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Isolation, characterization and idiotype of Lewis rat antibodies against peptide 68–88 of guinea pig myelin basic protein
,Mole
I’). No 5. pp. 665 670. 1982
0161-5X90,82.050665-06$03 0O;O 0 IYX? Pcrgamon Press Ltd
ISOLATION, CHARACTERIZATION AND IDIOTYPE OF LEWIS RAT ANTIBODIES AGAINST PEPTIDE 68-88 OF GUINEA PIG MYELIN BASIC PROTEIN ROBERT Departments
B. FRITZ, ANNE E. DESJARDINS
of Microbiology
(Receioed
and
RAYMOND
and Biochemistry, Emory University GA 30322, U.S.A. 30 Sepremher
School
SHAPIRA of Medicine,
Atlanta,
~lccepted30 Ocroher 1981)
1981;
Abstract--Antibodies against the 19 amino acid encephalitogenic peptide (residues 68-88) of guinea pig myelin basic protein (GPBP) were raised in Lewis (Le) rats. Anti-peptide antibodies were isolated from immune ascitic fluids by affinity chromatography using peptide 43-88%Sepharose 4B. The purified antibodies were characterized by sodium dodecyl sulphateepolyacrylamide gel electrophoresis and isoelectric focusing. Immunoglobulin class was determined by radioimmunoassay. Anti-idiotypic (anti-ID) antibodies were raised in a rabbit using purified anti-peptide antibodies from a single rat. The results of these experiments showed antibody heterogeneity both within an individual anti-peptide antiserum and between antisera from different rats. Antibody activity was found in IgGl, IgG2 and IgE immunoglobulin classes. Isoelectric focusing revealed multiple bands within a population of purified antibodies with significant pattern variation from one antiserum to another. Idiotypic characterization showed various levels of cross-reactive idiotypes present in some sera while these were absent in others.
INTRODUCTION
Immunization of Le rats with GPBP peptide 68-88 in complete Freund’s adjuvant (CFA)* induces antibody to the peptide (Kibler et al., 1977). Cell-mediated immunity as manifested by EAE in vivo, and a LPR and production of macrophage migration inhibition factor when lymphocytes from sensitized animals are cultured in vivo in the presence of peptide are also induced in this manner (Chou et al., 1979; Waxman et al., 1980). A major epitope reactive with antibody is located in the region of residues St%38 (Fritz et al., 1979). The latter studies utilized serum antibodies and showed heterogeneity of fine specificity among individual rat antisera. Since peptide 68-88 is a small immunogen and likely has only a single B-cell epitope, it was of interest to determine whether the antibody response to the immunogen was restricted with respect to the number of distinct antibodies produced as well as with respect to idiotype.
In order to answer this question anti-peptide antibodies were purified from immune ascitic fluids (Tung et al., 1976) and characterized by SDS-gel electrophoresis, and isoelectric focusing. Idiotypic heterogeneity was assessed by production of rabbit anti-ID antibody. The anti-ID antiserum was then tested against a panel of anti-peptide antibodies. The results of these studies showed that the anti-peptide antibodies were heterogeneous with respect to immunoglobulin class, charge and idiotype.
MATERIALS
AND METHODS
Animals Female Le rats were obtained from Charles River Breeding Laboratories (North Wilmington, Massachusetts), or from Microbiological Associates (Bethesda, Maryland), and were used at 3-5 months of age. Four to six months old random bred female New Zealand White rabbits were obtained from local suppliers.
*Abbreviations: GPBP, guinea pig basic protein; Le, Lewis; anti-ID, anti-idiotypic; CFA, complete Freund’s adjuvant; EAE, experimental allergic encephalomyelitis; LPR, lymphocyte proliferative response; BBS, borate buffered saline; RIA, radioimmunoassay; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis; IEF, isoelectric focusing; IFA, incomplete Freund’s adjuvant; i.p., intraperitoneal.
Antigens GPBP fragments 68-85, 68-88 and 79-88 were supplied by Drs Frank Chou and C.-H. Jen Chou and were prepared as previously described (Chou et a/., 1977, 1979). Purity of the 665
666
ROBERT
B. FRITZ.
ANNE E. DESJARDINS
fragments was assessed by amino acid analysis and peptide mapping.
and RAYMOND
labeled with “‘1 by the lactoperoxidase method of Marchalonis (1969). Solid-phase
Rat antibodies and immune ascites to peptide 68-88 were prepared as described by Fritz et (11.( 1979).
Rat antibodies to peptide 68-88 were purified by passage of antisera or immune ascitic fluids through a 1.5 x 2.0cm column of peptide 43-88 coupled to Sepharose-4B prepared according to the general procedure of Cuatrecasas & Anfinsen (1971). After adsorption, the column was washed with 50&2000 ml of 0.15 M BBS, pH 8.1. Adsorbed material was eluted with 3 M MgCl,, and the column was rewashed with BBS. In order to preserve antibody activity, fractions eluted from the column were collected directly into dialysis bags immersed in BBS at 4°C. After overnight dialysis, the fractions containing immunoglobulin were pooled and concentrated by vacuum dialysis. Protein content of the eluates was determined by optical density measurement at 280nm using an extinction coefficient of 14 for a 1% solution. Binding of normal serum proteins to fragment 43-88 was assessed by the same procedure. Rabbit anti-rat, goat anti-rabbit and rabbit anti-goat gamma globulin were purified by adsorption and elution from rat, rabbit or goat gamma globulins covalently coupled to Sepharose-4B. Five to 20 ml of the appropriate immune serum were added to a column (lx2cm) of insolubilized antigen. The column was washed with 0.02 M sodium phosphate buffered saline, pH 7.5, or 0.15 M BBS, pH 8.1, until the absorbance at 280 nm became zero. The adsorbed anti-globulin was then eluted with 2 M potassium iodide or 3 M MgCl,, the eluate dialysed against BBS, concentrated by vacuum dialysis and frozen until used. The protein content of eluaies was determined by OD measurement at 280 nm using an extinction coefticient of 14 for a l”i, solution. Radioiodination
Peptide 43-88, rabbit anti-rat gamma globulin, goat anti-rabbit gamma globulin, and rabbit anti-goat gamma globulin were radio-
SHAPIRA
r~dioi~n~l~no~.~s~~ (RIA)
Immunoglobulin class and subclass of rat antibodies to peptide 68-88 were measured by a modification (Fritz et al., 1979) of the solidphase RIA of Randolph et al. (1977). Normal sera bound 3”/, (anti-a), 5% (anti-E), 5% (anti-& 3% (anti-y,), 8% (anti-y,,) and 3% (anti-y,,) of radioactivity added. The SD was 5--10X, of the mean. The class and subclass specific antisera were the kind gift of Dr Herve Bazin.
