antibody’ in the anti-galactosylceramide sera while essentially n o reduction in the final fluorescence occurred when the EAE serum was pretreated with galactosylceramide.
TABLE 7. ‘ANTI-MYELIN ANTIBODY’
IN RABBIT ANTI-GALACTOSYLCERAMlDE SERUM
Serum
Fluorescence
EAE rabbit serum Normal control rabbit serum Rabbit anti-galactosylceramide serum 1 Rabbit anti-galactosylceramide serum 2
1690 120 1470 1780
Rabbits were inoculated with galactosylceramide according to FRYet ol. (1974).
TABLE 8. ABSORPTIONOF ‘ANTIMYELIN ANTIBODY’ BY GALACTOSYLCERAMIDE Serum EAE rabbit serum
Rabbit anti-galactosylceramide serum
~~~
Pretreatment
Fluorescence
% Change
None Lecithin + cholesterol on cellulose Lecithin + cholesterol + galactosylceramide on cellulose Bovine myelin
1060 1060
-
I020 216
-4 - 74
None Lecithin + cholesterol on cellulose Lecithin + cholesterol + galactosylceramide on cellulose Bovine myelin
1050 I350
f27
316 208
- 70 - 80
0
-
~
The immunoabsorbent was prepared by suspending 100 mg of acid- and base-washed cellulose powder in chloroformmethanol (2:l, v/v) containing 10 mg lecithin, 5 mg cholesterol and 10 mg galactosylceramide. The mixture was vacuumdried. For the control absorbent, galactosylceramide was omitted. Ten mg of the absorbents were mixed with 0.3 ml of test serum and incubated at 37°C for 60 min. Absorption by bovine myelin was done as described for Table 5. After elimination of the absorbents by centrifugation, ‘anti-myelin antibody’ was assayed according to the standard procedure.
350
K. SUZUKI, M. 8. BORNSTEINand CAROL w. TIFFANY
DISCUSSION The present standard assay procedure has several inThe spectrofluorimetric estimation of ‘anti-myelin herent, as well as practical, weaknesses and limitations antibodies’ described in this report is essentially an that must be kept in mind. First, the procedure is adaptation of a long-established histochemical finding. quantitative only in the relative sense. The FITC-conThe presence of myelin-binding antibody in EAE rab- jugated antibody is the IgG fraction of goat or sheep bit serum can be demonstrated by the indirect im- anti-rabbit immunoglobulin. The potency of the antimunofluorescent technique (BEUTNERrt al., 1958; sera and also the ratio of conjugated FITC to protein SHERWIN et al., 1961; RAUCH& RAFFEL,1964; APPEL vary from one preparation to another. Fluorescence in& BORNSTEIN, 1964). However, quantitative estimation tensity of conjugated FITC becomes weaker as the is difficult with the histochemical technique. The relative amount of conjugated protein increases. present procedure takes advantage of the relative Therefore, the values are quantitatively comparable ease with which highly purified CNS myelin can be only within a series of assays in which a single FITCconjugated antibody preparation is used. A satisfacprepared. The ‘anti-myelin antibody’ measured by this pro- tory compromise appears possible in this regard, howcedure appears to be reasonably organ-specific and ever, if a few standard test serum samples are set aside species-independent. These are the properties expected and used to standardize the variations. For each for the circulating antibodies in EAE rabbit serum FITC-conjugated preparation, a correction factor can which are responsible for the various biological activi- be developed so as to bring the reading of the standard ties. For example, APPEL & BORNSTEIN (1964) showed test sera to arbitrary standard values. Another potential limitation inherent in this prothat the demyelinating activity of EAE serum was abolished by absorption with CNS tissues of several cedure is the possible unavailability to antibodies of mammalian species but not with non-neural organs. certain myelin antigens in the intact myelin sheath. While known biological activities of EAE serum are Any anti-myelin antibody directed to a myelin constilikely to be due to humoral antibodies against consti- tuent that is buried in the myelin membrane and is intuents of the myelin sheath, it is not known whether accessible immunologically will not be detected by