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Isolation and characterization of rabbit antibodies to bovine myelin basic protein
ISOLATION
AND CHARACTERIZATION OF RABBIT TO BOVINE MYELIN BASIC PROTEIN* A. DESJARDINS Departments
WALLACE.
of Biochemistry Atlanta.
R. SHAPIRA
and Microbiology, GA 30322. U.S.A.
ANTIBODIES
and R. B. FRITZ Emor)
University.
Abstract---Injection of a wide variety of animal species with homologous or heterologous myelin basic protein (BP) emulsified with complete Freund’s adjuvant (CFA) produces the autoimmune demyehnnting disease, experimental allergic encephalomyelitis (EAE). As a first step in the study of antigenic determinants of bovine BP (BBP). specific antibodies were purified from rabbit serum b\i adsorption on SepharoseeBBP atfinity columns and elution with 0.1 ,V acetic acid. Yields of 0.0233.50 mg protein per milliliter of serum were obtained from the acetic acid eluates. lmmunoelectrophoresis and polyacrylamide gel clectrophoresis indicated that the acid eluted material was I&G. The acid eluted IgG formed stable precipitin bands with BBP in agarose gels and also demonstrated anti-BBP activit) in a sodium sulfate precipitation radioimmunoassay and in a solid phase radioimmunoassay. Quantitative precipitation of purified antibodies was used to estimate the number of antigenic determinants in the BBP molecule. From a plot of the antibody to antigen ratio of precipitates vs the log of antigen in the valence of approximately four was indicated. precipitates at antibody excess, an effective antigenic
MATERIALS
INTRODUCTIO’V
Basic protein (BP) of central nervous system myelin was first purified and characterized by Kies and coworkers (1969). Although its functions irl rice remain unknown, it is clear that myelin BP is the major antigen responsible for the demyelinating disease, experimental allergic encephalomyelitis (EAE) (RobozEinstein ef ul., 1962). Physico-chemical studies of myelin BP have shown little secondary folding of the molecule in aqueous solution. Sedimentation equilibrium studies indicate a frictional ratio of IO:1 (Eylar & Thompson, 1969), and circular dichroism and optical rotatory dispersion argue for a complete lack of alpha-helix or betapleated sheet structure (Palmer & Dawson. 1969). Recent studies on small angle X-ray scattering conflict and suggest either a highly asymmetric prolate ellipsoidal conformation (Epand et LII.. 1974) or an open coil (Krigbaum SC Hsu, 1975) in aqueous solution. The lack of significant conformation implies that antigenie determinants of BP are formed by short linear sequences of amino acids, and that the determinants may be delineated by studies of BP fragments. Furthermore, since the biological and immunological activities of basic proteins isolated from different species may vary greatly while the primary structure of these proteins varies to a lesser extent, it should be possible to identify critical amino acid residues required for biological and immunological function. As a first step in these studies, we have determined the conditions necessary for the preparation of specific anti-BBP antibodies by affinity chromatography. These antibodies have been characterized and used to determine the effective antigenic valence of bovine myelin BP. * Research was supported by Public Health grants NS 08278, NS 11418 and NS 10721 from the National lnstitutc of Neurological and Communicative Disorders and Stroke. 17
AND
METHODS
Four- to five-month-old random bred female New Zcaland white rabbits were obtained from a local supplier
(Hiram Davies. Stockbridge. GA. U.S.A.) and immunized with a single footpad injection. Each rabbit rcccived 600 /~g bovine BP (BBP) and 6 mg of !21!,c,oh~lcf~,~il~~~l hrrr!nc~~m (CFA) (Sigma Chemical Co.. St. Louis. MO. U.S.A.) in a O.l-ml emulsion. Ammals were bled from the ear artery before immunization and after onset of EAE. Scra were centrifuged and stored frozen. Onset of EAE approximately 3 weeks after injection was indicated by tail Ilaccldity followed by hind leg weakness. incontinence. and. in some cases, severe paralysis and death. Day of onset and severity of clinical symptoms varied from animal to animal. EAE was confirmed by histological studies.
BBP was purilied by the procedure of Humlenn! 01 trl. (1974). Bovine cord or brain. l2Og. was homoge&ed in 3520 ml of cold butanol:acetic acid:watcr (4: I :2) in a I-gal Waring bl:nder. The solution was filtered to rcmobc debris and IOXOml of water were added with stirring. The two layers were separated bq centrifugation at 4000 ret min for I hr at 2 C. The aqueous solution wah conccntratcd to lOOmI m an Amicon DC2 dlalyrer concentrator with a HIDP IO cartridge. washed with 500 ml of 0.1 ,21 acetic acid. and lyophiliaed. The crude protein. approx I g, was purified by preparative electrophoresis on IS”,, polyacrylamide gels in 0.5 M acetic acid followed by chromntography on carboxymethylccllulose (Whatman Inc., Clifton. NJ. U.S.A.) in 8 ,cI urea at pH 10.4. and was desalted by chromatography on Scphadcx G-25 (Pharmacia Fine Chemicals Inc.. Piscataway. NJ, U.S.A.) in 0.1 .Vf acetic acid. Homogeneity was determined by electrophoresis on polyacrylamide gels at pH 4.0. BBP peptide fragments l-37. 44-89 and 90-l 70 were obtained through the courtesy of Dr. C.-H. Jen Chou. These fragments were purified and characterized as previously published (Chou cr trl., 1977).
Rabbit IgG was purified by a modification of the proccdure of Sela and Mo7cs C1966). Fort! milliliters of 1.O,4f
4x
A. DESJARDINS
WALLACE.
K. SHAPIRA
sodium sulfate were added dropwisc with stlrring to 70 ml of normal rabbit serum at room tcmperaturc. The prccipitate was allowed to form for 30 min. then centrifuged for 30 min at 1400 re\.min. The pellet was dirsol\ed in 0.17 ,I! borate buffered saline pH 8.0 (BBS) and dialyzed overnight. The dialysate was centrifuged for clarification and applied to a column of DEAE-celluiosc (Whatman inc.. Clifton. NJ. l.J.S..A.). The ~ow-tllrot]gl~ peak ohtaincd by ehttton with 0.02 A,l phosphate bufti-r pH X.0 \sas l~~~pl~ili~c~i. Purity was dctermincd by itnmunoclectropl~t~re~is and poljacrylamide gel electrophorosis with 2nd l’iilhout sodium dodecyl sulfate (SDS). About 200 mg of lgG were ohtaincd from 20 ml of serum.
Five milliliters of 27”,, sodium sulfate were added to 5 ml of normal rabbit serum. the mixture was allowed to stand for 30 min at room temperature then centrifuged for 31 min at 3000 rev:min. The pellet was dis5olva.I in 3 ml of BBS and dialyired against BBS overnight. To the supernatant fluid from the preceding step were added 5 ml of 17”,, sodium sulfate. the mixture was allowed to stand for 30 min and centrifuged as above. The pellet and sup~rn~~t~llt Iiyuid were dialq-zed against BBS overnight. The fractions were tested for abrlity to inhibit direct binding in the soiid phase assay and for protein composition hy immunoclectrophoresis.
BBP. 20 mg. was coupled to IO ml of cyanogen bromideactivated Sepharose 4B (Pharmacia Fine Chemicals Inc.. Piscataaay. NJ. U.S.A.) at pH 9 by the method of Cuatrecasas and Anfinsen (1971). Amino acid analysis of the washed immunoadsorbent indicated that appros 2 mg of BBP were bound per ml of Sepharose. Immunondsorbent. 2 or 5 ml in 7 x 20%mm glass columns. was pre-washed with BBS and 0.1 .21 acetic acid until protein no longer eluted. then stored at 4 C. A volume of R- 30 ml of rabbit anti-BBP serum wan slowly passed through a 7 or 5 ml-Seph:rrose -BBP column at 4 C. The column was exhttusti\cly wnshed with BBS until the optical density (O.D.) at 2XOnm of the ettluent solution became zero. Adsorbed mntcrial was elutcd with 0.1 hl acetic acid and the column was rewashed with BBS. The acid cluate was immediately neutralized with S”,, potassium borate or 2 hf NaOH. The BBS and acid washes were each pooled and concentrated by vacuum dialysis or lyophilizution. The concentrated solutions were dialyzed against 0.01 M phosphate buffered saline (PBS) pH 7.4. clarified by low speed centrifugation. assayed for protein (Lowry it tri.. 1952) and for anti-BBP acticity. and stored frozen.