Sodium s~rlfite RIA
Rat antibodies to peptide 68-88 were measured by a modification (Kibler et a/., 1977) of the sodium sulfate precipitation procedure of Day & Pitts (1974). The ability of peptides 68-88 and 79-88 to inhibit binding of antibody to radioiodinated peptide 43-88 was determined as previously described (Fritz rt N/., 1979). SDS-PAGE
SDS-PAGE was performed by the procedure of Laemmli (1970) at 2 mA per gel for approximately 5 hr at room temperature. Samples were heated in a solution of 0.06 M Tris, 0.7 &I ~-mercaptoethanol, 10% glycerol at 100°C for 30 set prior to application to the gel column. Following electrophoresis the gels were immersed in 150/ trichloroacetic acid for 20 min, and then stained with 0.25% Coomassie Blue in 50% methanol, 77; acetic acid for 2 hr. After destaining densitometric scanning of the stained proteins was done at 600nm in a Beckman Model 25 spectrophotometer equipped with a gel scanner. Mol. wts were estimated from concurrently run protein standards. IEF
IEF was done using horizontal 5% polyacrylamide gel slabs in an LKB 2117 Multiphore apparatus. The gel solution consisted of 7.5 g sucrose, 10 ml of 29.10,;;acrylamide, 10 ml of 0.9% bisacrylamide, 3.0 ml of ampholines, pH 3.5-10, 0.4 ml of ampholines, pH 9-l 1, and 0.4 ml of 0.004% riboflavin. The gel was photopolymerized using U.V. light. Samples.
Rat Anti-encephalitogenic
25100 ,ul, were applied to 0.75 cm squares of filter paper and placed on the gel surface. Following electrofocusing the pH gradient was determined at 1 cm intervals. The gel was then immersed overnight in a solution containing 22.5 g of sulfosalicylic acid, 75 g of trichloroacetic acid, 0.75 g of Coomassie Brilliant Blue R-250 in a mixture of 225 ml of methanol and 465 ml of distilled water. The next day the gel was destained with several changes of destaining solution (8 : 3 : 1 water: ethanol : acetic acid). The individual tracks were then sliced out of the gel and scanned at 600 nm.
Anti-ID
antiserum
A single rabbit was immunized by injection of purified anti-peptide antibody from a single rat. The primary immunization consisted of 1OOpg of protein emulsified in CFA. Booster injections at 3 and 6 weeks were given with a similar amount of protein emulsified in IFA. The rabbit antiserum was exhaustively absorbed by repeated passage over a column of Sepharose 4B to which was conjugated a preparation of crude Le rat globulin. RIA for anti-ID antibodies were carried out as follows. Fifty microliters of absorbed anti-ID or preimmune serum were mixed with 50 ~1 of rat anti-peptide antibody. After incubation at 4°C overnight 20 ~1 of this mixture were added to 200 ~1 of a 1:3 dilution of normal rabbit serum. To this mixture was added 20~1 of a 10m6 M solution of radioiodinated peptide 43-88 (4 x 104-6 x 104cpm). After 90min, immune complexes were precipitated from this mixture by addition of 2 vol 1.9 M sodium sulfate. The precipitate was removed by centrifugation, washed 3 x with 1.27 M Na,SO, and assayed for radioactivity. Anti-ID activity was expressed as the per cent inhibition of binding of radiolabeled peptide as compared to those tubes which contained preimmune serum in the initial mixture. All anti-peptide antisera were titrated prior to the RIA to the point where 10-15x of the radiolabeled peptide was bound to insure that the assay was carried out in antigen excess.
RESULTS
Le rats were challenged and boosted with peptide 68-88 of GPBP. Although this strain is highly inbred, previous experiments showed
Peptide
Antibodies
667
variation in antibody fine specificity from rat to rat, thus use of pooled sera for antibody characterization was undesirable. In order to obtain large amounts of antibody, immune ascitic fluids were induced by repeated intraperitoneal (i.p.) injections of antigen and complete adjuvant (Tung et al., 1976). Since the possibility existed that antibody quantity, specificity, or major class and subclass might differ in ascites fluids and sera, preliminary experiments were done in order to investigate this point. When anti-peptide 68-88 antisera and immune ascitic fluids obtained on the same day from several rats were titrated for antibody content and specificity by sodium sulfate RIA, the two fluids were found to be very similar. The distribution of antibody activity among the various classes of immunoglobulins was examined using class or subclass-specific antisera in a solid-phase RIA. The results of these experiments showed that the distribution of antibody activity was similar in immune serum and ascites, and that antibody activity resided primarily in the IgG2 and IgE immunoglobulin classes. Affinity chromatography, which had been used for isolation of antibodies to myelin basic protein (Wallace et al., 1978) was used to purify antibodies to peptide 68888. In general, antisera or immune ascites fluids were added to a small column of Sepharose-4B-peptide 43-88, allowed to incubate in the cold for 1 hr and then exhaustively washed with BBS. Bound proteins were then eluted with 15 ml of 3 M MgCl,. After dialysis and concentration, the eluted proteins were tested for antibody activity by means of the sodium sulfate RIA. The results of affinity chromatography are shown in Table 1. The data were obtained by titration of each serum or eluate to the point of maximum antigen binding in antigen excess, and the eluates are corrected to starting volume for comparison with the original antisera. As shown in Table 1, there was extensive variation in recovery from serum to serum. The best and worst cases have been presented in order to illustrate the wide range of recovery. For example, serum E consistently gave poor recoveries over a period of several months with a usual recovery of approximately 20%. Serum D generally gave recoveries of S&950/, over the same period under identical conditions. When eluates and precolumn antisera were compared with respect to class and subclass of immunoglobulin (Table 2) the compositions of each
ROBERT
668
Table
B. FRITZ,
I. Binding
ANNE
activity
E. DESJARDINS
of affinity-purified antibodies pmoles
Rat anti-peptide 68-88
rat anti-peptide
of ‘Z51-peptide 43388 bound milliliter”~b
Original
J K L C D E
and RAYMOND
per
342 295 77 300 726 116
“Determined by titration in antigen excess. ‘Normal Le rat serum controls bound 3 10 pmoles 43-88 per milliliter.