this each of the biological activities is caused by a specific procedure. As mentioned above, several pragmatic antibody or whether any one antibody is the cause of considerations were also incorporated in choosing the more than one biological activity. It is even conceiv- conditions for the standard assay system. The exciable that different antibodies against different myelin tation wavelength of 295 nm as described above was constituents might exert the same biological effect, also a practical compromise because the spectrosuch as demyelination of CNS cultures. Since the mye- fluorometer in our laboratory is equipped with a merlin sheath contains a number of known and potential cury lamp, which, by its nature, is extremely uneven in antigens, including the basic protein and galactosylcer- light intensity at different wavelengths. This precluded amide, the composition of circulating antibodies in the use of the optimal excitation wavelength for FITC. serum of animals inoculated with whole spinal cord or Also, we had to set both slits for the excitation and even with purified myelin is likely to be highly com- emission beams at the widest opening because of the plex. Therefore, the ‘anti-myelin antibodies’ measured relative insensitivity of the equipment. A more efficient by this assay procedure are expected to be highly het- excitation wavelength and narrower slit widths should erogeneous including different proportions of anti- be possible with a spectrofluorometer equipped with a bodies to different myelin constituents. It should also xenon lamp and a more powerful and stable amplifier be stressed that although there was a reasonably good to increase the sensitivity. Despite these limitations and despite the necessity to correlation between demyelinating activity of a serum sample and fluorescence, a strict one-to-one corre- eventually correlate different ‘anti-myelin antibodies’ spondence has not been established. For example, FRY with biological activities, the spectrofluorometric proet a!. (1974) were able to absorb a substantial portion cedure described here appears sufficiently simple, senof demyelinating activity of EAE serum with galacto- sitive, and quantitative to be used for further explosylcerarnide, but we did not observe a reduction in ration of the immunological properties of sera of anifluorescence after pretreatment of EAE serum with mals inoculated with whole CNS tissue, myelin, myelin galactosylceramide. By virtue of its simple and quanti- basic protein, or galactosylceramide, and also of sera tative nature, the assay procedure should be useful not of human patients with demyelinating disorders. only for assessing the total ‘anti-myelin antibodies’ in test sera, but also for further characterization in which REFERENCES a test serum is fractionated by varieties of biochemical APPEL S. H. & BORNSTEIN M. B. (1964) J . cup. M r d . 119, and immunological means. 303-3 12.
Anti-myelin antibody assay BELlTNliK E. H., WITEBSKY E., ROSE N. R. & GERBASI J. R. (1958) Proc. Soc. rxp. Biol. M r d . 97, 712-716. BORNSTEINM. B. & APPELS. H. (1961) J. Neuropath. exp. N r u i ~ l20, . 141-157. BOKNSTEIN M. B. & CRAINS. M. (1965) Science 148, 12421244.
BORNSTEIN M. B. & RAINEC. S. (1970)Lab. Invest. 23, 5 3 6 542.
DUBOIS-DALCQ M., NIEDIECK B. & BUYSEM. (1970) Pathol. Europ. 5 , 3 3 1-347. FRYJ. M., LEHRER G. M. & BORNSTEIN M. B. (1972) Science 175, 192-194.
FRYJ. M., WEISSBARTH S., LEHRERG. M. & BORNSTEIN M. B. (1974) Science 183,54&542.
351
KIESM. W. (1974) in Biology of Brairi Dysfitnctiori (GALJLL G. E., ed.), Vol. 2, pp. 185-224. Plenum Press, New York. NORTONW. T. & Poous~oS. E. (1973) J . N ~ u r o c h ~ w 21, ~. 749-757.
RALJCH H. C. & RAFFELS. (1964) J . I m m i m l . 92, 452--455.
S~ILF.J..FALKG.A.,KIESM.W.&ALVORDE.C.(~~~~)E Neurol. 22,545-555. SHERWIN A. L.. RICHTERM., COSGROVE J. B. & Rost B. (1961) Science 134, 137O-1372. M. B. & TIFFANY C. W. (1974) Trciris. SUZUKI K., BORNSTEIN Am. SOC.Neurochem. 5, 162. YONEZAWA T., ISHIHARA Y.& SATOY. (1969) J . Neuropurii. exp. Neuro[. 28, 1XO- 1X 1.