BBP and peptic fragments 1 37. 44-XY and 90-l 70 were r~~dioio~~iil~~tcd as described by Marchalonis (196’)). Equimolar amounts of BBP (2 mg). fragment I 37 (0.4 mg). fragment 44. XY (0.5 mg) or fragment YO 171) (0.9 mg), were dissolved in 100 ,II of SO m&I PBS pH 7.2. and mixed with 40 1~1of I2 m&l potassium iodide.‘ To this mixture were added the neccssarv amount of Izil (usuallv 100 400 rc<‘i) or “’ I (20 /Ki) (Amcrsham Scarlr. Arlington Heights: IL. U.S.A.). IO 111of lactopcroxidase (I mg,‘ml) (Sigma Chcmical Co.. St. Louis. MO, U.S.A.), and IO 111of fresh hydrogen at room peroxlde, 0.03”,, in PBS. After a 2-min incubation temperature, the reaction was stopped bv the addition of 500 itl of 5 m&f cyst&e. The reaction mjxture &tab added to a 7 x ?_itO-mm glass column containing Scphadcx G’S and cluted with IO m&I HCl. Appr~~~ii~~~~tcspecific active-
and
R. B. FRITZ
AntIbodIes to BBP purified from clght immune rabbit sera were pooled. lyophilized and chromatographed on Scphadex G-SO in SO mM PBS. The tlow-through protein peak from this column was iodinated as described above. Addition of 50 IKi of ‘liI to 700 b$g of antibodies in I ml of 50 m.%l PBS resulted in a specific activity of 5.6 !tCi mg protein. Rabbit anti-BBP sera and the imrnu~loglobuli[l fractions of goat ~~ilti-r~bbit IgG and rabbit anti-BBP were iodinated by the method of Greenwood CI tri. ( I Xi?). To a glass test tube were added I:! 111of the protein (4 mg/ml) in Jistilled water. 20 /iI of 500 m,ZI sodium phosphate butfer pH 7.5. and the necessary amount of “‘1 (usually I mCi). Ten microliters of freshly dissolved chloramlne T I IOmg, 20 ml) in 50 m&I phosphate huller were added. and the reaction uas allowed to proceed for 30 set before termination by the addition of 50 1~1ol” sodium mctabisultite ( 12.5mg:S.O ml) in SO m!LI phosphate bufIer. Unreactcd iodine was removed by exhaustive dialysis against PBS pH 7.4.
Immunoclectrophoresis was carried out on microscope slidcs coated with l”,, agarose dissolved in barbital buffer pH 8. 1’ = 0.0. Samples were diluted in barbitai buflcr and clectrophoresis was performed at 2OOV for i hr at room tempcraturc. Immunoelrctrophoresis patterns were devclopcd by incubation with goat anti-rabbit IgG and goat anti-rabbit strum (Cappel Laboratories. Inc.. Downington. PA. U.S.A.) or BBP. Slides were stained m a solution of 4S”,, methanol. 35”,, water. IO”,, acetic acid and O.l”,, Amide blue black.
Polyacrylamide gel electrophoresis was performed as described by Shapiro @I crl. (1967). The Y-cm gels contained 5”_ acrylamide, 0.13”,, bis-ncrylamide. 0. I”,, ammonium persulfate. and 0.05”,, (siv) TEM ED suspended in 3 rnnf Tris plycine buffer. pH 8.2--X.4. Samples of St& lOi)/~g of protein were added to a l-ml Sephadcx G-25 stacking gel in water and t).Ol”,, bromnphenol blue. and clectrophoresis wac performed at room temperature in Tris glycine buffer at 1.5 to 2 mA per gel for Z-3 hr. Samples to be analyzed on sodium dodtcyl sulfate gels were heated with 2”,, SDS with or without I”,, mercaptoethanol (Z-ME) for 2 min in a holhng water bath prior to clectrophoresis. Gels were stained by the method of Fairbanks et trl. (lY71) in a solution containine 25” isopropyl alcohol, IO”,, acetic acid and 0.003”,, Coo&ass& blue for several hours. Molecular weights were &mated from concurrently run SO ~18 samples of horse heart cytochromc oxidase (I 1.700). thymotrypsrnopcn A (25.0001. aldolase sub-unit (40.000). ov;;l~XI~III (45.000). beta-galactosidase (9 1.0001 and phosphor>last H (lX5.tifX)f. Noimal rabbit IgG was run’in &r&i with ail samples. Gels were scanned m a Beckman DU 0.L). convertcr at ~pectr~~pl~otornetcr with a Gilfat-d 540 nm.
Anti-BRP antibodies were assayed by two methods of rudioimmunoassay: (A) a modification of the sodium sulfate precipitation method of Day and Pltts (1974). and (8) the solid phase assay of Randolph cf ol. (1977). In the sodium sulfate assay. 100 /II of normal rabbit serum diluted I :3 in 0.01 Al PBS pH 7.2 were added to I2 x 75-mm disposable pyres test tuhcs. Triplicate samples of antiserum. 35 or 50 /‘I, were added to the tubes. followed by 25 1’1 of 12’1 HBP. 1p!f in PBS. The contcnts were mIxed and allowed to stand for 5 min at room temperature. A volume of 0.5 ml of 1.9 1\1 sodium sulfate HIS added to each tube and irnitledi~ltely mixed. Aftcr 1 hr at room tcnip~r~~tllre. the suspensions were centrifuged at 7_400 rcv*‘min for 30 mm The %upernntant Ruids were asplratcd and the pellets
Rabbit were twice resuspended in 1.27 M sodium sulfate and centrifuged as above. After removal of the supernatant tlulds. the pellets were dissolved in 4 x 0.3 ml washes of PBS which were pooled and assayed for radioactivity for one minute in a Packard model 3002 gamma s~~~~tiIl~~tionspectrometcr. Antibody levels were expressed as I()() x
radioacttvity precipitated - normal serum control -. _- -. _ ~- .-.. ~ ~ _ _ total radioactivity added
Results were normalized to original protein concentration and corrected for activity of pre-imlnui~i~ation sera of individual rabbits (usually 5-l@‘,,). The standard error was routinely Z-Y’,, of the mean. In the solid phase assay, 100 !(I of a freshly prepared solution of BBP or one of Its peptic fragments in BBS were added direclly to the bottom of 12 x 75 mm glass test tubes, and the tubes were rotated for 1 hr at room temperature in a nearly horizontal position at a rate of I rev:5 min (ML~ltipurpose rotator. Model I50 TC. Scien~ific Industries Inc., Spring~eld, MA, U.S,A.). The protein coated tubes were washed with 2 x I ml of OS’,, ovalhumin in BBS (EA-BBSI. Experiments with “‘I-labeled proteins indicated that the percentages of added radioactivity remaining in the tubes after washing were X5”,,. 70”,,. 66”,, and 71”,, for BRP and fragments l-37, 4G-89 and O&170. respectively. Triplicate 100 jll samples of anti-serum. diluted l:IOO to I :50.000 in EA-BBS. were added to the tubes. which wcrc covered and rotated as above at room temperature overnight. After 2 x I ml washes with EA-BBS, I ml of lz51 goat anti-rabbit gamma globulin in EA BBS was added to each tube and agitated at a rate of I40 shakes;min (Eberback Corp. Shaker. Ann Arbor. MI, U.S.A.). Each tube was washed with I x I ml of BBS and assayed for radioactivity for IO min in a Packard model 3002 gamma s~i~~t~ll~~t~o~~ spectrometer. Antibody levels were expressed as radioactivity
100 x ~~- ----
-
bound
~
- normal
--
total radioactivity
serum control added
Results were normalized to original protein concentration and corrected for activity of pre-immunization sera of individual rabbits (usu;~lly O.?,,). The standard error was routinely S-lo”,, of the mean. In studies on inhibitory elects of whole rabbit serum in the solid phase assay. a direct bindtnc method was used. To test tub& coated with BBP as described above were added 1.0.ml aliquots of the “‘I-labeled gamma globulin fraction of rabbit ,-lnti-BBP in EA-BBS. The tubes were shaken. washed and assayed for radioactivity as above.
49
Antibodies
Sepharose-BBP column, the column was exhaustively washed with BBS and the adsorbed protein was then eluted with 0.1 M acetic acid. Protein concentration was assessed by O.D. measurements at 280nm. The first and largest protein peak eluted with BBS was divided into two pools; fractions with an O.D. at 280 nm > 0.02 (fractions I-13) constituted pool 1. and fractions with an O.D. < 0.02 (fractions 1450) constituted pool 2. Protein eluted with 0.1 M acetic acid was designated pool 3 (fractions 56-65). Representative recoveries of protein from pool 3 of IO samples of immutle sera and pooled normal sera are given in Table I. The recovery from the ten immune sera ranged from 0.02 to OSOmg per ml starting serum. The majority of sera from bleedings after the onset of EAE yielded values in the range of 0.20-0.5Omg per mf of serum: two sera from day twelve bleedings gave much lower yields, No detectable protein was recovered in pool 3 of pooled normal rabbit serum, Immunoelectrophoretic analysis of the eluates using goat anti-rabbit serum and goat anti-rabbit IgG indicated that the pool 3 protein was IgG (Figs. 2A and 2B). Occasionally, a trace of a second more negatively charged protein was seen (Fig. 2B). In order to determine whether pool 3 contained precipitating antibody reactive with BBP, reverse immunoeiectrophoresis was performed. A representative pattern is shown in Fig. 3. The eluates were capable of forming a stable precipitin band with antigen showing that they retained immunological activity, and that they recognized multiple antigenic determinants on BBP. The acid-eluted IgG was further tested for immunologic activity by two other methods, the sodium sulfate precipitation procedure of Day and Pitts (1974) and the solid phase radioimmunoassay developed in this laboratory (Randolph CTirl., in press). These two assays were used since the former procedure gives semi-quantitative data on antibodies while the latter is highly sensitive and avoids the destruction of antigenie determinants caused by external labeling proccdures. The solid phase radioimmunoassay showed a recovery of more than loo”,, of the activity of the original sera in pool 3 (Table 2). Study of this phenomenon showed a loss of inhibitory material from the serum during the purification process. Inhib~tory activity reappeared when pool 3 was com-
Aliquots (lOO$) of I’5 I-labeled. pooled anti-BBP antibodies, 0.40 m&ml in 5 rn:W phosphate butfer pH 7.5. were added to siliconizetl I x IO-cm glass test tubes. From 0.08 to 32/q amounts of ‘“‘I-labeled BBP were also added to the tubes. resulting in total volumes of less than 200 III per tube. The tubes were incubated for 24 hr at 4 C.. then the pr~cipltates were washed wvlth 3 x I ml of cold phosphate buffer by centrifugation at 3500 rcv;min for 30 min at 4 c‘. The precipitates and washes were assayed for radioactivity in a Packard model 3002 dual channel gamma scintillation spectrometer. Data were corrected for “I I contribution to the “‘I channel. RESULTS
Nineteen samples of immune serum from It rabbits were passed through Sepharose-BBP columns which were then sequentially washed with BBS and 0.1 M acetic acid. A Lepresentative pattern of one of these samples is shown in Fig. 1. In this experiment 1.5ml ot antiserum (rabbit No. 4) were added to a ?-ml
.IOM acetfc acid J,
.,
Pool 2
0
IO
20
30 FRACTION
40
50
Pool 3,
60
70
NUMBER
Fip. I. Aflinity chromatography of immune serum. Fifteen milliliters of immune serum (rabbit No. 4) were added to a I-ml column of Scpharose-BBP which was then washed with BBS and 0.1 hl acetic acid. Fraction volume: 3 ml; flow rate: 0.6 mlimin
A. DESJARDINS Table
I. Yields of protein
Rabbit
No.