Table 2. Immunoglobulin fluid and affinity-purified
68 88
3 M MgCl,
788 864 247 453 139 673
were similar with the exception of loss of IgM antibodies in the eluates. The results of the affinity chromatography experiments suggest heterogeneity of binding affinity from one serum to another. However, this procedure did not select with respect to class since the eluates were very similar to the starting material in this respect. In order to determine whether the eluates were contaminated with non-immunoglobulin proteins, each preparation was analysed by IEP against goat anti-rat serum. By this method all preparations were shown to contain IgG2 and many had IgGl antibodies as well. The solid-phase RIA (Table 2) showed other immunoglobulin classes to be present. However, our goat anti-rat serum apparently did not detect these by immunoelectrophoresis. Analysis of the eluates by SDS-PAGE under reducing conditions in 10% gels confirmed the earlier observations. The major polypeptides observed correspond to light chains and gamma heavy chains (Fig. 1). With several eluates a peak at mol. wt 70,000 was observed, likely representing epsilon chain, and often a
SHAPIRA
of ‘z51-peptide
second distinct band was noted in the lightchain region. Under identical conditions our DEAE-cellulose-purified IgG2 standard gave a single light-chain band and two heavy-chain bands estimated to have mol. wts of 22,000, 50,000 and 55,000, respectively. All eluates also gave two heavy-chain bands corresponding to the two latter mol. wts. In order to assess charge heterogeneity of the affinity-purified anti-peptide antibodies, preparations of MgCl,-eluted proteins were analysed by IEP in pH 3.5-10 gradients (Fig. 2). Also shown in this figure is a pattern obtained with DEAE-cellulose-purified IgG2 analysed under identical conditions. The latter preparation gave approximately 20 distinct bands. Affinity-purified preparations often gave patterns which were more complex than the standard although the majority of the proteins focused in the pH range of 778. However, affinity-purified preparations also contained a number of additional proteins which focused at pH less than 7.
class and subclass of rat ascitic antibodies to peptide 68888
Net “/, bound of anti-class or subclass antiserum added” Class or subclass Alpha Epsilon Mu Gamma Gamma Gamma
Ascitic fluid
1 2A 2C
“Determined
3 M MgCI,
15 34 10 24 51 21 by solid-phase
22 29 0 39 50 18 radioimmunoassay.
eluate
Fig. 1. SDSPAGE pattern of MgCl,-affinity-purified rat anti-peptide 68-88 stained with Coomassie Blue. Acrylamide concentration was lo:;,. L, OVA, H and BSA refer to rat immunoglobulin light chain, ovalbumin rat immunoglobulin G heavy chains, and bovine serum albumin, respectively, used as mol wt markers. Migration is from right to left.
Rat Anti-encephalitogenic
Peptide
669
Antibodies
detect those idiotypes associated with the antigen binding site. Thus, total idiotypic crossreactivity might be somewhat greater. DISCUSSION
I
These experiments were designed to evaluate the degree of heterogeneity of the antibody response to encephalitogenic peptide 68-88 in the Le rat. Three levels of heterogeneity were assessed; class heterogeneity, charge heterogeneity and idiotypic heterogeneity. Since immune ascitic fluids were the starting material for purification by affinity chromatography, preliminary experiments compared antibody levels and class in these fluids with serum antibodies. No significant differences were noted. Antibody activity was found predominantly in the IgG2 and IgE class with some animals having significant levels of IgGl antibodies as well. Although the anti-peptide response is considerably more restricted than the antibody response to basic protein (Fritz et al., 1979) it is still heterogeneous with respect to class and subclass. SDS-PAGE of the purified antibodies showed that they consisted predominantly of polypeptide chains of mol. wts 22,000 and 50,000. Occasionally, a second peak was seen in the light chain region and. in all instances. two bands of approximately equal staining density were seen in the gamma heavy-chain region. In many preparations a less densely stained band of mol. wt approx. 70,000 was seen. Since IgE was present at significant levels in the anti-peptide 684% antibodies in this as well as previous studies (Fritz et al., 1979). the latter band likely corresponds to epsilon heavy chain.
Table Fig. 2. Densitometric tracings of Coomassie Blue stained MgCI,-affinity-purified rat anti-peptide 68-88 antibodies after IEF. Panels A and B represent two different affinitypurified antibodies; panel C is a tracing of DEAE-purified normal Le rat immunoglobulin.
In order to test for idiotypic cross-rectivity, rabbit anti-ID antiserum was tested against a panel of 10 individual Le rat anti-peptide antibodies. The results showed significant idiotypic cross-reactivity with only two antisera (Table 3). It should be emphasized that the assay used to assess idiotypic cross-reaction would only
3. Idiotypic cross-reactivity of Le rat anti-peptide 68-88 antisera
Anti-peptide 68-88 antiserum A B C D E F G H I J
“() inhibltion 0 1 0 90” 53 7 0 0 0 34
“Homologous reaction-antiserum used for immunization of rabbit.