SERUM ‘ANTI-MYELIN ANTIBODIES : A SPECTROFLUOROMETRIC ASSAY PROCEDURE’ K. SUZUKI, M. B. BORNSTEIN and CAROLW. TIFFANY The Saul R. Korey Department of Neurology, and the Rose F. Kennedy Center for Research in Mental Retardation and Human Development, Albert Einstein College of Medicine, Bronx, NY 10461, U.S.A. (Received 12 April 1974. Accepted I 1 July 1974)
Abstract-A simple spectrofluorometric procedure has been devised to determine serum antibodies, directed to constituents of the myelin sheath. It is an adaptation of the indirect immunofluorescent technique. A suspension of highly purified bovine myelin is incubated successively with a test rabbit serum and fluoresceinisothiocyanate-conjugatedanti-rabbit gamma-globulin. Intensity of fluorescence in the final myelin suspension is determined spectrofluorometrically. Sera from rabbits with experimental allergic encephalomyelitis, induced by whole bovine spinal cord, generally gave fluorescence at least 10 times that of normal rabbit serum. Fluorescence of sera with high demyelinating activity was more intense than that of sera with equivocal demyelinating activity. The assay is specific for immunoglobulins directed to myelin constituents, organ-specific and species-independent. Rabbit anti-galactosylceramide serum with known demyelinating activity gave high fluorescence similar to that in sera of rabbits inoculated with whole spinal cord. Galactosylceramide could absorb a substantial portion of ‘anti-myelin antibodies’ of the anti-galactosylceramide serum but it did not absorb ‘anti-myelin antibodies’ of serum of rabbits with whole tissue-induced experimental allergic encephalomyelitis. This assay system may be useful for further studies of ‘antimyelin antibodies’.
I
IT HAS long been known that serum from animals with experimental allergic encephalomyelitis (EAE), induced by intradermal inoculation of whole bovine spinal cord and Freund’s adjuvant, contains circulating antibodies directed to the myelin sheath. They can be demonstrated histologically by the indirect fluorescent antibody technique (SHERWIN et al., 1961 ; APPEL & BORNSTEIN,1964). Serum from animals with EAE also possesses other biological activities. It has the capacity to cause demyelination in myelinated cultures of rat and mouse cerebellum (BORNSTEIN & APPEL, 1961). This demyelinating factor requires complement and is abolished by absorption of EAE serum with brain tissues. EAE serum also causes inhibition of bioelectric activities of cultured mouse cerebellum (BORNSTEIN & CRAIN,1965), and inhibits initiation of myelination by apparently delaying maturation of oli-
’
godendroglial cells (BORNSTEIN & RAINE,1970). This delayed oligodendroglial maturation appears to be reflected also in reduced formation of sulphatide in cultures continuously treated with EAE serum from the time of explantation (FRY et al., 1972). It is well recognized that the antigen responsible for the induction of EAE with characteristic clinical and histological features is the basic protein of the myelin sheath (KIES,1974). While sera of EAE animals inoculated with whole white matter consistently show high demyelinating activities, the demyelinating capacity of sera of EAE animals that received purified basic protein appears to be less consistent and is a subject of controversy (SEILet al., 1968; YONEZAWA et al., 1969). In 1970, DUBOIS-DALCQ et al. reported, on the other hand, that antiserum to galactosylceramide has the capacity to demyelinate CNS cultures. This finding was expanded more recently by FRYet al. (1974) who showed that antiserum to galactosylceramide not only causes demyelination but also inhibits initiation of myelination with reduced sulphatide synthesis. Furthermore, suggestive evidence exists to indicate that inhibition of bioelectric activity is caused by a factor different from the demyelinating factor. All of these findings indicate the highly complex nature of the immunological properties of the serum of animals inoculated with whole white matter, basic protein or galactosylceramide.
This investigation was supported by research grants, 433-D-10 and 834-C-3 from the National Multiple Sclerosis Society, and NS-10885, NS-06735, NS-03356 and HD-01799 from the United States Public Health Service. Presented in part at the 5th Annual Meeting of the American Society for Neurochemistry, New Orleans, La., March l(r14. 1974, and published in an abstract form in the Transaction of the meeting (SUZUKI et al., 1974). Abbreviations used: FITC, fluoresceinisothiocyanate; EAE, experimental allergic encephalomyelitis. 345
346
K. SUZUKI, M. B. BORNSTEIN and CAROLW. TIFFANY
Biochcmical or immunological characterization of thcse biological 'factors' has been slow, due in part to thc bioassay technique by which these biological activitics a r e measured. In general elaborate facilities and specialized techniques are required for CNS culture, thc method is inherently slow, and the results obtained are at best semiquantitative. It would be desirable to devise a simpler and more quantitative procedure that can be used routinely for many test samples in order to further characterize the biological factors in EAE serum. This report describes one such attempt. The procedure appears to be sufficiently simple, quantitative and specific for the purpose for which it is designed.