IS I5 15 15 30
5 5 6 7 8 9* 9 lo*
I0 normal
serum
R. SHAPIRA
from Sepharose-BBP
Serum volume (ml)
4
Pooled
WALLACE.
and
affinity
Mg protein Total 6.5 7.1 5.7 5.8 7.2
16
5.9
15 34 IS 15 ‘5
0.3 2.4 0.4 2.2 0
R. 9. FRITZ columns”
isolated per ml serum 0.43 0.47 0.38 0.39 0.24 0.37 0.02 0.07 0.02 0. I 5 0
” Samples of rabbit serum obtained at 12(*) or about 20 days after immunization wcrc applied to Sepharose- BBP columns and washed as described in Methods. The yields shown arc representative of all samples tested.
Fig. 2. Immunoelectrophoresis of protein eluted from Sepharose-BBP alfinity columns. The lower trough contains goat anti-rabbit serum; the upper trough contains goat anti-rabbit gamma-globulin. (A) immune serum rabbit No. 5; (B) immune serum rabbit No. 6. Anode is to the right
:A)
(9)
Fig. 3. lmmunoelectrophoresis of protein eluted from Sepharose-BBP affinity columns. The upper and lower troughs contain BBP. 0.25 and 0.50 mg/ml, respectively. (A) immune serum rabbit No. 5; (9) immune serum rabbit No. 6. Anode is to the right.
bined with pool I from the same immune serum, with pool 1 from other immune sera, or with normal sera from rabbit or guinea-pig. Normal chicken, calf, horse and rat sera were without inhibitory effect on pool 3 suggesting that the effect was not due to enzymatic degradation of BBP during radioimmunoassay. To further study the inhibitory effect, the ability of normal rabbit serum, heated (56”C, 30 min) normal rabbit serum and three salt-precipitated fractions of normal rabbit serum to inhibit binding of the ‘2’I-labeled gamma globulin fraction of rabbit antiBBP to BBP-coated tubes was tested. As shown in Table 4, heated normal serum and the supernatant fraction of normal rabbit serum retained inhibitory activity comparable to that of normal rabbit serum, while the two salt-precipitated fractions showed little inhibition. lmmunoelectrophoresis indicated that the supernatant fraction contained primarily albumin and alpha globulins. In order to determine whether the acid elution procedure had selectively eluted antibodies reactive with certain regions of BBP while leaving other antibodies on the column, the original sera and the acid eluates were tested for reactivity against BBP fragments I 37. 44-89 and 9G-170 by means of solid phase radioimmunoassay (Table 5). These data show that each serum and eluate was reactive with BBP and its three peptic fragments. Three of the four sera showed approximately the same degree of reactivity with the fragments before and after elution. The fourth serum (rabbit No. 4) showed some loss of reactivity to fragments 1-37 and 44-89. Radioimmunoassay by sodium sulfate precipitation showed that up to 7lY,, of the activity of the original sera was recovered in pool 3 (Table 3). The data in this table indicate that little significant activity remained in pools 1 and 2 due to the high efficiency of this type of column for antibody adsorption. It is of interest that rabbit serum IO. from which only small amounts of protein were recovered in the eluate, had good binding activity. Since the original activity of serum 10 was comparable to sera 7 and 8. this would imply that there was more inactive antibody protein in eluates 7 and 8. In two further experiments,
Rabbit
51
Antibodies
Table 2. Activity of BBP with immune sera and eluates from SepharoseeBBP columns as determined by solid phase radioimmunoassay”
Rabbit
No.
7 8 9 10
Serum 1:lO I:100
Pool I 1:lO 1:lOO
Pool 2 1: 100 I:10
1.4 2.0 0.9 0.9
0.1 0. I 0.1 0
1.0
0.2
3.1 3. I
I.1
a Sera and eluates
1.7 3.0 2.2 1.0
were assayed
0.2 0. I 0.2 0.1
at dilutions
0.4 0.3
I.6
of I : 10 and
afiinity
Pool 3 I:10 I : 100 4.3 6.3 5.1 6.6
3.5 4.8 3.7 4.6
1: 100.
Table 3. Activity of BBP with immune sera and cluates from Sepharosee BBP affinity columns as determined by radioimmunoassay using I.27 iz;I sodium sulfate
Rabbit
Sample Source
No.
Activity
Serum activity recovered in eluate (“,J
serum pool 1 pool 2 pool 3
34 3 16
9 3 47
serum pool 1 pool 2 pool 3
48 3 3 I7
6 6 36
strum pool 1 pool 2 pool 3
36 3 23
8 3 61
serum pool I pool 2 pool 3
50 6
I2
36
72
I
Table 4. The inhibitory effect of normal rabbit serum binding of anti-BBP antibodies to BBP as determined radioimmunoassay
“‘I Treatment Untreated Normal rabbit Heated normal Sodium sulfate Sodium sulfate Supernate from
serum rabbit serum pellet No. 1 pellet No. 2 pellet No. 2
proteins on the by solid phase
counts, IO min 8190 1065 1179 7233 7640 I807
Aliquots of the ‘251-labeled gamma globulin fraction of rabbit antiBBP were treated with equal volumes of the above materials diluted 1:10 in BBS and assayed in triplicate as described in Methods.
pool 3 from Sepharose-BBP adsorbed radioiodinated anti-BBP serum was added to a second column of Sepharose-BBP and washed with BBS. In both experiments approximately 60% of the pool 3 material retained the ability to bind to immobilized antigen. In order to study the nature of the inactive antibody in pool 3, polyacrylamide gel electrophoresis
was performed on seven acid eluates. In the presence of SDS and 2-ME, two bands of approx 50.000 and 23,000 daltons were observed (Figs. 4A and B). These molecular weights are consistent with the sizes of rabbit IgG heavy and light chains. However, when electrophoresis was performed in the presence of SDS alone, the same seven eluates produced a major band
A. DESJARDINS Table 5. Activity
WALLACE,
R. SHAPIRA
R. B. FRITZ
of BBP and three peptic fragments with immune sera and eluates from SepharoseeBBP affinity columns as determined by solid phase radioimmunoassay. Activity
Rabbit No.
and
Sample Source
with fragment
t activity
with BBP x 100
Activity with BBP
I-37
44 89
90~ 170
2.3 4. I 2.4 4.2 2.7 3.7 1.1 4.2
22 17 35 8 20 Ih ?? 16
36 17 20 7 9 12 34 27
2x 37 43 26 27 ?X 31 30
Serum Pool 3 Serum Pool 3 Serum Pool 3 Serum Pool 3
Total X6 71 98 31 56 66 S8 7?
determine the elfective antigenic valence of BBP by means of paired-label quantitative precipitation. In two experiments. ‘“‘I-labeled antigen was used in the precipitation reaction with ‘Z”I-labeled pooled antibody. The antibody was over 90”,, precipitable at equivalence. The antibody-antigen ratios of the precipitates in antibody excess were determined and a plot made in which effective antigenic valence was determined by extrapolation of the antibody to antigen ratio to infinite antibody excess (Fritz et ul..
of approx 170,000 daltons and four other bands of approximately 120,000. 80.000, 50,000 and 20,000 daltons when compared to standard proteins (Fig 4C). Normal rabbit IgG did not show significant amounts of these smaller components after treatment with 0.1 M acetic acid (Fig. 4D). Although some sera were obtained as early as 12 days after immunization, IgM antibodies were not detected in the acid eluates. Anti-BBP IgG purified by immunoadsorption and Sephadex-G-50 chromatography was then used to
B
A 2.0 -
g s
II
5
123456709
“0r
I*
2 I.
3
4
Fig. 4. Polyacrylamide gel Immune serum (rabbit No. (treated with 0.1 M acetic C
5
6 .