D
670
ROBERT
B. FRITZ,
ANNE
E. DESJ.~RDlNS
We have noted two heavy-chain bands of mol. wts 50,000 and 55,000 in all of our normal rat IgG standards. The larger of the two bands is reduced in amount when DEAE-purified rat IgG is analysed under these conditions implying that this polypeptide chain is associated with a more negatively charged molecule. Charge heterogeneity of the eluates was revealed by electrofocusing. Affinity-purified anti-peptide 68-88 antibodies often gave patterns which were more complex than the IgG standard. This is not surprising in light of the heterogeneity observed in the solid-phase RIAs. By the latter method the affinity-purified preparations were found to contain IgGl, IgG2 and IgE, although the levels of these immunoglobulins varied from serum to serum. The fact that the antibody response to this small defined antigen was quite heterogenous is not surprising in view of the data showing extensive variation of binding afinities (Eisen & Siskind, 1964) of anti-hapten antibodies in the course of the immune response. An earlier study from our laboratory had shown heterogeneity of antibodies raised by the immunization of Le rats with peptide 43-88 from myelin basic protein (Fritz et al., 1978). Kreth & Williamson (1973) have estimated that CBA/8 mice are potentially capable of producing 8000 different anti-NIP (4-hydroxy-5-iodo-3-tlitrophenacetyl) antibodies. The results of testing 10 anti-peptide antibodies for idiotypic cross-reactivity showed that with this anti-idiotypic antiserum only a minority of the rat antisera carried cross-reactive idiotopes. It should be emphasized that these results were obtained only with a single anti-ID antiserum. The number and location of the idiotopes recognized by this antiserum are unknown although they are located at or in close proximity to the antigen binding site. However, it is apparent that with respect to the idiotopes measured by this RIA, there is considerable variation from one rat anti-peptide antiser~im to another as well as within a single antiserum. In summary, rat anti-peptide 68.--88 antibodies were found to be heterogeneous within a single antiserum with respect to class. charge and idiotype. Antibody activity was found in the IgGl. IgG2 and IgE immunoglobulin
and RAYMOND
SHAPIRA
classes. IEF revealed multiple bands within a single antiserum. Comparison of different antipeptide antisera by IEF revealed both common and unique immunoglobulin species. Crossreacting idiotopes were present in some antipeptide antisera. Ackno,~lc~df/ert?~,~~f,s This work was supported Grants NS 1141X and NS 10721.
by USPHS
REFERENCES
Chou F. C.-H.. Chou C.-H. J.. Kowalski T. J.. Shapira R. & Kibler R. F. (1977) The major site of guinea pig myelin basic protein and encephalitogcnic in Lewis rats. f. N~~ur~f+m. 28, 1 i5- 119. Chou C.-H. J., Fritz R. B., Chou F. C.-H. & Kibler R. F. (1979) The immune response of Lewis rats to peptide 6X-88 of guinea pig myelin basic protein. 1. T cell determinarlts. J. ~~?z~I~~~I. i23? 1540 1543. Cuatrecasas P. & Anfinsen C. B. (1971) Afiinity chromatography. In Mrrhod.s it1 En-?moloy~ (Edited by Colowick S. P. & Kaplan N. 0.). Vol. 22. p. 345. Academic Press, New York. Day E. D. & Pitts 0. M. (1974) R~idioimmun(~~~ssay of myelin basic protein in sodium sull’atc. Irnnirir?tit~fipnii.srr) II, 651 659. Eisen H. N. & Siskind G. W. (lY64) Variations in alfinities of antibodies during the immune response. BIO~/?P~PG\~PJ 3, 99& 1008. Fritz R. B.. Chou F. C.-H., Chou C.-H. J. & Kibler R. F. (1979) The immune response of Lewx rats to peptide 6X XX of guinea pig myelin basic protein. II. B cell determinants. J. Immztn. 123, 1544-l 547. Fritz R. B.. Chou C.-H. J., Randolph D. H., Desjardins A. E. & Kibler R. F. (1978) Specificity of antisera from Lewis rats immunized with encephalitogemc fragment 43-88 of guinea pig myelin basic protein. J. lmn~un. 121, 1X65-1869. Kibler R. F.. Fritz R. B.. Chou F. C.-H.. Chou C.-H. J., Pcacockc N. Y., Brown N. M. & McFarlin D. E. (1977) Immune response of Lewis rati to peptide Cl (residues 68 88) of guinea pig and rat my&n basic protein. J. e’r,~. M&. 146, 1323 1331. Kreth H. W. & WIlliamson A. R. 11973) The extent of diversity of ~lnti-hapten antibodies‘in &red mice: antiNIP (4-hydroxy-S-iodo-3-nitrophenacetvl) antibodies in CBA:H mice. Eur. J. Immun. 3; I41 -14;. Marchalonis J. J. (1969) An enzymatic method for the trace iodination of immunoglobulins and other proteins. Biocf7m. J. 113, 299 30s. Randolph D. H.. Kibler R. F. & Frit/ R. 9. (1977) Solidphase radioimmunoassay for detection of antibodies to myelin basic protein. J. Imniu~?. Merit. 18, 215. 224. Tung A. S.. Ju S.. %itO S. & Nisonolf A. (1976) Production of large amounts of antibodies in individtiai mice. J. Irnmu,i. 116,676 6X i. Wallace A. D., Shapira R. & Fritz R. B. (197X) Isolation and characterization of rabbit antibodies to bovine myelin basic protein. Immunochemistrr 15. 47-54. Waxman F.. Fritz R. B. & Hinrichs D. J. (IYXO) The presence of specific antigen-reactive cells during the induction. recovery and resistance phases of experimental allergic enccphalomyelitis. Ccl/. Imtmrn. 49, 34-42.
