- Email: [email protected]
Radioimmunoassays for myelin basic protein
Clinical Immunology Newsletter May 7, 1982
Vol. 3, No. 9
Radioimmunoassays for Myelin Basic Protein Eugene D. Day, Ph.D. Departments of Microbiol~gy&nmunology and Surger3, Duke University~Medical Center Durhanl, North Carolina 27710
The C o n f l i c t By means of a solid-phase radioimmunoassay (RIA) based upon '2q-protein-A detection of adsorbed immunoglobulin in myelin basic protein (MBP)-coated wells o f plastic microtiter plates (30), Bernard et al. (3) have been able to detect anti-MBP antibody in extracts o f multiple sclerosis (MS) brain. The authors were therefore able to explain and support the findings of Panitch et al. (37) that the cerebrospinal fluids (CSFs) o f progressive MS patients often contain anti-MBP antibodies measurable by solid-phase techniques. These findings would appear to be in direct conflict with other lines of evidence. For example, Cohen and Gutstein (12) had been able to show that in the spinal fluids of MS patients there were no detectable anti-MBP antibodies even though, by their liquid-phase RIA technique, they were easily able to measure antiMBP activity in the CSFs of sheep afflicted with experimental allergic encephalomyelitis (EAE). Cohen and his colleagues, after developing an effective liquid-phase RIA for measuring MBP in biologic fluids (14), had been able to show in a blind study of 303 CSFs from patients with a variety of neurologic
disorders that those patients with active demyelinating disease not only did not contain detectable antibody but actually registered relatively high levels of MBP (13). To punctuate this fact Cohen et al. (I 1) had been able to demonstrate similar findings in an additional 394 patients. I m m u n o c h e m i c a l F e a t u r e s o f the MBP Molecule The findings of Panitch et al. (37) and Cohen et al. (l l) are not in conflict, however; the data from both groups may be taken as correct and definitive, and the issue can easily be resolved. The reasons why free anti-MBP antibody may be found by one group while free antigen may be found by another stem from some highly interesting features o f the MBP molecule. Therefore, before discussing any further the clinical findings of MBP a n d / o r its fragments in CSFs, it would be well to review some of the known immunochemical features o f the MBP molecule as it appears in liquid and solid phases. Since a number o f reviews concerning the immunologic, biologic, and chemical aspects of MBP have recently appeared (4, 7, 8, 15, 28, 34), it will not be necessary to present more than a capsular view of the molecule. When the two complete amino acid sequences for bovine and human myelin are compared (Table I), using the original sequence o f Eylar et al. (26) as a frame of
reference to establish the numbering system,* the molecule falls naturally into three segments that are joined by two phenylalanine doublets: A, residues 1-43; B, residues 44-89; and C, residues 90-170. Segments A and C, the N- and C-terminal portions, respectively, have a higher degree of homology'than segment B, one of the many facts that contrast the evolutionary aspects o f MBP with most other proteins. The regions where gap events and interchanges disrupt homologies more frequently are the N-terminal and C-terminal ends (23). At residues 99-t01 in segment C, there is a highly conserved knot of three prolines that was once thought to form a U-type p bend in the molecule, thus simulating a *A different sequence for bovine MBP has also appeared (5) in ~vhichthe serine at position #2 is deleted. In some immunologic studies (e.g., 27, 50) this Brostoff sequence is used, causing segments A, B, and C to be numbered 1-42, 43-88, and 89-169, respectively, and in one review (15) a numbering system based on kno~vnmaximum homology among the species is presented, causing segments A, B, and C to be numbered 1-44, 45-90, and 91-171, respectively.
In This Issue Radioimmunoassays for Myelin Basic Protein . . . . . . . . . . . . . . . . . . . . . . . 53 The present state of the art and a dilemma Guest Editorial . . . . . . . . . . . . . . . . . 59 Clinical applications o f radioimmunoassays for myelin basic protein
hairpin (5), but Martenson (31) disagrees. In the highly conserved C-terminal leg, an internal deletion o f ,.,40 residues, 118-158, occurs in two-thirds of the MBP molecules in the rat. Other Myomorpha (mouse, hamster) as well as Scittromorpha (prairie dog, woodchuck, squirrel) also bear this deletion (presumably the same sequence) to produce in those mammals a mixture o f small and large MBP (33). The hystricomorphs (guinea pig, chinchilla) as well as other vertebrates (rabbit, sheep, chicken, frog) retain the full framework of the C segment of the bovine and human sequences. Microheterogeneity (see Table 1, third footnote) undoubtedly' blays at least a small role in immunologic responses and reactivities, but it is believed that by far the greatest contributor to immunologic differences among MBP molecules of different species is conformational, thus causing differences even in homologous portions o f the MBP molecule.