7 8 DISTANCE I.
1
C1123456709 D
9 0 I 2 FROM TOP OF GEL (cm)
3
4
5
6
7
0
9
electrophorcsis of protein cluted from a Sepharose-BBP affinity column. 5) with SDS and 2-ME (A) and with SDS only (C). Normal rabbit IgG acid) with SDS and Z-ME (B) and with SDS only (D). Patterns A and are representative of the seven acid eluates tested.
5?
Rabbit Antibodies
I
1
I
2
3
4
ANTIGEN
5
6
7
8
ADDED (yg)
Fig. 5. Quantitative precipitation of ‘251-labeled anti-BBP IgG and r3rI-labeled BBP. The points shown are the means
of triplicate
determinations
1967). The results of the experiments are shown in Fig. 5. From these data it may be seen that the effective antigenic valence of BBP is approximately four. DISCUSSION
Anti-BP antibodies of guinea-pigs and rabbits have been partially characterized. Lisak et (11.(1969) found 7S antibody in 13:~ of guinea-pigs after injection with homologous BP and CFA, by radioimmunoelectrophoresis of immune sera with specific anti-guinea-pig antisera. Using a gel filtration assay and Z-ME sensitivity, Lennon et (11.(1971) found mainly IgM in the serum of rabbits injected with human BP and CFA. although some IgG was found twenty days after the rabbits were boosted. Pitts et al. (1976) used BP coupled to Sepharose to adsorb antibodies from immune rat sera in their study of the cross-reactivity of rat antibodies to BP from beef, pig, rabbit, guineapig and rat, but they did not report characterization of the antibodies. In the present study, SepharoseBBP columns were used to adsorb anti-BBP activity from immune rabbit sera, and antibody recovered in a 0.1 M acetic acid wash (pool 3) was active in a sodium sulfate precipitation radioimmunoassay (Day & Pitts, 1974) and in a solid phase radioimmunoassay (Randolph et u/., in press). A normal rabbit serum fraction containing primarily albumin and alpha globulins inhibited binding of antibodies to BBP in the solid phase RIA. It is not clear why this inhibitory effect was not observed with the salting-out method of RIA, although it is possible that the high Na,SO, concentration of the assay decreases binding of the inhibitor(s) to BP. The major antibody component isolated in pool 3 was of the IgG class as shown by immunoelectrophoresis and polyacrylamide gel electrophoresis. It is possible that these procedures were not sufficiently sensitive to detect small amounts
and the bars indicate
standard
error.
of IgM antibodies. The results of analysis of pool 3 by polyacrylamide gel electrophoresis were interesting in that upon denaturation and reduction only two bands were observed and the molecular weights of the polypeptides in the bands were consistent with rabbit IgG light and heavy chains; while upon denaturation with SDS alone, several bands appeared in the gels suggesting disaggregation of non-covalently bound IgG complexes. The molecular weights of these components were consistent with various combinations of light and heavy immunoglobulin chains. The number and location of humoral antigenic determinants in the BP molecule have been studied by several different methods of immunization and assay. Using passive hemagglutination, Hashim and Eylar (1969), Burnett and Eylar (1971) and Eylar et ul. (1971) found no activity to BBP fragment 117-170 in guinea-pigs and none in BBP fragment 44-89 in rabbits. McFarlin et (11.(I 975) used a double antibody radioimmunoassay and found no activity to BBP fragment 45-88 in Lewis rats injected with the whole protein. In rabbits injected with BBP coupled to rabbit serum albumin, BBP fragments l-43, 9&170 were active and 44-89 inactive when studied by quantitative complement fixation hapten inhibition (Whittaker ef al., 1975). In partial contrast, Driscoll et al. (1974) found three antigenic sites, in fragments 44-89, 9&l 16 and 117-170, in guinea-pigs injected with GP BP and CFA. In our studies, sera and acid eluted antibodies with high anti-BBP activity were tested for ability to bind to tubes coated with the BBP fragments, l-37, 4489 and 9@170. All sera and eluates tested demonstrated activity with all three fragments with fragment 90-170 usually more active than fragments l-37 and 44-89. These results suggest the presence of at least one antigenic site in each fragment, and possibly more in fragment 90-170. From the quantitative precipitation data of Webb et al. (1973)
54
A. DESJARDINS
WALLACE.
where BBP was added to the serum of rabbits immunized with a phosvitin-BBP complex, Day and Pitts (1974) estimated that there were three antigenic determinants in the BBP molecule. In our studies a pool of immunospecifi~dlly purified anti-BBP IgG was used to estimate the number of humoral detcrminants in the molecule. Extrapolation of the data to infinite antibody excess indicated that the effective antigenic valence of BBP is approximately 4 which is in agreement with data obtained with other proteins of similar molecular weight (Ginader & Pearce, 19561. REFFRERiCES 1 1
Burnett P. R. & Eylar
1
E. H. (1971) J. hiol. Chem. 246, 3425. Carnegie P. R. (1971) Biochern. J. 123, 57. Chou F. C.-H., Chou C.-H. Jen, Shapira R. & Kibier R. F. (1976) J. hiof. CCwm. 251, 2671. Chou F. C.-H., fhou C.-H. Jen, Shapira R. & Kibler R. F. (1977)J. Newociiem. 28, 115. Cinader B. & Pearce J. H. (1956) Br. J. ~.yp. Puth 37, 541. Cuatrecasas P. & Anfinsen C. B. (1971) Methodsin EKJmoloyy(Edited by Jakoby W. B.). Vol. 22, p. 345. Academic Press, New York. Day E. D. & Pitts 0. M. (1974) Itnnru,locllrmisfrg fl, 651. Driscoll 3. F., Kramer A. J. & Kies M. W. (1974) Science 184, 13. Epand R. H., Moscarello M. A.. Zierenberg B. & Vail W. J. (1974) Biochemistry 13, 1264. Eylar E. H. (1970) Proc. mm. Acud. SC.. C’.S.A. 67. 1425. Eylar E. H. & Thompson M. (1969) Archs Biochrm. Binwph~x 129. 468. Evlar E. H.. Westall F. C. & Brostoff S. (1971), J. hi& ‘Chem. 246. 3418. Fairbanks G.. Steele T. L. & Wallack D. F. (1971) Biockenri.sfrl~
IO.
2XOh.
R. SHAPIRA
and
R. B. FRITZ
Fritz R. B., Lassiter S. & Day E. D. (1967) J. //wtwr. 98. 1713. Greenwood F. c’.. Hunter W. M. & Clover J. S. (1963) Biochlwf. J. 89, 114.
Humienny M. E., McKncally S., Kibler R. F. 61 Shapiro R. (I 9741 ‘I‘rilr~s. .1/n Sot. !Vrwoc/w/,~. 5. 141. Kies M.. Murphy J. B. & Alvord E. C. (1960) FL’&. Proc.. Fedrl. Aw. Soc,s e.\-p. Biol. 19, 207. Krigbaum W. R. & Hsu T. S. (1975) Biochemistry 14, 1975. Lennon V. A.. Whittil~gl~~lrn S.. Carnegie P. R.. McPherson T. A. & Mackay I. R. (1971) J. ~~~~~~f/l.107. 1971. Lisak R. P.. Hcinzc R. G.. Kies M, W. & Alvord E. C. (1969) Prw. So<,. r’\-,‘. Uicji. M~vl. 130. X14. Lowry 0. H., Rosebrough N. 1.. Farr A. L. & Randall R. j. (1951) J. hiol. Cl&. 193, 265. Marchalonis J. J. (1969) Biocl~rt~ .I. 113. 299 McFarlin D. E.. l&u S. C.-L.. Slcmenda S. B.. Chou F. C.-H. & Kibler R. F. (1975) J. r.up. hfcd. 141, 72. Palmer F. B. Cp Dawson R. M. C. (1969) ~i~~~~7~,~~~. J. 111, 629. Pltts 0. M.. Variteck V. A. & Day E. D. (1976) Irnrwrwc~/le,,listr~ 13, 307. Randolph D. H. (1976) M. S. thesis. Emory University, Atlanta. Georgia. Randolph D. H.. Kibler R. F. & i:rit7 R. B. (1977) J. irw flw~. Mc~tli. (in press). Rohoz-Einstein E., Robertson D.. DiCaprto J. & Moore W. / 1962) J. Mvurochrr,~. Sela M. & Mazes E. (1966) Proc. 1wrt1. Actrll. Sci., C’.S.A. 55, 445. Shapiro A. W.. Vinuela E. 6i Maigel J. (1967) Biochwt. hiopl~.vs. Res. Comtw~i. 28, Xt 5. Webb C.. Teitelbaum D., Arnon R. & Sela M. (1973) Eur. J. l~~~~~ll/l~. 3, 179. Whittaker J. N.. Chou C.-H. Jcn, Chou F. C.-H. R: tiibler R. F. ( 1975) ,I. hiol. C/wnt. 250, 9106.