I’). No 5. pp. 665 670. 1982
0161-5X90,82.050665-06$03 0O;O 0 IYX? Pcrgamon Press Ltd
ISOLATION, CHARACTERIZATION AND IDIOTYPE OF LEWIS RAT ANTIBODIES AGAINST PEPTIDE 68-88 OF GUINEA PIG MYELIN BASIC PROTEIN ROBERT Departments
B. FRITZ, ANNE E. DESJARDINS
of Microbiology
(Receioed
and
RAYMOND
and Biochemistry, Emory University GA 30322, U.S.A. 30 Sepremher
School
SHAPIRA of Medicine,
Atlanta,
~lccepted30 Ocroher 1981)
1981;
Abstract--Antibodies against the 19 amino acid encephalitogenic peptide (residues 68-88) of guinea pig myelin basic protein (GPBP) were raised in Lewis (Le) rats. Anti-peptide antibodies were isolated from immune ascitic fluids by affinity chromatography using peptide 43-88%Sepharose 4B. The purified antibodies were characterized by sodium dodecyl sulphateepolyacrylamide gel electrophoresis and isoelectric focusing. Immunoglobulin class was determined by radioimmunoassay. Anti-idiotypic (anti-ID) antibodies were raised in a rabbit using purified anti-peptide antibodies from a single rat. The results of these experiments showed antibody heterogeneity both within an individual anti-peptide antiserum and between antisera from different rats. Antibody activity was found in IgGl, IgG2 and IgE immunoglobulin classes. Isoelectric focusing revealed multiple bands within a population of purified antibodies with significant pattern variation from one antiserum to another. Idiotypic characterization showed various levels of cross-reactive idiotypes present in some sera while these were absent in others.
INTRODUCTION
Immunization of Le rats with GPBP peptide 68-88 in complete Freund’s adjuvant (CFA)* induces antibody to the peptide (Kibler et al., 1977). Cell-mediated immunity as manifested by EAE in vivo, and a LPR and production of macrophage migration inhibition factor when lymphocytes from sensitized animals are cultured in vivo in the presence of peptide are also induced in this manner (Chou et al., 1979; Waxman et al., 1980). A major epitope reactive with antibody is located in the region of residues St%38 (Fritz et al., 1979). The latter studies utilized serum antibodies and showed heterogeneity of fine specificity among individual rat antisera. Since peptide 68-88 is a small immunogen and likely has only a single B-cell epitope, it was of interest to determine whether the antibody response to the immunogen was restricted with respect to the number of distinct antibodies produced as well as with respect to idiotype.
In order to answer this question anti-peptide antibodies were purified from immune ascitic fluids (Tung et al., 1976) and characterized by SDS-gel electrophoresis, and isoelectric focusing. Idiotypic heterogeneity was assessed by production of rabbit anti-ID antibody. The anti-ID antiserum was then tested against a panel of anti-peptide antibodies. The results of these studies showed that the anti-peptide antibodies were heterogeneous with respect to immunoglobulin class, charge and idiotype.
MATERIALS
AND METHODS
Animals Female Le rats were obtained from Charles River Breeding Laboratories (North Wilmington, Massachusetts), or from Microbiological Associates (Bethesda, Maryland), and were used at 3-5 months of age. Four to six months old random bred female New Zealand White rabbits were obtained from local suppliers.
*Abbreviations: GPBP, guinea pig basic protein; Le, Lewis; anti-ID, anti-idiotypic; CFA, complete Freund’s adjuvant; EAE, experimental allergic encephalomyelitis; LPR, lymphocyte proliferative response; BBS, borate buffered saline; RIA, radioimmunoassay; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis; IEF, isoelectric focusing; IFA, incomplete Freund’s adjuvant; i.p., intraperitoneal.
Antigens GPBP fragments 68-85, 68-88 and 79-88 were supplied by Drs Frank Chou and C.-H. Jen Chou and were prepared as previously described (Chou et a/., 1977, 1979). Purity of the 665
666
ROBERT
B. FRITZ.
ANNE E. DESJARDINS
fragments was assessed by amino acid analysis and peptide mapping.
and RAYMOND
labeled with “‘1 by the lactoperoxidase method of Marchalonis (1969). Solid-phase
Rat antibodies and immune ascites to peptide 68-88 were prepared as described by Fritz et (11.( 1979).
Rat antibodies to peptide 68-88 were purified by passage of antisera or immune ascitic fluids through a 1.5 x 2.0cm column of peptide 43-88 coupled to Sepharose-4B prepared according to the general procedure of Cuatrecasas & Anfinsen (1971). After adsorption, the column was washed with 50&2000 ml of 0.15 M BBS, pH 8.1. Adsorbed material was eluted with 3 M MgCl,, and the column was rewashed with BBS. In order to preserve antibody activity, fractions eluted from the column were collected directly into dialysis bags immersed in BBS at 4°C. After overnight dialysis, the fractions containing immunoglobulin were pooled and concentrated by vacuum dialysis. Protein content of the eluates was determined by optical density measurement at 280nm using an extinction coefficient of 14 for a 1% solution. Binding of normal serum proteins to fragment 43-88 was assessed by the same procedure. Rabbit anti-rat, goat anti-rabbit and rabbit anti-goat gamma globulin were purified by adsorption and elution from rat, rabbit or goat gamma globulins covalently coupled to Sepharose-4B. Five to 20 ml of the appropriate immune serum were added to a column (lx2cm) of insolubilized antigen. The column was washed with 0.02 M sodium phosphate buffered saline, pH 7.5, or 0.15 M BBS, pH 8.1, until the absorbance at 280 nm became zero. The adsorbed anti-globulin was then eluted with 2 M potassium iodide or 3 M MgCl,, the eluate dialysed against BBS, concentrated by vacuum dialysis and frozen until used. The protein content of eluaies was determined by OD measurement at 280 nm using an extinction coefticient of 14 for a l”i, solution. Radioiodination
Peptide 43-88, rabbit anti-rat gamma globulin, goat anti-rabbit gamma globulin, and rabbit anti-goat gamma globulin were radio-
SHAPIRA
r~dioi~n~l~no~.~s~~ (RIA)
Immunoglobulin class and subclass of rat antibodies to peptide 68-88 were measured by a modification (Fritz et al., 1979) of the solidphase RIA of Randolph et al. (1977). Normal sera bound 3”/, (anti-a), 5% (anti-E), 5% (anti-& 3% (anti-y,), 8% (anti-y,,) and 3% (anti-y,,) of radioactivity added. The SD was 5--10X, of the mean. The class and subclass specific antisera were the kind gift of Dr Herve Bazin.