ASSAY F O R "ANTI-MYELIN ANTIBODY" Tnt &It
1a~m (0.1 ml) +purlfled bovlne mydin (2 mg in 0.1 ml saline)'
I n c h t a at
3P for 60 min
I I
w a h h l c o with saline ond cemtrlfugotlon
R u * p n d myelln In 0.1 ml wllne + 0.05 ml of fluoresceinconlugaid gcai a n t i 4 I t ganmo-globulin ~rnuboteat
3P form mln w&
h l c a wlth :din. and c n M h g a t i a n
S u p m d mydln In 1 ml of O.ZM glycina buffar, pH 9.8. Intntlty of fluorrcenca d e t e n l n d wlth excltotlon rmala&~ 295 nm m d rmlulm wavalmgth 51 7 nm.
MATERIALS AND METHODS Wmk hbm contaln 0.1 ml aallna in place of the tast swum. Randomly-bred rabbits were used for induction of EAE. They received a single challenge of approximately 10 mg of FIG. I. The standard assay procedure for 'anti-myelin antibody'. lyophili7ed bovine spinal cord emulsified in complete t'rcund's adjuvant. injected intradermally in all 4 foot-pads. Animals werc hled 2 weeks after inoculation. Demyelinating activities werc assayed as described previously (BORNSTEIN once without additional saline, and the supernatant & APPLL.1961). In some experiments, stored frozen EAE decanted. The washed myelin pellets were suspended in 0.1 rabbit scra. previously prepared and assayed for demye- ml saline with momentary sonication with a probe-sonicdtor h a t i n g activities, were used. Induction of EAE was essen- at a very low energy setting (Kontes Glass Co., Vineland, tially identical in all instances. NJ). Then, 50 pl of FITC-conjugated anti-rabbit gammaFluoresceinisothiocyanate (F1TC)-conjugated IgG frac- globulin was added, and the tubes were incubated once tions prepared from either sheep or goat anti-rabbit more for 60 min at 37°C with gentle shaking. Myelin was gamma-globulin (or its subfractions) serum were purchased then washed twice with saline by vigorous suspension and from either Miles Labs, Inc., Elkhart, IN, or Cappel Labs, centrifugation as before. The blank tubes were subjected to Inc.. Downingtown, PA. Unlabelled goat anti-rabbit the same washing procedure a s sample tubes. The final myegamma-globulin and rabbit anti-human gamma-globulin lin pellets were suspended in 1.0 ml of 0 2 M-glycine-NaOH werc also products of the Miles Labs. Guinea-pig comple- buffer, p H 9.8. by sonication. Intensity of fluorescence was ment was from Grand Island Biological Co., Grand Island, determined directly on the final myelin suspension in an NY. Complete Freund's adjuvant was purchased from Difco Aminco-Bowman SPF-125 spectrofluororneter (American Labs. Detroit, MI. Galactosylceramide, lecithin and choles- Instrument Co., Silver Spring, MD) at an excitation wavetcrol were from Applied Science Labs, State College, PA. length of 295 nm and a n emission wavelength of 517 nm. Their purity was ascertained by TLC. Bovine and human Sample readings were corrected for those of the blank tubes. myelin fractions were prepared according to the method of Assays were always carried out in duplicate. NORTON& PODUSLO (I973). The staiidard assay systain. Lyophilized bovine myelin RESULTS was weighed and suspended in 150 mM-NaC1 at a conStandardization of the assay procedure. The standard centration of 20 mg/ml. The standard assay procedure for assay procedure described in Methods was adopted thc rabbit serum 'anti-myelin antibody' is shown in Fig. I. The first incubation was carried out with a mixture of 0.1 after a series of experiments to obtain the optimum ml each of the above myelin suspension and test serum in conditions. When the amount of test serum was varied a 15 ml round-bottom Corex centrifuge tube with a rubber in the assay system, there were corresponding changes stopper. Blank tubes were incubated with 0.1 ml of the mye- in the final fluorescence readings. The changes, howlin suspension and 0.1 ml of physiological saline. The tubes ever, were not quite linear against the amount of the were gently shaken for 60 min at 37°C. At the end of incuba- serum (Fig. 2). By practical considerations of obtaining tion, the myelin suspensions were shaken vigorously with a sufficiently intense fluorescence without using excesvortcx stirrer with the addition of a few ml of saline. Saline was further added to approximately 10 ml, and the tubes sive amounts of test serum, 0.1 ml was chosen for the centrifuged at 18,000 g for 10 min in a Sorval high-speed standard system. Maximum fluorescence was obtained ccntrifuge. The supernatant was decanted and the myelin with 2 mg of myelin (Fig. 3). Since increase in the pellets were washed once more with saline. The blank tubes amount of myelin caused increases in the blank reading, 2 mg of myelin was chosen for the standard assays. containing only myelin and saline were centrifuged only
Anti-myelin antibody assay
0
20
50
200
100
Amount of test serum,
347
pl/tube
FIG.2. Effect of the amount of test serum. The ‘anti-myelin antibody’ was assayed according to the standard procedure described in the text except that the volume of the test serum was varied as indicated. Appropriate volumes of saline were added to keep the total volume of all tubes constant. In this experiment, the vohme of the initial incubation with the test serum was 0.3 ml, instead of 0.2 ml, in order to permit assays with up to 0.2 ml of the test serum.
