Radioimmunoassay for detection of anti-oligodendrocyte antibodies

/vk,uresdence Letters, 23 (DSI) 143-t48 © Eisevier/No]~h-HoI|and Scientific Publishers Ltd.

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gAD~O~gMUNOASSAY F O R DETECTgON O F ANTI-OUGODENDROC~rg'E

ANT~DDIES*

ABDOLMOHAMMAD ROSTAMI**, DA~ ,-a E. PLEASURE, ROBERT P. LISAK, DONALD H. SILBERBERG, ODED ABRAMSKY and S. MICHAEL PHILLIPS

Department of Neurology and (S.M.P.) Section oj Allergy-lmmunology, Department of Medwme and Multiple Sclerosis Research, Center of the Umversity of Pennsylvania - W~starInstitute, Phdadelph~a, PA (U.S.A.) (Received October 17th, I980; Accepted January 30th, 1981)

A solid phase radioimmunoassay (RIA) for d e ~ f i o n and quantitadon of rabbit anu-ohgodendrocyte antibody has been developed using bovine oliurdendroglia preparation. The assay ~s simple, rap~d, reproducible ~nd economical. It is appro×imateIy 150 × as sensitive as immunofluorescencc. Spec~ficuy has been established by using different bovine tissue antigens as substrate and absorption s:udies. "-h~ assay represents a potentially powerful to31 for ~he detect;on and quanfitat~on of ant~-ofigod~ndroghal antibodies and oligodendroglial amigens in serum and CSF of man and experimenta~ animals.

And-oligodendroglial antibodies have been demonstrated in rabbitsimmuw_zed with bulk-isolated otigodendrogliaI preparations by immunofluorescence (IF) and complement fixation studies |2, 3, 7, 10]. hlcreased bi~ding to oIigodendrog!ial ce~h by serum of padems with multiple sclerosis (MS), subacute sc[erosing panencephalitis (SSPE) and acute disseminated encephalomyelitis (ADE~) has been demonstrated by some |t], bm not alI groups [9, Ill. Since these ceils are known to produce mye|in in the central nervous system (CNS), the significarr~ce of antibody against them is of potential clinical concern. However, work in thi~ area has been hampered because of the relative insensitivity of the immunofluore,~cence

and complement fixation techniques. In order to overcome problems of specificity and sensitivity, we have devcic.ped a sensitive and specific sofid phase radioimmunoassay (RIA) to detect antibodies to oligodendroglia. It is anticipated that this methc~,;Ioiogy will permit the st~.~dy of oligodendroglia membrane components in mature and deveioping brains and ¢aable precise quantitative studies of kinetics and specificity. *Pre~nted in part at the 3!st Annua! Meeting of The American Academy of Neurology, Chicago, April, 1979. **To whom correspondence and reprint requests shoul~ be addressed at: Departmem ot ~.cw ~;.og~'~ Hospital of the University of Pennsylvania, 3400 Spruce Street, PhiIadetp~a~ PA i9~34, U.~;.A