Sodium s~rlfite RIA
Rat antibodies to peptide 68-88 were measured by a modification (Kibler et a/., 1977) of the sodium sulfate precipitation procedure of Day & Pitts (1974). The ability of peptides 68-88 and 79-88 to inhibit binding of antibody to radioiodinated peptide 43-88 was determined as previously described (Fritz rt N/., 1979). SDS-PAGE
SDS-PAGE was performed by the procedure of Laemmli (1970) at 2 mA per gel for approximately 5 hr at room temperature. Samples were heated in a solution of 0.06 M Tris, 0.7 &I ~-mercaptoethanol, 10% glycerol at 100°C for 30 set prior to application to the gel column. Following electrophoresis the gels were immersed in 150/ trichloroacetic acid for 20 min, and then stained with 0.25% Coomassie Blue in 50% methanol, 77; acetic acid for 2 hr. After destaining densitometric scanning of the stained proteins was done at 600nm in a Beckman Model 25 spectrophotometer equipped with a gel scanner. Mol. wts were estimated from concurrently run protein standards. IEF
IEF was done using horizontal 5% polyacrylamide gel slabs in an LKB 2117 Multiphore apparatus. The gel solution consisted of 7.5 g sucrose, 10 ml of 29.10,;;acrylamide, 10 ml of 0.9% bisacrylamide, 3.0 ml of ampholines, pH 3.5-10, 0.4 ml of ampholines, pH 9-l 1, and 0.4 ml of 0.004% riboflavin. The gel was photopolymerized using U.V. light. Samples.
Rat Anti-encephalitogenic
25100 ,ul, were applied to 0.75 cm squares of filter paper and placed on the gel surface. Following electrofocusing the pH gradient was determined at 1 cm intervals. The gel was then immersed overnight in a solution containing 22.5 g of sulfosalicylic acid, 75 g of trichloroacetic acid, 0.75 g of Coomassie Brilliant Blue R-250 in a mixture of 225 ml of methanol and 465 ml of distilled water. The next day the gel was destained with several changes of destaining solution (8 : 3 : 1 water: ethanol : acetic acid). The individual tracks were then sliced out of the gel and scanned at 600 nm.
Anti-ID
antiserum
A single rabbit was immunized by injection of purified anti-peptide antibody from a single rat. The primary immunization consisted of 1OOpg of protein emulsified in CFA. Booster injections at 3 and 6 weeks were given with a similar amount of protein emulsified in IFA. The rabbit antiserum was exhaustively absorbed by repeated passage over a column of Sepharose 4B to which was conjugated a preparation of crude Le rat globulin. RIA for anti-ID antibodies were carried out as follows. Fifty microliters of absorbed anti-ID or preimmune serum were mixed with 50 ~1 of rat anti-peptide antibody. After incubation at 4°C overnight 20 ~1 of this mixture were added to 200 ~1 of a 1:3 dilution of normal rabbit serum. To this mixture was added 20~1 of a 10m6 M solution of radioiodinated peptide 43-88 (4 x 104-6 x 104cpm). After 90min, immune complexes were precipitated from this mixture by addition of 2 vol 1.9 M sodium sulfate. The precipitate was removed by centrifugation, washed 3 x with 1.27 M Na,SO, and assayed for radioactivity. Anti-ID activity was expressed as the per cent inhibition of binding of radiolabeled peptide as compared to those tubes which contained preimmune serum in the initial mixture. All anti-peptide antisera were titrated prior to the RIA to the point where 10-15x of the radiolabeled peptide was bound to insure that the assay was carried out in antigen excess.
RESULTS
Le rats were challenged and boosted with peptide 68-88 of GPBP. Although this strain is highly inbred, previous experiments showed
Peptide
Antibodies
667
variation in antibody fine specificity from rat to rat, thus use of pooled sera for antibody characterization was undesirable. In order to obtain large amounts of antibody, immune ascitic fluids were induced by repeated intraperitoneal (i.p.) injections of antigen and complete adjuvant (Tung et al., 1976). Since the possibility existed that antibody quantity, specificity, or major class and subclass might differ in ascites fluids and sera, preliminary experiments were done in order to investigate this point. When anti-peptide 68-88 antisera and immune ascitic fluids obtained on the same day from several rats were titrated for antibody content and specificity by sodium sulfate RIA, the two fluids were found to be very similar. The distribution of antibody activity among the various classes of immunoglobulins was examined using class or subclass-specific antisera in a solid-phase RIA. The results of these experiments showed that the distribution of antibody activity was similar in immune serum and ascites, and that antibody activity resided primarily in the IgG2 and IgE immunoglobulin classes. Affinity chromatography, which had been used for isolation of antibodies to myelin basic protein (Wallace et al., 1978) was used to purify antibodies to peptide 68888. In general, antisera or immune ascites fluids were added to a small column of Sepharose-4B-peptide 43-88, allowed to incubate in the cold for 1 hr and then exhaustively washed with BBS. Bound proteins were then eluted with 15 ml of 3 M MgCl,. After dialysis and concentration, the eluted proteins were tested for antibody activity by means of the sodium sulfate RIA. The results of affinity chromatography are shown in Table 1. The data were obtained by titration of each serum or eluate to the point of maximum antigen binding in antigen excess, and the eluates are corrected to starting volume for comparison with the original antisera. As shown in Table 1, there was extensive variation in recovery from serum to serum. The best and worst cases have been presented in order to illustrate the wide range of recovery. For example, serum E consistently gave poor recoveries over a period of several months with a usual recovery of approximately 20%. Serum D generally gave recoveries of S&950/, over the same period under identical conditions. When eluates and precolumn antisera were compared with respect to class and subclass of immunoglobulin (Table 2) the compositions of each
ROBERT
668
Table
B. FRITZ,
I. Binding
ANNE
activity
E. DESJARDINS
of affinity-purified antibodies pmoles
Rat anti-peptide 68-88
rat anti-peptide
of ‘Z51-peptide 43388 bound milliliter”~b
Original
J K L C D E
and RAYMOND
per
342 295 77 300 726 116
“Determined by titration in antigen excess. ‘Normal Le rat serum controls bound 3 10 pmoles 43-88 per milliliter.