AND CHARACTERIZATION OF RABBIT TO BOVINE MYELIN BASIC PROTEIN* A. DESJARDINS Departments
WALLACE.
of Biochemistry Atlanta.
R. SHAPIRA
and Microbiology, GA 30322. U.S.A.
ANTIBODIES
and R. B. FRITZ Emor)
University.
Abstract---Injection of a wide variety of animal species with homologous or heterologous myelin basic protein (BP) emulsified with complete Freund’s adjuvant (CFA) produces the autoimmune demyehnnting disease, experimental allergic encephalomyelitis (EAE). As a first step in the study of antigenic determinants of bovine BP (BBP). specific antibodies were purified from rabbit serum b\i adsorption on SepharoseeBBP atfinity columns and elution with 0.1 ,V acetic acid. Yields of 0.0233.50 mg protein per milliliter of serum were obtained from the acetic acid eluates. lmmunoelectrophoresis and polyacrylamide gel clectrophoresis indicated that the acid eluted material was I&G. The acid eluted IgG formed stable precipitin bands with BBP in agarose gels and also demonstrated anti-BBP activit) in a sodium sulfate precipitation radioimmunoassay and in a solid phase radioimmunoassay. Quantitative precipitation of purified antibodies was used to estimate the number of antigenic determinants in the BBP molecule. From a plot of the antibody to antigen ratio of precipitates vs the log of antigen in the valence of approximately four was indicated. precipitates at antibody excess, an effective antigenic
MATERIALS
INTRODUCTIO’V
Basic protein (BP) of central nervous system myelin was first purified and characterized by Kies and coworkers (1969). Although its functions irl rice remain unknown, it is clear that myelin BP is the major antigen responsible for the demyelinating disease, experimental allergic encephalomyelitis (EAE) (RobozEinstein ef ul., 1962). Physico-chemical studies of myelin BP have shown little secondary folding of the molecule in aqueous solution. Sedimentation equilibrium studies indicate a frictional ratio of IO:1 (Eylar & Thompson, 1969), and circular dichroism and optical rotatory dispersion argue for a complete lack of alpha-helix or betapleated sheet structure (Palmer & Dawson. 1969). Recent studies on small angle X-ray scattering conflict and suggest either a highly asymmetric prolate ellipsoidal conformation (Epand et LII.. 1974) or an open coil (Krigbaum SC Hsu, 1975) in aqueous solution. The lack of significant conformation implies that antigenie determinants of BP are formed by short linear sequences of amino acids, and that the determinants may be delineated by studies of BP fragments. Furthermore, since the biological and immunological activities of basic proteins isolated from different species may vary greatly while the primary structure of these proteins varies to a lesser extent, it should be possible to identify critical amino acid residues required for biological and immunological function. As a first step in these studies, we have determined the conditions necessary for the preparation of specific anti-BBP antibodies by affinity chromatography. These antibodies have been characterized and used to determine the effective antigenic valence of bovine myelin BP. * Research was supported by Public Health grants NS 08278, NS 11418 and NS 10721 from the National lnstitutc of Neurological and Communicative Disorders and Stroke. 17
AND
METHODS
Four- to five-month-old random bred female New Zcaland white rabbits were obtained from a local supplier
(Hiram Davies. Stockbridge. GA. U.S.A.) and immunized with a single footpad injection. Each rabbit rcccived 600 /~g bovine BP (BBP) and 6 mg of !21!,c,oh~lcf~,~il~~~l hrrr!nc~~m (CFA) (Sigma Chemical Co.. St. Louis. MO. U.S.A.) in a O.l-ml emulsion. Ammals were bled from the ear artery before immunization and after onset of EAE. Scra were centrifuged and stored frozen. Onset of EAE approximately 3 weeks after injection was indicated by tail Ilaccldity followed by hind leg weakness. incontinence. and. in some cases, severe paralysis and death. Day of onset and severity of clinical symptoms varied from animal to animal. EAE was confirmed by histological studies.
BBP was purilied by the procedure of Humlenn! 01 trl. (1974). Bovine cord or brain. l2Og. was homoge&ed in 3520 ml of cold butanol:acetic acid:watcr (4: I :2) in a I-gal Waring bl:nder. The solution was filtered to rcmobc debris and IOXOml of water were added with stirring. The two layers were separated bq centrifugation at 4000 ret min for I hr at 2 C. The aqueous solution wah conccntratcd to lOOmI m an Amicon DC2 dlalyrer concentrator with a HIDP IO cartridge. washed with 500 ml of 0.1 ,21 acetic acid. and lyophiliaed. The crude protein. approx I g, was purified by preparative electrophoresis on IS”,, polyacrylamide gels in 0.5 M acetic acid followed by chromntography on carboxymethylccllulose (Whatman Inc., Clifton. NJ. U.S.A.) in 8 ,cI urea at pH 10.4. and was desalted by chromatography on Scphadcx G-25 (Pharmacia Fine Chemicals Inc.. Piscataway. NJ, U.S.A.) in 0.1 .Vf acetic acid. Homogeneity was determined by electrophoresis on polyacrylamide gels at pH 4.0. BBP peptide fragments l-37. 44-89 and 90-l 70 were obtained through the courtesy of Dr. C.-H. Jen Chou. These fragments were purified and characterized as previously published (Chou cr trl., 1977).
Rabbit IgG was purified by a modification of the proccdure of Sela and Mo7cs C1966). Fort! milliliters of 1.O,4f
4x
A. DESJARDINS
WALLACE.
K. SHAPIRA
sodium sulfate were added dropwisc with stlrring to 70 ml of normal rabbit serum at room tcmperaturc. The prccipitate was allowed to form for 30 min. then centrifuged for 30 min at 1400 re\.min. The pellet was dirsol\ed in 0.17 ,I! borate buffered saline pH 8.0 (BBS) and dialyzed overnight. The dialysate was centrifuged for clarification and applied to a column of DEAE-celluiosc (Whatman inc.. Clifton. NJ. l.J.S..A.). The ~ow-tllrot]gl~ peak ohtaincd by ehttton with 0.02 A,l phosphate bufti-r pH X.0 \sas l~~~pl~ili~c~i. Purity was dctermincd by itnmunoclectropl~t~re~is and poljacrylamide gel electrophorosis with 2nd l’iilhout sodium dodecyl sulfate (SDS). About 200 mg of lgG were ohtaincd from 20 ml of serum.
Five milliliters of 27”,, sodium sulfate were added to 5 ml of normal rabbit serum. the mixture was allowed to stand for 30 min at room temperature then centrifuged for 31 min at 3000 rev:min. The pellet was dis5olva.I in 3 ml of BBS and dialyired against BBS overnight. To the supernatant fluid from the preceding step were added 5 ml of 17”,, sodium sulfate. the mixture was allowed to stand for 30 min and centrifuged as above. The pellet and sup~rn~~t~llt Iiyuid were dialq-zed against BBS overnight. The fractions were tested for abrlity to inhibit direct binding in the soiid phase assay and for protein composition hy immunoclectrophoresis.
BBP. 20 mg. was coupled to IO ml of cyanogen bromideactivated Sepharose 4B (Pharmacia Fine Chemicals Inc.. Piscataaay. NJ. U.S.A.) at pH 9 by the method of Cuatrecasas and Anfinsen (1971). Amino acid analysis of the washed immunoadsorbent indicated that appros 2 mg of BBP were bound per ml of Sepharose. Immunondsorbent. 2 or 5 ml in 7 x 20%mm glass columns. was pre-washed with BBS and 0.1 .21 acetic acid until protein no longer eluted. then stored at 4 C. A volume of R- 30 ml of rabbit anti-BBP serum wan slowly passed through a 7 or 5 ml-Seph:rrose -BBP column at 4 C. The column was exhttusti\cly wnshed with BBS until the optical density (O.D.) at 2XOnm of the ettluent solution became zero. Adsorbed mntcrial was elutcd with 0.1 hl acetic acid and the column was rewashed with BBS. The acid cluate was immediately neutralized with S”,, potassium borate or 2 hf NaOH. The BBS and acid washes were each pooled and concentrated by vacuum dialysis or lyophilizution. The concentrated solutions were dialyzed against 0.01 M phosphate buffered saline (PBS) pH 7.4. clarified by low speed centrifugation. assayed for protein (Lowry it tri.. 1952) and for anti-BBP acticity. and stored frozen.