MBP Cross-Reactivity A m o n g the Species The differences among MBP sequences of the species do not engage the attention o f immunologists as much as the similarities-leading to a high degree of cross-reactivity and self-reactivity. This is not to say that there are not important differences that arise because o f nonhomologous determinants. Whitaker (46), for example, has measured such differences among basic proteins from human, monkey, dog, sheep, pig, cow, guinea pig, rat, rabbit, chicken, and turtle using the Wasserman-Levine method of quantitative microcomplement fixation. In a typical response, even though rabbit-anti-human MBP did not seem to be able to differentiate between human and monkey MBP, it reacted better with dog, sheep, pig, and guinea pig MBPs than with cow, rat (large), or rabbit, and reacted least well with rat (small), chicken or turtle. Even so, the reaction with rabbit MBP was still threequarters of the maximum, and the reaction with chicken MBP still over
54
a quarter. And even in the case of a rabbit-anti-chicken MBP antiserum (32), double immunodiffusion reactivity was obtained with a high degree of identity among precipitin lines and complete fusion among human, rabbit, guinea pig, and cow MBPs or among frog, turtle, chicken, and cow MBPs. In our laboratory (39) the question asked was "whether syngeneic rat antisera raised against Lewis rat MBP would be directed primarily against those parts o f the MBP molecule that were private to the species or whether they would cross-react fully with MBPs from other species, indicating an indifference to species in the Witebskian sense of organ specificity and autoimmunity." A panel of different MBPs coupled to Sepharose 4B was used to absorb several individual syngeneic antiMBP antisera from Lewis rats, and then the absorbed sera were compared with the unabsorbed sera with respect to their antigenic binding capacity for different '2Sl-labeled MBPs. More than 80% of the antibodies in each serum were found to be cross-reactive with the different MBPs, and less than 20% were specific only for the homologous '2q-MBP rat (large). Coupled with the fact that such syngeneic antibodies were also found to display relatively high affinities in their reactions with MBP (42), these findings were taken by Roset to "exclude the JemmersonMargoliash hypothesis (29) that the late evolving, species-restricted determinants are the ones most likely to be involved in autoimmune responses." In the world of clinical immunology, scientists may be used to thinking in terms of a thyroglobulin model (41), in which cross-reactions among the species play little part, but, as Roset has pointed out, "Thyroglobulin represents one end of the spectrum o f organ-specific antigens since there is relatively little cross-reaction among thyroglobulins of different tN. R. Rose. 1981. Personal communication.
species, whereas myelin basic protein typifies the other extreme, showing extensive cross-reactions. Obviously, the evolutionary concepts based on one antigen would not apply to the other." T h e A n t i g e n i c Valence o f M B P The first question might well be, " W h e r e are the antigenic determinants located in the MBP molecule that react and cross-react with anti-MBP antibodies? And how many determinants are there?" A valence of 3 is suggested by a Heidelberger-type treatment (20) of the quantitative precipitin reaction o f a published individual rabbit antiserum to bovine M B P , , but a valence of 4 or more is suggested by Wallace et al. (43), who carried out a liquid-phase Heidelberger-type analysis of one of their rabbit antisera to bovine MBP. Wallace et al. also measured the distribution of solid-phase adsorption activity among the three segments of MBP referred to in Table 1 and found a fairly uniform concentration of antibodies against each of the three parts with a tendency to react somewhat more with the C segment, somewhat less with the A segment, and less uniformly with segment B. Driscoll et al. (25) analyzed seven syngeneic guinea pig (strain 13) antiMBP antisera with respect to the distribution o f activity among six fragments, 1-19, 1-89, 44-88, 1-I 16, 90-170, and 117-169, as compared to whole-molecule MBP, finding that none reacted with 1-19, two with all five of the active fragments (a valence of at least 5 with " b r o a d specificity"), and five with only some o f the active fragments ("narrow specificity"). Whitaker et al. (49) compared rabbit and guinea pig antisera for their reactivities with peptides 90-170, 1-39, and 44-89, finding that all guinea pig antisera preferred 90-170, whereas rabbit antisera reacted with 1-39 as well. Low and variable reactivity with 44-89 was obtained with either type of antiserum. ~:J. Boggs. 1981. Personal communication.