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Preparation of oiigodendrogtial cells. Oligodendroglial cells were isolated according to the method of Poduslo and Norton with modifications as previously described [6, 8]. White matter was aseptica'fly prepared in phosphate buffer saline (FB$) ~pH 6.0) media containing 5070 glucose, 5~/a fru~ose, bovine serum albumin (BSAL penicillin, streptomycin and amphoteficin B. The crude white matter was in:ubated at 37°C in 0.25a70 trypsin for 75 rain. Following the addition of calf serum to inactivate the trypsin, it was washed and sieved successive!y through nylon monofilament and stainless steel screens. The resultant cell suspension was layered on a 0.9 M, 1.4 M/1.55 M discontinuous sucrose gradient and centrifuged at 36 × g for 10 rain. The oligodendroglial cell layer, localized at the 1.55 M sucrose interface was collected, washed and lyophifized. Ant#oligodendroglial antiserum preparation [2~ 7]. A total of 5 × 1 ~ lyophilized oligodendroglial cells were suspended in 0.25 ml of PBS (pH 7.4), emulsified in 0.75 ml of complete Freund's adjuvant (CFA), and injected into the foot pads of male albino rabbits weighing 2.5-3 kg. The animals were boosted one month later with identical injections and subsequently were injected intraperitoneally at monthly intervals with the oligodendroglial cells in PBS. Sera were obtained via ear bleed~ two weeks after each booster and stored at - 7 0 ° C until use. Radioimmunaassay [12]. An aqueous suspension of lyophilized oligodendrogliocytes was prepared. Ten #1 of this suspension conzaining the equivale; t of 2 x l0 s cells was placed in each well of a polyvinyl microtiter plate (Dynatech Laboratories, Alexandria, VA). Following drying at 37°C, the wells were incubated with 10 #1 of 1°70 BSA in PBS for 30 rain and washed with the same solution. Then, 10 #1 of the test serum was added. Following incubation at 37°C for o0 rain the plates were washed with PBS 5 times and 10 ~1 of the second antibody, [~2~l]ccnjugated goat anti-rabbit IgG ~Cappel Laboratories), was added and incubated for an additional 90 rain at 37°C. The iodinated anti-lgG was prepared using chloramine-T and established methodology [4]. The wells were then washed thoroughly with PBS. The plates were dried at room temperature and individual wells were assessed for the ame~at of bound radioactivity utilizing a Packard Auto-Gamma counter (Fig. I). SensitP,ity of the assay. Anti-oligodendroglia antisera (RAP) (both primary and secondary responses) from one of several rabbits were compared with the preimmunization serum from the same animal (Fig. 2). Approximately a 4-fold increase in absolute binding activity with R a P during the primary response and an ~-fold increase in the secondary response was demonstrated in this rabbit. Significant differences between immune and normal sera was detectable at dilutions of up to 1/128,000. The standard deviations were very small. The sensitivity was approximately 15C times greater than that demonstrated on the identical sera when indirect immunofluorescence was used (U80). In other sensitized rabbits, titers ranged from I:3200 to 1:6400 by RIA. In these sera the RIA was 125 times as ~nsitive. D~lutions of 1/400 and 1/800 were optimal for the demonstration of differences between RaO antisera and normal sera for the number of

t45

R!A

0LIGODENDROCYTES

MICROTITERPLATE DRY ADD BSA WASH ADD TEST SERUM WASH

ADD125I- GOATANTI-RABBITIGG ~I

WASH

GAMMACOUNTER Fig. 1. Experimental deslgnsfor the radioimmunoassay (RIA), details m the text.

I000

2" Response

200 L.)

|o Response NRS

'

a/400

I/

h0t)

;

I/ 600

I;3200 . . . . . 1/6400 . . . . . .1/12BO0

D,luhon of Anhsera

Fig. 2. Sensitivity of the RIA in detecting anti-oligodendroglial antibodies. Significant differences between normal and immune sera can be seen at dilations of up to 1/12,800 for this rabbit. Abc~ssa ~s log cpm binding of [nSI]goat-anu-rabbit lgG to the rabbit anti-oligodendroc} te antibody-oligode~drocyte complex. The CPM were not corrected for background binding. NRS is normal rabbit serum.

otigodendroglia used in RIA (t × 105 !yophilized cells/wel~). These dih~tions were, therefore, employed in subsequent experiments to demonstrate speciFcity (Fig. 2). Specificity. To demonstrate the specificity of the RIA, the binding of RaO andsera to oligodendroglia preparation was compared wi~.h binding against minced and lyophi|ized bovine liver powder and bovine serum albumin (Fig. 3). There were