BBP and peptic fragments 1 37. 44-XY and 90-l 70 were r~~dioio~~iil~~tcd as described by Marchalonis (196’)). Equimolar amounts of BBP (2 mg). fragment I 37 (0.4 mg). fragment 44. XY (0.5 mg) or fragment YO 171) (0.9 mg), were dissolved in 100 ,II of SO m&I PBS pH 7.2. and mixed with 40 1~1of I2 m&l potassium iodide.‘ To this mixture were added the neccssarv amount of Izil (usuallv 100 400 rc<‘i) or “’ I (20 /Ki) (Amcrsham Scarlr. Arlington Heights: IL. U.S.A.). IO 111of lactopcroxidase (I mg,‘ml) (Sigma Chcmical Co.. St. Louis. MO, U.S.A.), and IO 111of fresh hydrogen at room peroxlde, 0.03”,, in PBS. After a 2-min incubation temperature, the reaction was stopped bv the addition of 500 itl of 5 m&f cyst&e. The reaction mjxture &tab added to a 7 x ?_itO-mm glass column containing Scphadcx G’S and cluted with IO m&I HCl. Appr~~~ii~~~~tcspecific active-
and
R. B. FRITZ
AntIbodIes to BBP purified from clght immune rabbit sera were pooled. lyophilized and chromatographed on Scphadex G-SO in SO mM PBS. The tlow-through protein peak from this column was iodinated as described above. Addition of 50 IKi of ‘liI to 700 b$g of antibodies in I ml of 50 m.%l PBS resulted in a specific activity of 5.6 !tCi mg protein. Rabbit anti-BBP sera and the imrnu~loglobuli[l fractions of goat ~~ilti-r~bbit IgG and rabbit anti-BBP were iodinated by the method of Greenwood CI tri. ( I Xi?). To a glass test tube were added I:! 111of the protein (4 mg/ml) in Jistilled water. 20 /iI of 500 m,ZI sodium phosphate butfer pH 7.5. and the necessary amount of “‘1 (usually I mCi). Ten microliters of freshly dissolved chloramlne T I IOmg, 20 ml) in 50 m&I phosphate huller were added. and the reaction uas allowed to proceed for 30 set before termination by the addition of 50 1~1ol” sodium mctabisultite ( 12.5mg:S.O ml) in SO m!LI phosphate bufIer. Unreactcd iodine was removed by exhaustive dialysis against PBS pH 7.4.
Immunoclectrophoresis was carried out on microscope slidcs coated with l”,, agarose dissolved in barbital buffer pH 8. 1’ = 0.0. Samples were diluted in barbitai buflcr and clectrophoresis was performed at 2OOV for i hr at room tempcraturc. Immunoelrctrophoresis patterns were devclopcd by incubation with goat anti-rabbit IgG and goat anti-rabbit strum (Cappel Laboratories. Inc.. Downington. PA. U.S.A.) or BBP. Slides were stained m a solution of 4S”,, methanol. 35”,, water. IO”,, acetic acid and O.l”,, Amide blue black.
Polyacrylamide gel electrophoresis was performed as described by Shapiro @I crl. (1967). The Y-cm gels contained 5”_ acrylamide, 0.13”,, bis-ncrylamide. 0. I”,, ammonium persulfate. and 0.05”,, (siv) TEM ED suspended in 3 rnnf Tris plycine buffer. pH 8.2--X.4. Samples of St& lOi)/~g of protein were added to a l-ml Sephadcx G-25 stacking gel in water and t).Ol”,, bromnphenol blue. and clectrophoresis wac performed at room temperature in Tris glycine buffer at 1.5 to 2 mA per gel for Z-3 hr. Samples to be analyzed on sodium dodtcyl sulfate gels were heated with 2”,, SDS with or without I”,, mercaptoethanol (Z-ME) for 2 min in a holhng water bath prior to clectrophoresis. Gels were stained by the method of Fairbanks et trl. (lY71) in a solution containine 25” isopropyl alcohol, IO”,, acetic acid and 0.003”,, Coo&ass& blue for several hours. Molecular weights were &mated from concurrently run SO ~18 samples of horse heart cytochromc oxidase (I 1.700). thymotrypsrnopcn A (25.0001. aldolase sub-unit (40.000). ov;;l~XI~III (45.000). beta-galactosidase (9 1.0001 and phosphor>last H (lX5.tifX)f. Noimal rabbit IgG was run’in &r&i with ail samples. Gels were scanned m a Beckman DU 0.L). convertcr at ~pectr~~pl~otornetcr with a Gilfat-d 540 nm.
Anti-BRP antibodies were assayed by two methods of rudioimmunoassay: (A) a modification of the sodium sulfate precipitation method of Day and Pltts (1974). and (8) the solid phase assay of Randolph cf ol. (1977). In the sodium sulfate assay. 100 /II of normal rabbit serum diluted I :3 in 0.01 Al PBS pH 7.2 were added to I2 x 75-mm disposable pyres test tuhcs. Triplicate samples of antiserum. 35 or 50 /‘I, were added to the tubes. followed by 25 1’1 of 12’1 HBP. 1p!f in PBS. The contcnts were mIxed and allowed to stand for 5 min at room temperature. A volume of 0.5 ml of 1.9 1\1 sodium sulfate HIS added to each tube and irnitledi~ltely mixed. Aftcr 1 hr at room tcnip~r~~tllre. the suspensions were centrifuged at 7_400 rcv*‘min for 30 mm The %upernntant Ruids were asplratcd and the pellets
Rabbit were twice resuspended in 1.27 M sodium sulfate and centrifuged as above. After removal of the supernatant tlulds. the pellets were dissolved in 4 x 0.3 ml washes of PBS which were pooled and assayed for radioactivity for one minute in a Packard model 3002 gamma s~~~~tiIl~~tionspectrometcr. Antibody levels were expressed as I()() x
radioacttvity precipitated - normal serum control -. _- -. _ ~- .-.. ~ ~ _ _ total radioactivity added
Results were normalized to original protein concentration and corrected for activity of pre-imlnui~i~ation sera of individual rabbits (usually 5-l@‘,,). The standard error was routinely Z-Y’,, of the mean. In the solid phase assay, 100 !(I of a freshly prepared solution of BBP or one of Its peptic fragments in BBS were added direclly to the bottom of 12 x 75 mm glass test tubes, and the tubes were rotated for 1 hr at room temperature in a nearly horizontal position at a rate of I rev:5 min (ML~ltipurpose rotator. Model I50 TC. Scien~ific Industries Inc., Spring~eld, MA, U.S,A.). The protein coated tubes were washed with 2 x I ml of OS’,, ovalhumin in BBS (EA-BBSI. Experiments with “‘I-labeled proteins indicated that the percentages of added radioactivity remaining in the tubes after washing were X5”,,. 70”,,. 66”,, and 71”,, for BRP and fragments l-37, 4G-89 and O&170. respectively. Triplicate 100 jll samples of anti-serum. diluted l:IOO to I :50.000 in EA-BBS. were added to the tubes. which wcrc covered and rotated as above at room temperature overnight. After 2 x I ml washes with EA-BBS, I ml of lz51 goat anti-rabbit gamma globulin in EA BBS was added to each tube and agitated at a rate of I40 shakes;min (Eberback Corp. Shaker. Ann Arbor. MI, U.S.A.). Each tube was washed with I x I ml of BBS and assayed for radioactivity for IO min in a Packard model 3002 gamma s~i~~t~ll~~t~o~~ spectrometer. Antibody levels were expressed as radioactivity
100 x ~~- ----
-
bound
~
- normal
--
total radioactivity
serum control added
Results were normalized to original protein concentration and corrected for activity of pre-immunization sera of individual rabbits (usu;~lly O.?,,). The standard error was routinely S-lo”,, of the mean. In studies on inhibitory elects of whole rabbit serum in the solid phase assay. a direct bindtnc method was used. To test tub& coated with BBP as described above were added 1.0.ml aliquots of the “‘I-labeled gamma globulin fraction of rabbit ,-lnti-BBP in EA-BBS. The tubes were shaken. washed and assayed for radioactivity as above.
49
Antibodies
Sepharose-BBP column, the column was exhaustively washed with BBS and the adsorbed protein was then eluted with 0.1 M acetic acid. Protein concentration was assessed by O.D. measurements at 280nm. The first and largest protein peak eluted with BBS was divided into two pools; fractions with an O.D. at 280 nm > 0.02 (fractions I-13) constituted pool 1. and fractions with an O.D. < 0.02 (fractions 1450) constituted pool 2. Protein eluted with 0.1 M acetic acid was designated pool 3 (fractions 56-65). Representative recoveries of protein from pool 3 of IO samples of immutle sera and pooled normal sera are given in Table I. The recovery from the ten immune sera ranged from 0.02 to OSOmg per ml starting serum. The majority of sera from bleedings after the onset of EAE yielded values in the range of 0.20-0.5Omg per mf of serum: two sera from day twelve bleedings gave much lower yields, No detectable protein was recovered in pool 3 of pooled normal rabbit serum, Immunoelectrophoretic analysis of the eluates using goat anti-rabbit serum and goat anti-rabbit IgG indicated that the pool 3 protein was IgG (Figs. 2A and 2B). Occasionally, a trace of a second more negatively charged protein was seen (Fig. 2B). In order to determine whether pool 3 contained precipitating antibody reactive with BBP, reverse immunoeiectrophoresis was performed. A representative pattern is shown in Fig. 3. The eluates were capable of forming a stable precipitin band with antigen showing that they retained immunological activity, and that they recognized multiple antigenic determinants on BBP. The acid-eluted IgG was further tested for immunologic activity by two other methods, the sodium sulfate precipitation procedure of Day and Pitts (1974) and the solid phase radioimmunoassay developed in this laboratory (Randolph CTirl., in press). These two assays were used since the former procedure gives semi-quantitative data on antibodies while the latter is highly sensitive and avoids the destruction of antigenie determinants caused by external labeling proccdures. The solid phase radioimmunoassay showed a recovery of more than loo”,, of the activity of the original sera in pool 3 (Table 2). Study of this phenomenon showed a loss of inhibitory material from the serum during the purification process. Inhib~tory activity reappeared when pool 3 was com-
Aliquots (lOO$) of I’5 I-labeled. pooled anti-BBP antibodies, 0.40 m&ml in 5 rn:W phosphate butfer pH 7.5. were added to siliconizetl I x IO-cm glass test tubes. From 0.08 to 32/q amounts of ‘“‘I-labeled BBP were also added to the tubes. resulting in total volumes of less than 200 III per tube. The tubes were incubated for 24 hr at 4 C.. then the pr~cipltates were washed wvlth 3 x I ml of cold phosphate buffer by centrifugation at 3500 rcv;min for 30 min at 4 c‘. The precipitates and washes were assayed for radioactivity in a Packard model 3002 dual channel gamma scintillation spectrometer. Data were corrected for “I I contribution to the “‘I channel. RESULTS
Nineteen samples of immune serum from It rabbits were passed through Sepharose-BBP columns which were then sequentially washed with BBS and 0.1 M acetic acid. A Lepresentative pattern of one of these samples is shown in Fig. 1. In this experiment 1.5ml ot antiserum (rabbit No. 4) were added to a ?-ml
.IOM acetfc acid J,
.,
Pool 2
0
IO
20
30 FRACTION
40
50
Pool 3,
60
70
NUMBER
Fip. I. Aflinity chromatography of immune serum. Fifteen milliliters of immune serum (rabbit No. 4) were added to a I-ml column of Scpharose-BBP which was then washed with BBS and 0.1 hl acetic acid. Fraction volume: 3 ml; flow rate: 0.6 mlimin
A. DESJARDINS Table
I. Yields of protein
Rabbit
No.