Table 2. Immunoglobulin fluid and affinity-purified
68 88
3 M MgCl,
788 864 247 453 139 673
were similar with the exception of loss of IgM antibodies in the eluates. The results of the affinity chromatography experiments suggest heterogeneity of binding affinity from one serum to another. However, this procedure did not select with respect to class since the eluates were very similar to the starting material in this respect. In order to determine whether the eluates were contaminated with non-immunoglobulin proteins, each preparation was analysed by IEP against goat anti-rat serum. By this method all preparations were shown to contain IgG2 and many had IgGl antibodies as well. The solid-phase RIA (Table 2) showed other immunoglobulin classes to be present. However, our goat anti-rat serum apparently did not detect these by immunoelectrophoresis. Analysis of the eluates by SDS-PAGE under reducing conditions in 10% gels confirmed the earlier observations. The major polypeptides observed correspond to light chains and gamma heavy chains (Fig. 1). With several eluates a peak at mol. wt 70,000 was observed, likely representing epsilon chain, and often a
SHAPIRA
of ‘z51-peptide
second distinct band was noted in the lightchain region. Under identical conditions our DEAE-cellulose-purified IgG2 standard gave a single light-chain band and two heavy-chain bands estimated to have mol. wts of 22,000, 50,000 and 55,000, respectively. All eluates also gave two heavy-chain bands corresponding to the two latter mol. wts. In order to assess charge heterogeneity of the affinity-purified anti-peptide antibodies, preparations of MgCl,-eluted proteins were analysed by IEP in pH 3.5-10 gradients (Fig. 2). Also shown in this figure is a pattern obtained with DEAE-cellulose-purified IgG2 analysed under identical conditions. The latter preparation gave approximately 20 distinct bands. Affinity-purified preparations often gave patterns which were more complex than the standard although the majority of the proteins focused in the pH range of 778. However, affinity-purified preparations also contained a number of additional proteins which focused at pH less than 7.
class and subclass of rat ascitic antibodies to peptide 68888
Net “/, bound of anti-class or subclass antiserum added” Class or subclass Alpha Epsilon Mu Gamma Gamma Gamma
Ascitic fluid
1 2A 2C
“Determined
3 M MgCI,
15 34 10 24 51 21 by solid-phase
22 29 0 39 50 18 radioimmunoassay.
eluate
Fig. 1. SDSPAGE pattern of MgCl,-affinity-purified rat anti-peptide 68-88 stained with Coomassie Blue. Acrylamide concentration was lo:;,. L, OVA, H and BSA refer to rat immunoglobulin light chain, ovalbumin rat immunoglobulin G heavy chains, and bovine serum albumin, respectively, used as mol wt markers. Migration is from right to left.
Rat Anti-encephalitogenic
Peptide
669
Antibodies
detect those idiotypes associated with the antigen binding site. Thus, total idiotypic crossreactivity might be somewhat greater. DISCUSSION
I
These experiments were designed to evaluate the degree of heterogeneity of the antibody response to encephalitogenic peptide 68-88 in the Le rat. Three levels of heterogeneity were assessed; class heterogeneity, charge heterogeneity and idiotypic heterogeneity. Since immune ascitic fluids were the starting material for purification by affinity chromatography, preliminary experiments compared antibody levels and class in these fluids with serum antibodies. No significant differences were noted. Antibody activity was found predominantly in the IgG2 and IgE class with some animals having significant levels of IgGl antibodies as well. Although the anti-peptide response is considerably more restricted than the antibody response to basic protein (Fritz et al., 1979) it is still heterogeneous with respect to class and subclass. SDS-PAGE of the purified antibodies showed that they consisted predominantly of polypeptide chains of mol. wts 22,000 and 50,000. Occasionally, a second peak was seen in the light chain region and. in all instances. two bands of approximately equal staining density were seen in the gamma heavy-chain region. In many preparations a less densely stained band of mol. wt approx. 70,000 was seen. Since IgE was present at significant levels in the anti-peptide 684% antibodies in this as well as previous studies (Fritz et al., 1979). the latter band likely corresponds to epsilon heavy chain.
Table Fig. 2. Densitometric tracings of Coomassie Blue stained MgCI,-affinity-purified rat anti-peptide 68-88 antibodies after IEF. Panels A and B represent two different affinitypurified antibodies; panel C is a tracing of DEAE-purified normal Le rat immunoglobulin.
In order to test for idiotypic cross-rectivity, rabbit anti-ID antiserum was tested against a panel of 10 individual Le rat anti-peptide antibodies. The results showed significant idiotypic cross-reactivity with only two antisera (Table 3). It should be emphasized that the assay used to assess idiotypic cross-reaction would only
3. Idiotypic cross-reactivity of Le rat anti-peptide 68-88 antisera
Anti-peptide 68-88 antiserum A B C D E F G H I J
“() inhibltion 0 1 0 90” 53 7 0 0 0 34
“Homologous reaction-antiserum used for immunization of rabbit.