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significant differences between the binding of RaO to bovine oligodendroglia when compared to bovine liver and BSA (P < 0.001 in all instances). To further evaluate the specificity, absorption studies were performed. Bovine myelin [5], liver, oligodendrocytes, or white matter powders ~,ere hic~ubated with the antisera (20°70 v/v); first at 37°C for 1 i~ and then a~ 4°C overnight. The absorbed sera were filtered through a 0.22 MiUipore membrane to remove the antigenantibody complexes and then tested in the RIA. The anti-oligodendroglial activiz, was significantly reduced by pre-incubadon of the serum with oligodendroglia or bovine white matter but not by pre-incubation with purified myelin (Fig. 4). In addition, pre-incubadon of cells with normal rabbit serum did not affect the binding of RaO to the oligodendreglia, suggesting that binding was not due to Fc receptor activity. I000 EO0 "m'l~

=.~

80hgo

=====================

200

B Liver

100 .........................

O. ¢.)

"~

. . . .

,~oo

B SA

i ....

,/8oo Ddut~on of Anhsero

Fig. 3. Specificity of the RIA, demonst-ated by using different bovine tissues as substrate. Bovine oligodendrocytes show the highest binding capacity to the anti-oligodendrocyte serum. P < 0.00].

4000~ None B Myehn

7 ~

~ ~"DDUHI0m=J~SHI.Imal00 'q00H||ie~HaU0OBDnW,I|H~Ih~

a. 20001 . . . . . . . . . . . . . . . . . . . . . .

.~

B While Motter B Ohgodendrocvte

I000

0

1/400 U800 Dduhon ~f Adsorbed Antlsero

Fig. 4. Specificity of the RIA, demonstrated by absorption studies. Absorption of the antisera with oligodendrocytes and white matter eontaimng oligodendrocytes results in a marked decrease in the binding activity of the antisera to the oligodendrocyte. Absorption with myelin does not decrease this activity to a significant degree.

147 TABLE I R E P R O D U C I B I L I T Y O F R I A FOR A N T I - O L i G O D E N D R O G L I A A N T I B O D I E S Antiserum dilutions o f 1/400 and 2 x IGs oligodendroeytes per well were used. lmr ~, e sera are taken after the second booster. Pre-immunization serum C P M binding

Post-immunization serum C P M binding

Rabbit 1 Experiment 1 Experiment 2 Experiment 3

95 _+ 7.3 a 93 +_ 7.3 107 _+ 4.2

783 ___45 698 ___44.3 733 _+ 49.5

Rabbit 2 Experiment I Experiment 2 Experiment 3

168 + 8.2 113 +_.4.6 102 + 4.9

1819 __+_93.9 1385 + 55.4 1176 _+ 8.1

aMean o f 4 replicates _+.S.D.

Reproducibility. Reproducibility of the RIA proved to be excellent7 Similar results were oi:,tained when sera were tested repeat.edly using the same radiola~'elled reagent. It can be clearly seen from Table 1 that the results obtained on different days, some of them months apart, were very consistent. These studies establish the feasibility of measuring anti-oligodendroglial activity through the use of an RIA. The sensitivity :ff this RIA is e~tremely high compared to other techniques currently used for detection of ~ nti-oligodendroglia! antibody such as immunofluorescence Gr complement fixation. Therefore, the detection and quantification of minute amounts of such anti-o~igodendroglial a~tibodies in experimental and human diseases, will be much easier. The specificity of anti-oligodendroglial serum, whi:h has beer., shown previously [3, 10], was also found in this assay. The marked reduction in anti-oligodendrocyte activity by absorption with oUgodendrocytes suggests that much of the antibody activity of anti-oligodendroglial serum is dir~ted against oligodendrocytes. The residual antibody activity seen after absorption with oligodendrocyte could be due to the fact that the oligodendrocytes in BSA are used for anti-oligodendroglial serum production and, hence, some anti-BSA activity is present in antioligodendroglial serum. Since BSA was also used for coating the microtiter plate wells to decrease non-specific and anti-bovine hir~ding, *.hebinding of anti-3SA with a small amouxxt of BSA in wells could be responsible for increased background activity. Addition~ly, experimental antisera may have other non-specific antibodies related to some small amount of polyclonal activation. Absorption of the antioligodendroglial serum with white matter b,at not with purified bovine myeI~n sigrfificantly reduced the anti-oligodendrc~cyte activity. This may be due to the fact that the antigen responsible for anti-oligodevdrocyte activity is not present in the