IS I5 15 15 30
5 5 6 7 8 9* 9 lo*
I0 normal
serum
R. SHAPIRA
from Sepharose-BBP
Serum volume (ml)
4
Pooled
WALLACE.
and
affinity
Mg protein Total 6.5 7.1 5.7 5.8 7.2
16
5.9
15 34 IS 15 ‘5
0.3 2.4 0.4 2.2 0
R. 9. FRITZ columns”
isolated per ml serum 0.43 0.47 0.38 0.39 0.24 0.37 0.02 0.07 0.02 0. I 5 0
” Samples of rabbit serum obtained at 12(*) or about 20 days after immunization wcrc applied to Sepharose- BBP columns and washed as described in Methods. The yields shown arc representative of all samples tested.
Fig. 2. Immunoelectrophoresis of protein eluted from Sepharose-BBP alfinity columns. The lower trough contains goat anti-rabbit serum; the upper trough contains goat anti-rabbit gamma-globulin. (A) immune serum rabbit No. 5; (B) immune serum rabbit No. 6. Anode is to the right
:A)
(9)
Fig. 3. lmmunoelectrophoresis of protein eluted from Sepharose-BBP affinity columns. The upper and lower troughs contain BBP. 0.25 and 0.50 mg/ml, respectively. (A) immune serum rabbit No. 5; (9) immune serum rabbit No. 6. Anode is to the right.
bined with pool I from the same immune serum, with pool 1 from other immune sera, or with normal sera from rabbit or guinea-pig. Normal chicken, calf, horse and rat sera were without inhibitory effect on pool 3 suggesting that the effect was not due to enzymatic degradation of BBP during radioimmunoassay. To further study the inhibitory effect, the ability of normal rabbit serum, heated (56”C, 30 min) normal rabbit serum and three salt-precipitated fractions of normal rabbit serum to inhibit binding of the ‘2’I-labeled gamma globulin fraction of rabbit antiBBP to BBP-coated tubes was tested. As shown in Table 4, heated normal serum and the supernatant fraction of normal rabbit serum retained inhibitory activity comparable to that of normal rabbit serum, while the two salt-precipitated fractions showed little inhibition. lmmunoelectrophoresis indicated that the supernatant fraction contained primarily albumin and alpha globulins. In order to determine whether the acid elution procedure had selectively eluted antibodies reactive with certain regions of BBP while leaving other antibodies on the column, the original sera and the acid eluates were tested for reactivity against BBP fragments I 37. 44-89 and 9G-170 by means of solid phase radioimmunoassay (Table 5). These data show that each serum and eluate was reactive with BBP and its three peptic fragments. Three of the four sera showed approximately the same degree of reactivity with the fragments before and after elution. The fourth serum (rabbit No. 4) showed some loss of reactivity to fragments 1-37 and 44-89. Radioimmunoassay by sodium sulfate precipitation showed that up to 7lY,, of the activity of the original sera was recovered in pool 3 (Table 3). The data in this table indicate that little significant activity remained in pools 1 and 2 due to the high efficiency of this type of column for antibody adsorption. It is of interest that rabbit serum IO. from which only small amounts of protein were recovered in the eluate, had good binding activity. Since the original activity of serum 10 was comparable to sera 7 and 8. this would imply that there was more inactive antibody protein in eluates 7 and 8. In two further experiments,
Rabbit
51
Antibodies
Table 2. Activity of BBP with immune sera and eluates from SepharoseeBBP columns as determined by solid phase radioimmunoassay”
Rabbit
No.
7 8 9 10
Serum 1:lO I:100
Pool I 1:lO 1:lOO
Pool 2 1: 100 I:10
1.4 2.0 0.9 0.9
0.1 0. I 0.1 0
1.0
0.2
3.1 3. I
I.1
a Sera and eluates
1.7 3.0 2.2 1.0
were assayed
0.2 0. I 0.2 0.1
at dilutions
0.4 0.3
I.6
of I : 10 and
afiinity
Pool 3 I:10 I : 100 4.3 6.3 5.1 6.6
3.5 4.8 3.7 4.6
1: 100.
Table 3. Activity of BBP with immune sera and cluates from Sepharosee BBP affinity columns as determined by radioimmunoassay using I.27 iz;I sodium sulfate
Rabbit
Sample Source
No.
Activity
Serum activity recovered in eluate (“,J
serum pool 1 pool 2 pool 3
34 3 16
9 3 47
serum pool 1 pool 2 pool 3
48 3 3 I7
6 6 36
strum pool 1 pool 2 pool 3
36 3 23
8 3 61
serum pool I pool 2 pool 3
50 6
I2
36
72
I
Table 4. The inhibitory effect of normal rabbit serum binding of anti-BBP antibodies to BBP as determined radioimmunoassay
“‘I Treatment Untreated Normal rabbit Heated normal Sodium sulfate Sodium sulfate Supernate from
serum rabbit serum pellet No. 1 pellet No. 2 pellet No. 2
proteins on the by solid phase
counts, IO min 8190 1065 1179 7233 7640 I807
Aliquots of the ‘251-labeled gamma globulin fraction of rabbit antiBBP were treated with equal volumes of the above materials diluted 1:10 in BBS and assayed in triplicate as described in Methods.
pool 3 from Sepharose-BBP adsorbed radioiodinated anti-BBP serum was added to a second column of Sepharose-BBP and washed with BBS. In both experiments approximately 60% of the pool 3 material retained the ability to bind to immobilized antigen. In order to study the nature of the inactive antibody in pool 3, polyacrylamide gel electrophoresis
was performed on seven acid eluates. In the presence of SDS and 2-ME, two bands of approx 50.000 and 23,000 daltons were observed (Figs. 4A and B). These molecular weights are consistent with the sizes of rabbit IgG heavy and light chains. However, when electrophoresis was performed in the presence of SDS alone, the same seven eluates produced a major band
A. DESJARDINS Table 5. Activity
WALLACE,
R. SHAPIRA
R. B. FRITZ
of BBP and three peptic fragments with immune sera and eluates from SepharoseeBBP affinity columns as determined by solid phase radioimmunoassay. Activity
Rabbit No.
and
Sample Source
with fragment
t activity
with BBP x 100
Activity with BBP
I-37
44 89
90~ 170
2.3 4. I 2.4 4.2 2.7 3.7 1.1 4.2
22 17 35 8 20 Ih ?? 16
36 17 20 7 9 12 34 27
2x 37 43 26 27 ?X 31 30
Serum Pool 3 Serum Pool 3 Serum Pool 3 Serum Pool 3
Total X6 71 98 31 56 66 S8 7?
determine the elfective antigenic valence of BBP by means of paired-label quantitative precipitation. In two experiments. ‘“‘I-labeled antigen was used in the precipitation reaction with ‘Z”I-labeled pooled antibody. The antibody was over 90”,, precipitable at equivalence. The antibody-antigen ratios of the precipitates in antibody excess were determined and a plot made in which effective antigenic valence was determined by extrapolation of the antibody to antigen ratio to infinite antibody excess (Fritz et ul..
of approx 170,000 daltons and four other bands of approximately 120,000. 80.000, 50,000 and 20,000 daltons when compared to standard proteins (Fig 4C). Normal rabbit IgG did not show significant amounts of these smaller components after treatment with 0.1 M acetic acid (Fig. 4D). Although some sera were obtained as early as 12 days after immunization, IgM antibodies were not detected in the acid eluates. Anti-BBP IgG purified by immunoadsorption and Sephadex-G-50 chromatography was then used to
B
A 2.0 -
g s
II
5
123456709
“0r
I*
2 I.
3
4
Fig. 4. Polyacrylamide gel Immune serum (rabbit No. (treated with 0.1 M acetic C
5
6 .