The volume of the fluorescent anti-rabbit immunoglobulin for the standard system is 50 pl, which, however, was insufficient to obtain the maximum fluorescence (Fig. 4). The choice of 50 p1 was dictated by a practical consideration of the expense and by the fact that the relative fluorescence of test samples to the blank tubes did not improve substantially because of increasing blank readings with increasing amounts of the FITCconjugated antibody. Forty-five min were sufficient for each incubation to obtain the maximum fluorescence yield. The final fluorescence was not affected substantially when the ionic strength of the glycine buffer was varied from 12.5 mM to 0.2 M,and the pH from 8.6 to 10.4. There was a slight decline in the fluorescence reading outside of this range. It should be noted, therefore, that the standard assay procedure incorporates several pragmatic compromises that must be kept in
Amount of FIR-conjugated antibody,
mind in its applications. Several attempts were also made to either dissolve the suspended myelin or to quantitatively dissociate the final antigen-antibody complex so that the fluorescence measurement could be made in a clear solution, but they were all unsatisfactory. Characterization of the assay system. The reaction does not require complement. An EAE serum sample was heated at 56°C for 25 min to inactivate endogenous complement and then assayed with or without additional exogenous guinea-pig complement. The heated serum without exogenous complement gave even slightly higher fluorescence than the unheated sample or the heated sample with exogenous complement (Table 1). The final fluorescence of EAE sera could be blocked substantially when the myelin suspension was exposed to unlabelled goat anti-rabbit gamma-globulin after the initial incubation with the test serum but before the second incubation with the FITC-conjugated goat anti-rabbit gamma-globulin (Table 2). Such blockage did not occur when the unlabelled antibody was rabbit TABLEI. EFFECTOF
COMPLEMENT ON ‘ANTI-MYELIN ANTIBODY’ ASSAY
Sample Untreated active serum Heated active serum* Heated active serum* 0.4
I
2
Amount of myelin,
mg /tube
FIG. 3. Effect of the amount of myelin. The ‘anti-myelin antibody’ was assayed according to the standard procedure described in the text except that the amount of myelin was varied as indicated.
pl/tube
FIG.4. Effect of the amount of FITC-conjugated anti-rabbit gamma-globulin. The ‘anti-myelin antibody’ was assayed according to the standard procedure described in the text except that the volume of the FITC-conjugated antibody for the second incubation was varied as indicated.
Exogenous complement
-
+
Fluorescence 1310 1650 1440
* Active EAE rabbit serum was heated at 56°C for 25 min to inactivate the endogenous complement. The heat-inactivated serum was assayed for the ‘anti-myelin antibody’ according to the standard procedure with or without additional 0.05 ml of guinea-pig complement during the initial incubation. Saline, 0.05 ml, was added to other samples instead of the complement.
K. SUZUKI. M. 9. BORNSTEINand CAROLW . TI1 FANY
34x
TABLE 2. BLOCKINGOF ‘ANTI-MYELIN’
FLUORESCENCE
T A B L4.~ MYLLIN OF
DIFFERENT
sri.riEs FOR ‘ANTI-MYELIN
ANTIBODY’ ASSAY
Test serum
Blocking agent
Fluorescence
..
1400
Test serum EAE rabbit EAE rabbit EAE rahbit Normal rabbit Normal rabbit Normal rabbit
~
Anti-rabbit gamma-globulin Anti -hu ma n gamma-globulin -
Anti-rabbit gamma-globulin An ti-human gamma-globulin
500 1410 24
Normal rabbit Normal rabbit EAE rabbit I EAE rabbit 1 EAE rabbit 2 EAE rabbit 2
64
anti-human gamma-globulin. This finding indicated that the fluorescence measured by the standard system is specifically due to rabbit gamma-globulin coupled on to myelin during the initial incubation. When FITC-conjugated antibodies specific toward different rabbit immunoglobulin classes were used for the second incubation, EAE sera gave much higher final fluorescence readings than normal control sera in all instances, suggesting that all immunoglobulin classes are involved in the reaction (Table 3). For practical purposes, however, FITC-conjugated anti-rabbit gamma-globulin or anti-rabbit IgC appears to be the choice because of the high fluorescence yields. The inyclin fraction purified from human cerebral whitc matter could replace bovine myelin in the standard system for assaying the serum ‘anti-myelin antibody’ in rabbits with EAE induced by whole bovine spinal cord (Table 4). This result suggested that the assay procedure measures antibodies specific to components of the myelin sheath but not those specific to
Bovine Human Bovine Human Bovine Human
48 475 575 475
25
.