D
670
ROBERT
B. FRITZ,
ANNE
E. DESJ.~RDlNS
We have noted two heavy-chain bands of mol. wts 50,000 and 55,000 in all of our normal rat IgG standards. The larger of the two bands is reduced in amount when DEAE-purified rat IgG is analysed under these conditions implying that this polypeptide chain is associated with a more negatively charged molecule. Charge heterogeneity of the eluates was revealed by electrofocusing. Affinity-purified anti-peptide 68-88 antibodies often gave patterns which were more complex than the IgG standard. This is not surprising in light of the heterogeneity observed in the solid-phase RIAs. By the latter method the affinity-purified preparations were found to contain IgGl, IgG2 and IgE, although the levels of these immunoglobulins varied from serum to serum. The fact that the antibody response to this small defined antigen was quite heterogenous is not surprising in view of the data showing extensive variation of binding afinities (Eisen & Siskind, 1964) of anti-hapten antibodies in the course of the immune response. An earlier study from our laboratory had shown heterogeneity of antibodies raised by the immunization of Le rats with peptide 43-88 from myelin basic protein (Fritz et al., 1978). Kreth & Williamson (1973) have estimated that CBA/8 mice are potentially capable of producing 8000 different anti-NIP (4-hydroxy-5-iodo-3-tlitrophenacetyl) antibodies. The results of testing 10 anti-peptide antibodies for idiotypic cross-reactivity showed that with this anti-idiotypic antiserum only a minority of the rat antisera carried cross-reactive idiotopes. It should be emphasized that these results were obtained only with a single anti-ID antiserum. The number and location of the idiotopes recognized by this antiserum are unknown although they are located at or in close proximity to the antigen binding site. However, it is apparent that with respect to the idiotopes measured by this RIA, there is considerable variation from one rat anti-peptide antiser~im to another as well as within a single antiserum. In summary, rat anti-peptide 68.--88 antibodies were found to be heterogeneous within a single antiserum with respect to class. charge and idiotype. Antibody activity was found in the IgGl. IgG2 and IgE immunoglobulin
and RAYMOND
SHAPIRA
classes. IEF revealed multiple bands within a single antiserum. Comparison of different antipeptide antisera by IEF revealed both common and unique immunoglobulin species. Crossreacting idiotopes were present in some antipeptide antisera. Ackno,~lc~df/ert?~,~~f,s This work was supported Grants NS 1141X and NS 10721.
by USPHS
REFERENCES
Chou F. C.-H.. Chou C.-H. J.. Kowalski T. J.. Shapira R. & Kibler R. F. (1977) The major site of guinea pig myelin basic protein and encephalitogcnic in Lewis rats. f. N~~ur~f+m. 28, 1 i5- 119. Chou C.-H. J., Fritz R. B., Chou F. C.-H. & Kibler R. F. (1979) The immune response of Lewis rats to peptide 6X-88 of guinea pig myelin basic protein. 1. T cell determinarlts. J. ~~?z~I~~~I. i23? 1540 1543. Cuatrecasas P. & Anfinsen C. B. (1971) Afiinity chromatography. In Mrrhod.s it1 En-?moloy~ (Edited by Colowick S. P. & Kaplan N. 0.). Vol. 22. p. 345. Academic Press, New York. Day E. D. & Pitts 0. M. (1974) R~idioimmun(~~~ssay of myelin basic protein in sodium sull’atc. Irnnirir?tit~fipnii.srr) II, 651 659. Eisen H. N. & Siskind G. W. (lY64) Variations in alfinities of antibodies during the immune response. BIO~/?P~PG\~PJ 3, 99& 1008. Fritz R. B.. Chou F. C.-H., Chou C.-H. J. & Kibler R. F. (1979) The immune response of Lewx rats to peptide 6X XX of guinea pig myelin basic protein. II. B cell determinants. J. Immztn. 123, 1544-l 547. Fritz R. B.. Chou C.-H. J., Randolph D. H., Desjardins A. E. & Kibler R. F. (1978) Specificity of antisera from Lewis rats immunized with encephalitogemc fragment 43-88 of guinea pig myelin basic protein. J. lmn~un. 121, 1X65-1869. Kibler R. F.. Fritz R. B.. Chou F. C.-H.. Chou C.-H. J., Pcacockc N. Y., Brown N. M. & McFarlin D. E. (1977) Immune response of Lewis rati to peptide Cl (residues 68 88) of guinea pig and rat my&n basic protein. J. e’r,~. M&. 146, 1323 1331. Kreth H. W. & WIlliamson A. R. 11973) The extent of diversity of ~lnti-hapten antibodies‘in &red mice: antiNIP (4-hydroxy-S-iodo-3-nitrophenacetvl) antibodies in CBA:H mice. Eur. J. Immun. 3; I41 -14;. Marchalonis J. J. (1969) An enzymatic method for the trace iodination of immunoglobulins and other proteins. Biocf7m. J. 113, 299 30s. Randolph D. H.. Kibler R. F. & Frit/ R. 9. (1977) Solidphase radioimmunoassay for detection of antibodies to myelin basic protein. J. Imniu~?. Merit. 18, 215. 224. Tung A. S.. Ju S.. %itO S. & Nisonolf A. (1976) Production of large amounts of antibodies in individtiai mice. J. Irnmu,i. 116,676 6X i. Wallace A. D., Shapira R. & Fritz R. B. (197X) Isolation and characterization of rabbit antibodies to bovine myelin basic protein. Immunochemistrr 15. 47-54. Waxman F.. Fritz R. B. & Hinrichs D. J. (IYXO) The presence of specific antigen-reactive cells during the induction. recovery and resistance phases of experimental allergic enccphalomyelitis. Ccl/. Imtmrn. 49, 34-42.