7 8 DISTANCE I.
1
C1123456709 D
9 0 I 2 FROM TOP OF GEL (cm)
3
4
5
6
7
0
9
electrophorcsis of protein cluted from a Sepharose-BBP affinity column. 5) with SDS and 2-ME (A) and with SDS only (C). Normal rabbit IgG acid) with SDS and Z-ME (B) and with SDS only (D). Patterns A and are representative of the seven acid eluates tested.
5?
Rabbit Antibodies
I
1
I
2
3
4
ANTIGEN
5
6
7
8
ADDED (yg)
Fig. 5. Quantitative precipitation of ‘251-labeled anti-BBP IgG and r3rI-labeled BBP. The points shown are the means
of triplicate
determinations
1967). The results of the experiments are shown in Fig. 5. From these data it may be seen that the effective antigenic valence of BBP is approximately four. DISCUSSION
Anti-BP antibodies of guinea-pigs and rabbits have been partially characterized. Lisak et (11.(1969) found 7S antibody in 13:~ of guinea-pigs after injection with homologous BP and CFA, by radioimmunoelectrophoresis of immune sera with specific anti-guinea-pig antisera. Using a gel filtration assay and Z-ME sensitivity, Lennon et (11.(1971) found mainly IgM in the serum of rabbits injected with human BP and CFA. although some IgG was found twenty days after the rabbits were boosted. Pitts et al. (1976) used BP coupled to Sepharose to adsorb antibodies from immune rat sera in their study of the cross-reactivity of rat antibodies to BP from beef, pig, rabbit, guineapig and rat, but they did not report characterization of the antibodies. In the present study, SepharoseBBP columns were used to adsorb anti-BBP activity from immune rabbit sera, and antibody recovered in a 0.1 M acetic acid wash (pool 3) was active in a sodium sulfate precipitation radioimmunoassay (Day & Pitts, 1974) and in a solid phase radioimmunoassay (Randolph et u/., in press). A normal rabbit serum fraction containing primarily albumin and alpha globulins inhibited binding of antibodies to BBP in the solid phase RIA. It is not clear why this inhibitory effect was not observed with the salting-out method of RIA, although it is possible that the high Na,SO, concentration of the assay decreases binding of the inhibitor(s) to BP. The major antibody component isolated in pool 3 was of the IgG class as shown by immunoelectrophoresis and polyacrylamide gel electrophoresis. It is possible that these procedures were not sufficiently sensitive to detect small amounts
and the bars indicate
standard
error.
of IgM antibodies. The results of analysis of pool 3 by polyacrylamide gel electrophoresis were interesting in that upon denaturation and reduction only two bands were observed and the molecular weights of the polypeptides in the bands were consistent with rabbit IgG light and heavy chains; while upon denaturation with SDS alone, several bands appeared in the gels suggesting disaggregation of non-covalently bound IgG complexes. The molecular weights of these components were consistent with various combinations of light and heavy immunoglobulin chains. The number and location of humoral antigenic determinants in the BP molecule have been studied by several different methods of immunization and assay. Using passive hemagglutination, Hashim and Eylar (1969), Burnett and Eylar (1971) and Eylar et ul. (1971) found no activity to BBP fragment 117-170 in guinea-pigs and none in BBP fragment 44-89 in rabbits. McFarlin et (11.(I 975) used a double antibody radioimmunoassay and found no activity to BBP fragment 45-88 in Lewis rats injected with the whole protein. In rabbits injected with BBP coupled to rabbit serum albumin, BBP fragments l-43, 9&170 were active and 44-89 inactive when studied by quantitative complement fixation hapten inhibition (Whittaker ef al., 1975). In partial contrast, Driscoll et al. (1974) found three antigenic sites, in fragments 44-89, 9&l 16 and 117-170, in guinea-pigs injected with GP BP and CFA. In our studies, sera and acid eluted antibodies with high anti-BBP activity were tested for ability to bind to tubes coated with the BBP fragments, l-37, 4489 and 9@170. All sera and eluates tested demonstrated activity with all three fragments with fragment 90-170 usually more active than fragments l-37 and 44-89. These results suggest the presence of at least one antigenic site in each fragment, and possibly more in fragment 90-170. From the quantitative precipitation data of Webb et al. (1973)
54
A. DESJARDINS
WALLACE.
where BBP was added to the serum of rabbits immunized with a phosvitin-BBP complex, Day and Pitts (1974) estimated that there were three antigenic determinants in the BBP molecule. In our studies a pool of immunospecifi~dlly purified anti-BBP IgG was used to estimate the number of humoral detcrminants in the molecule. Extrapolation of the data to infinite antibody excess indicated that the effective antigenic valence of BBP is approximately 4 which is in agreement with data obtained with other proteins of similar molecular weight (Ginader & Pearce, 19561. REFFRERiCES 1 1
Burnett P. R. & Eylar
1
E. H. (1971) J. hiol. Chem. 246, 3425. Carnegie P. R. (1971) Biochern. J. 123, 57. Chou F. C.-H., Chou C.-H. Jen, Shapira R. & Kibier R. F. (1976) J. hiof. CCwm. 251, 2671. Chou F. C.-H., fhou C.-H. Jen, Shapira R. & Kibler R. F. (1977)J. Newociiem. 28, 115. Cinader B. & Pearce J. H. (1956) Br. J. ~.yp. Puth 37, 541. Cuatrecasas P. & Anfinsen C. B. (1971) Methodsin EKJmoloyy(Edited by Jakoby W. B.). Vol. 22, p. 345. Academic Press, New York. Day E. D. & Pitts 0. M. (1974) Itnnru,locllrmisfrg fl, 651. Driscoll 3. F., Kramer A. J. & Kies M. W. (1974) Science 184, 13. Epand R. H., Moscarello M. A.. Zierenberg B. & Vail W. J. (1974) Biochemistry 13, 1264. Eylar E. H. (1970) Proc. mm. Acud. SC.. C’.S.A. 67. 1425. Eylar E. H. & Thompson M. (1969) Archs Biochrm. Binwph~x 129. 468. Evlar E. H.. Westall F. C. & Brostoff S. (1971), J. hi& ‘Chem. 246. 3418. Fairbanks G.. Steele T. L. & Wallack D. F. (1971) Biockenri.sfrl~
IO.
2XOh.
R. SHAPIRA
and
R. B. FRITZ
Fritz R. B., Lassiter S. & Day E. D. (1967) J. //wtwr. 98. 1713. Greenwood F. c’.. Hunter W. M. & Clover J. S. (1963) Biochlwf. J. 89, 114.
Humienny M. E., McKncally S., Kibler R. F. 61 Shapiro R. (I 9741 ‘I‘rilr~s. .1/n Sot. !Vrwoc/w/,~. 5. 141. Kies M.. Murphy J. B. & Alvord E. C. (1960) FL’&. Proc.. Fedrl. Aw. Soc,s e.\-p. Biol. 19, 207. Krigbaum W. R. & Hsu T. S. (1975) Biochemistry 14, 1975. Lennon V. A.. Whittil~gl~~lrn S.. Carnegie P. R.. McPherson T. A. & Mackay I. R. (1971) J. ~~~~~~f/l.107. 1971. Lisak R. P.. Hcinzc R. G.. Kies M, W. & Alvord E. C. (1969) Prw. So<,. r’\-,‘. Uicji. M~vl. 130. X14. Lowry 0. H., Rosebrough N. 1.. Farr A. L. & Randall R. j. (1951) J. hiol. Cl&. 193, 265. Marchalonis J. J. (1969) Biocl~rt~ .I. 113. 299 McFarlin D. E.. l&u S. C.-L.. Slcmenda S. B.. Chou F. C.-H. & Kibler R. F. (1975) J. r.up. hfcd. 141, 72. Palmer F. B. Cp Dawson R. M. C. (1969) ~i~~~~7~,~~~. J. 111, 629. Pltts 0. M.. Variteck V. A. & Day E. D. (1976) Irnrwrwc~/le,,listr~ 13, 307. Randolph D. H. (1976) M. S. thesis. Emory University, Atlanta. Georgia. Randolph D. H.. Kibler R. F. & i:rit7 R. B. (1977) J. irw flw~. Mc~tli. (in press). Rohoz-Einstein E., Robertson D.. DiCaprto J. & Moore W. / 1962) J. Mvurochrr,~. Sela M. & Mazes E. (1966) Proc. 1wrt1. Actrll. Sci., C’.S.A. 55, 445. Shapiro A. W.. Vinuela E. 6i Maigel J. (1967) Biochwt. hiopl~.vs. Res. Comtw~i. 28, Xt 5. Webb C.. Teitelbaum D., Arnon R. & Sela M. (1973) Eur. J. l~~~~~ll/l~. 3, 179. Whittaker J. N.. Chou C.-H. Jcn, Chou F. C.-H. R: tiibler R. F. ( 1975) ,I. hiol. C/wnt. 250, 9106.