510
EAE was induced in rabbits with bovine spinal cord and complete Freunds adjuvant. ‘Anti-myelin antibody’ was assayed with the use of both bovine and human myelin. otherwise according to the standard assay procedure described in the text.
bovine species. Absorption experiments were carried out with purified bovine myelin. spinal cord of several mammalian species. and several bovinc organs. Absorption of active EAE serum with either purified inyelin or lyophilized spinal cord tissues prior to the assay procedure resulted in substantial decreases in thc final fluorescence readings while absorption with bovine non-neural tissues generally showed minor reductions in the final fluorescence (Table 5). These 2 experiments supportcd the organ-specific and speciesindependent nature of the ‘anti-myelin antibodies’ as measured by this procedure. The significance of the moderate absorption by bovine splecn is not clear at present. ‘Atiti-my/iti crritihodies’ in rahhit iriiwunc. s m m . Sera of EAE rabbits with known high demyelinating activities consistently gave high values in our assay system while normal control rabbit sera always gave very low values. Generally the intensity of fluorescence of normal serum was less than 10 pcr ccnt of sera with known high demyelinating activities. In order to assess the potential usefulness of the procedure in correlating the fluorescence intensity dnd the demyelinating activity. 2 EAE serum samples with known strong demyelinating
T A B L3.~ IMMUNOGLOBULIN CLASS1.S OF ‘ANTI-MYtLIN
Anti-gamma globulin Anti-gamma globulin Anti-IgC (heavy and light chain) Anti-IgC (hcavy and light chain) Anti-IgC (heavy chain) Anti-IgC (heavy chain) Anti-IgA An ti- IgA Anti-IgM Anti-lgM
Fluorescence
5
After the initial incubation with test sera, the myelin suspensions were treated with unlabelled goat IgG fraction specific to rabbit or human gamma-globulin prior to the incubation with the FITC-conjugated immunoglobulins. This intermediate incubation was carried out with a mixture of 0.1 ml myelin suspension in saline and 0.05 ml unlabelled immunoglobulin at 37°C for 60 min.
FITC-antibody
Myelin source
ANTIBOI>Y’
Test serum
Fluorescence
EA E/Normal
EAE Normal EAE Normal EAE Normal EAE Normal EAE Normal
1310 I05
12.5
878 XI 673 99
I05 0 298 57
10.8 6.X -
5.2
FITC-conjugated goat IgG, specific to different classes of rabbit immunoglobulins, were used for the assay of ‘anti-myelin antibody’ in EAE and normal rabbit sera.
Anti-myelin antibody assay TABLE 5. AILSORPTION OF ‘ANTI-MYELIN ANTIBODY’ BY
TISSUES
349
TABLE 6. CORRELATION OF THE DEMYELINATING FACTOR A N D THE ‘ANTI-MYELIN ANTIBODY’
Pretreatment
Fluorescence
% Change
2100 560 540 720 680 690 630 I780 1360 I990 2000
-
None Bovine myelin Bovine spinal cord Rabbit spinal cord Rat spinal cord Mouse spinal cord Guinea-pig spinal cord Bovine kidney Bovine spleen Bovine heart Bovine testis
Demyelinating activity
Serum
- 73 - 14 - 66 - 68 - 67 - 70 - 15 - 35 -5
Fluorescence
++ ++
EAE rabbit EAE rabbit EAE rabbit EAE rabbit Control rabbit Control rabbit
840 1430 348 568 59 68
k It
~~~
Four EAE and 2 control rabbit sera of known demyeh a t i n g activity were assayed blindly for the ‘anti-myelin antibody’ according to the standard procedure
-5
Ten mg of lyophilized tissues were incubated with 0.4 ml of the test serum at 37’C for 60 min with gentle shaking. The tissues were removed by centrifugation, and the supernatants were assayed for the ‘anti-myelin antibody’ according to the standard procedure as described in the text. activity. 2 EAE sera with equivocal demyelinating activity, a n d 2 normal rabbit sera were subjected to the assay procedure blindly. The results showed an apparent positive correlation between the demyelinating activities and the relative intensity of fluorescence by this procedure (Table 6). In view of the positive demyelinating activity of antiga1actosvlcer;~inidcserum. 2 samples of such scra wcrc subjected to the standard assay procedure. Both samples showed high fluorescence comparable to that obtained with sera with high demyelinating capacity from rabbits inoculated with whole spinal cord (Table 7). The fluorescence of both the EAE and anti-galactosylceramide sera could be absorbed by pretreatment with bovine myelin (Table 8). However, the difference in the nature of the fluorescence derived from the 2 dif-
ferent immune sera became apparent when the sera were pretreated with galactosylceramide adsorbed on cellulose powder. Galactosylceramide could absorb most of the ‘anti-myelin antibody’ in the anti-galactosylceramide sera while essentially n o reduction in the final fluorescence occurred when the EAE serum was pretreated with galactosylceramide.