148

purified myelin, may be hidden in it, or exists in amounts below the level demonstrable by this technique. Since we have used lyophillized oiigodendrocytes, the antigenic sites exposed to anti-oligodendroglial serum activity are likely composed of both membrane and cytoplasmic components. However, since the RIA readings correlated well with the result of IF done on the several rabbit sera tested, the antigens detected by the RIA appears to be primarily membrane components. Finally, it is hoped that by preparing a variety of oligoder.clroc:¢e component antigens, the hierarchy of the antigenicity of the oligodendrocyte components can be studied using competitive techniques. To further assess this question, we are currently also developing a technique for attaching live oligodendrocytes with intact membranes to the plastic wells for the assay. The RIA uses very small amounts oi the reagents and is very simple and rapid. Thus, it is economical and a large number of samples can be tested in a short time. Reproducibility was excellent and the endpoints not subjective, a defect of the immunofluorescence technique. Possessing these characteristics, the RIA promises t:~ be a powerful tool in the assessment of antibody to and antigens from oligodendroglia cells. Supported by Grant USPHS-NS-01137-07 and the National Multiple Sclerosis Society. 1 Abramsky, O., Lisak, R.P., Silberberg, D.H. and Pleasure, D., Antibodies to oligodendroglia in pa~mnts with multiple sclerosis, N. Engi. J. Med., 297 (1977) 1207-1211. 2 Abramsky, O., Lisak, R.P., Pleasure, D.E., Gilden, D.H. and Silberberg, D.H., Immunologic characteriza~lon of oligodendroglia, Neurosci. Lett. 8 (1978) 311-316. 3 Ahramsky, O., Lisak, R.P., bilberberg, D.H., Brenner, T. and Pleasure, D., Immune response to isolated oligodendrocytes, J. neurol. Sci., 43 (1979) 157-167. 4 Garvey, J.S., Cremer, N.E. and Sussdorf, D.H., Methods in Immunology, 3rd edn., W.A. Benjamin, Inc., 1977, pp. 178-181. 5 Norton, W.T. and Poduslo, S.E., Myelmation in rat brains: method of myelin isolation, J. Neurochem., 21 (1973) 749-757. 6 Pleasure, D.E., Abramsky, O., Silberberg, D., Quinn, B., Parris, J. and Saida, T., Lipid synthesis by an oligodendroglial fraction in suspension culture, Brain Res., 134 (1977) 377-382. 7 Poduslo, S.E., McFarland, H.F. and McKhann, G.M., Antiserum to neurons and to oligodendroglia from mammalian brain, Science, 197 (1977) 270-272. 8 Poduslo, S.E. and Norton, W.T., Isolation and some chemical properties of oligodendroglia from calf brains, J. Neurochem., 19 (1972) 727-736. 9 Steck, A.J. and Regli, F., Oligodendrocyte-bJnding antibodies in multiple sclerosis. 125I-protein A studies, Neurology (Minneap.), 30 (1980) 540-542. 10 Traugott, U., Snyder, D.S., Norton, W.E. and Raine, C.S., Characterization of anti-oligodendrocyte serum.. Arm. Neurol., 4 0978) 431-439. 11 Traugott, U., Snyder, D.S. and Raine, C.S., Non-specific staining of oligodendrocytes by multiple sclerosis and control sera, J. Neuropaah. exp. Neurol., 38 (1979) 345. 12 Wide, L., Solid phase antigen-antibody systems. In K.E. Kirkham and W.M. Hunter (Eds.), Radioimmunoassay Methods, Churchill Livingstone, I970, pp. 405-443.