TABLE 7. ‘ANTI-MYELIN ANTIBODY’
IN RABBIT ANTI-GALACTOSYLCERAMlDE SERUM
Serum
Fluorescence
EAE rabbit serum Normal control rabbit serum Rabbit anti-galactosylceramide serum 1 Rabbit anti-galactosylceramide serum 2
1690 120 1470 1780
Rabbits were inoculated with galactosylceramide according to FRYet ol. (1974).
TABLE 8. ABSORPTIONOF ‘ANTIMYELIN ANTIBODY’ BY GALACTOSYLCERAMIDE Serum EAE rabbit serum
Rabbit anti-galactosylceramide serum
~~~
Pretreatment
Fluorescence
% Change
None Lecithin + cholesterol on cellulose Lecithin + cholesterol + galactosylceramide on cellulose Bovine myelin
1060 1060
-
I020 216
-4 - 74
None Lecithin + cholesterol on cellulose Lecithin + cholesterol + galactosylceramide on cellulose Bovine myelin
1050 I350
f27
316 208
- 70 - 80
0
-
~
The immunoabsorbent was prepared by suspending 100 mg of acid- and base-washed cellulose powder in chloroformmethanol (2:l, v/v) containing 10 mg lecithin, 5 mg cholesterol and 10 mg galactosylceramide. The mixture was vacuumdried. For the control absorbent, galactosylceramide was omitted. Ten mg of the absorbents were mixed with 0.3 ml of test serum and incubated at 37°C for 60 min. Absorption by bovine myelin was done as described for Table 5. After elimination of the absorbents by centrifugation, ‘anti-myelin antibody’ was assayed according to the standard procedure.
350
K. SUZUKI, M. 8. BORNSTEINand CAROL w. TIFFANY
DISCUSSION The present standard assay procedure has several inThe spectrofluorimetric estimation of ‘anti-myelin herent, as well as practical, weaknesses and limitations antibodies’ described in this report is essentially an that must be kept in mind. First, the procedure is adaptation of a long-established histochemical finding. quantitative only in the relative sense. The FITC-conThe presence of myelin-binding antibody in EAE rab- jugated antibody is the IgG fraction of goat or sheep bit serum can be demonstrated by the indirect im- anti-rabbit immunoglobulin. The potency of the antimunofluorescent technique (BEUTNERrt al., 1958; sera and also the ratio of conjugated FITC to protein SHERWIN et al., 1961; RAUCH& RAFFEL,1964; APPEL vary from one preparation to another. Fluorescence in& BORNSTEIN, 1964). However, quantitative estimation tensity of conjugated FITC becomes weaker as the is difficult with the histochemical technique. The relative amount of conjugated protein increases. present procedure takes advantage of the relative Therefore, the values are quantitatively comparable ease with which highly purified CNS myelin can be only within a series of assays in which a single FITCconjugated antibody preparation is used. A satisfacprepared. The ‘anti-myelin antibody’ measured by this pro- tory compromise appears possible in this regard, howcedure appears to be reasonably organ-specific and ever, if a few standard test serum samples are set aside species-independent. These are the properties expected and used to standardize the variations. For each for the circulating antibodies in EAE rabbit serum FITC-conjugated preparation, a correction factor can which are responsible for the various biological activi- be developed so as to bring the reading of the standard ties. For example, APPEL & BORNSTEIN (1964) showed test sera to arbitrary standard values. Another potential limitation inherent in this prothat the demyelinating activity of EAE serum was abolished by absorption with CNS tissues of several cedure is the possible unavailability to antibodies of mammalian species but not with non-neural organs. certain myelin antigens in the intact myelin sheath. While known biological activities of EAE serum are Any anti-myelin antibody directed to a myelin constilikely to be due to humoral antibodies against consti- tuent that is buried in the myelin membrane and is intuents of the myelin sheath, it is not known whether accessible immunologically will not be detected by this each of the biological activities is caused by a specific procedure. As mentioned above, several pragmatic antibody or whether any one antibody is the cause of considerations were also incorporated in choosing the more than one biological activity. It is even conceiv- conditions for the standard assay system. The exciable that different antibodies against different myelin tation wavelength of 295 nm as described above was constituents might exert the same biological effect, also a practical compromise because the spectrosuch as demyelination of CNS cultures. Since the mye- fluorometer in our laboratory is equipped with a merlin sheath contains a number of known and potential cury lamp, which, by its nature, is extremely uneven in antigens, including the basic protein and galactosylcer- light intensity at different wavelengths. This precluded amide, the composition of circulating antibodies in the use of the optimal excitation wavelength for FITC. serum of animals inoculated with whole spinal cord or Also, we had to set both slits for the excitation and even with purified myelin is likely to be highly com- emission beams at the widest opening because of the plex. Therefore, the ‘anti-myelin antibodies’ measured relative insensitivity of the equipment. A more efficient by this assay procedure are expected to be highly het- excitation wavelength and narrower slit widths should erogeneous including different proportions of anti- be possible with a spectrofluorometer equipped with a bodies to different myelin constituents. It should also xenon lamp and a more powerful and stable amplifier be stressed that although there was a reasonably good to increase the sensitivity. Despite these limitations and despite the necessity to correlation between demyelinating activity of a serum sample and fluorescence, a strict one-to-one corre- eventually correlate different ‘anti-myelin antibodies’ spondence has not been established. For example, FRY with biological activities, the spectrofluorometric proet a!. (1974) were able to absorb a substantial portion cedure described here appears sufficiently simple, senof demyelinating activity of EAE serum with galacto- sitive, and quantitative to be used for further explosylcerarnide, but we did not observe a reduction in ration of the immunological properties of sera of anifluorescence after pretreatment of EAE serum with mals inoculated with whole CNS tissue, myelin, myelin galactosylceramide. By virtue of its simple and quanti- basic protein, or galactosylceramide, and also of sera tative nature, the assay procedure should be useful not of human patients with demyelinating disorders. only for assessing the total ‘anti-myelin antibodies’ in test sera, but also for further characterization in which REFERENCES a test serum is fractionated by varieties of biochemical APPEL S. H. & BORNSTEIN M. B. (1964) J . cup. M r d . 119, and immunological means. 303-3 12.
Anti-myelin antibody assay BELlTNliK E. H., WITEBSKY E., ROSE N. R. & GERBASI J. R. (1958) Proc. Soc. rxp. Biol. M r d . 97, 712-716. BORNSTEINM. B. & APPELS. H. (1961) J. Neuropath. exp. N r u i ~ l20, . 141-157. BOKNSTEIN M. B. & CRAINS. M. (1965) Science 148, 12421244.
BORNSTEIN M. B. & RAINEC. S. (1970)Lab. Invest. 23, 5 3 6 542.
DUBOIS-DALCQ M., NIEDIECK B. & BUYSEM. (1970) Pathol. Europ. 5 , 3 3 1-347. FRYJ. M., LEHRER G. M. & BORNSTEIN M. B. (1972) Science 175, 192-194.
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KIESM. W. (1974) in Biology of Brairi Dysfitnctiori (GALJLL G. E., ed.), Vol. 2, pp. 185-224. Plenum Press, New York. NORTONW. T. & Poous~oS. E. (1973) J . N ~ u r o c h ~ w 21, ~. 749-757.
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S~ILF.J..FALKG.A.,KIESM.W.&ALVORDE.C.(~~~~)E Neurol. 22,545-555. SHERWIN A. L.. RICHTERM., COSGROVE J. B. & Rost B. (1961) Science 134, 137O-1372. M. B. & TIFFANY C. W. (1974) Trciris. SUZUKI K., BORNSTEIN Am. SOC.Neurochem. 5, 162. YONEZAWA T., ISHIHARA Y.& SATOY. (1969) J . Neuropurii. exp. Neuro[. 28, 1XO- 1X 1.