Clinical ImmunologyNewsletter
Disparate RIAs At this juncture in our discussion, we should now focus upon two distinct observations that have emerged from RIA data in the MBP system: a) liquid-phase RIAs capture a smaller and possibly different spectrum o f anti-MBP antibodies than do solid-phase methods; b) small peptide fragments of MBP carry determinants that often do not cross-react with whole-molecule MBP and vice versa, even though the amino acid sequences of the fragments may be homologous to the parent whole molecule. The problems are, perhaps, best illustrated in the work of Fritz et al. (27), who were studying~the B-cell immune responses of Lewis rats to peptide 69-89 (referred to by them as 68-88) homologous to the sequence of guinea pig MBP for that region. In a liquid-phase sodium sulfate RIA with '~q-labeled guinea pig MBP, the rat antipeptide antiserum had no capacity to bind, yet in a solid-phase assay with guinea pig MBP adsorbed to glass, antibodies from the antipeptide antiserum did effectively bind. As possible explanations for this "unexpected finding," the authors suggested either "confotmational changes associated with adsorption o f the protein to a solid matrix" or "dissociation [of relatively low affinity complexes] in the presence of high concentrations of sodium sulfate." It had been noted previously that rabbit antisera to monkey or bovine MBP failed to react with the 44-89 peptide (47), but that if antisera were raised in rabbits to the 44-89 peptide linked to rabbit serum albumin (48) good reactions in a liquid R1A could be obtained with the peptide, even though these antipeptide antisera would not react with whole-molecule MBP. A difference in conformation between the peptide and the parent whole molecule ("molecular internalization" o f part or all o f the 44-89 region in whole MBP) was taken as the explanation. In our laboratory (19), it was shown that synthetic peptides from region 65-84
Copyright © 1982 by G. K. Hall & Co.
of bovine MBP would not react with antibodies in any of nine pools of syngeneic rat anti-MBP antisera, and only peptide $82 (bovine MBP residues 65-83 plus glycine) would react even to a Iimited extent with 14/15 rabbit antisera to MBPs from several species. At the same time (18), whole-molecule, parent bovine MBP would not react with any of 26 rat and rabbit antisera to peptides $82 and S81 (residues 68-83 plus glycine), even though the antipeptide antisera were highly reactive in liquid-phase RIAs with the labeled peptides. Dr. Joan Boggs of the Hospital for Sick Children in Toronto, Ontario, has informed us~ that an anti-S82 antiserum that does not interact with '2SI-MBP in solution (thus confirming that fact) does, however, precipitate lipid vesicles containing MBP in a sequestered form. Meanwhile, we have determined that the same anti-S82 antiserum has a major population o f antibodies that reacts with a conformational determinant of $82 but not with small overlapping sequences o f the same $82 region (17), and that a minor population of antibodies in that antiserum, reacting with a sequential determinant (16), is not involved in MBP cross-reaction. Recently, in our laboratory, K. J. Lazarus also found populations of antibodies in some rabbit antisera against whole-molecule MBP that react with synthetic peptide $24, which is half as large as $82 and representative o f its N-terminal half (residues 65-74 plus glycine), yet do not interact with intermediate $82. These results suggest that the 20-amino acid $82 peptide does indeed have a conformation all its own in solution that is not found in either whole-molecule MBP or the shorter I l-amino acid $24 peptide. The Consequences of MBP Catabolism Where does this leave us with respect to the interpretation of existing reports of immunologically identified MBP molecules and their :l:J. Boggs. 1981. Personal communication.
fragments in CSFs, sera, and other biologic fluids? We observe that MBP is a rather labile protein even in vivo and that it does tend to fragment, yet we find that much of the immunologic reactivity o f antibodies to the whole molecule is retained by reactivity with the large pieces. Consequently, it is sometimes impossible to determine whether a whole molecule or its pieces or both are involved in any one particular immunologic reaction. Although we know little about the conformation o f the large fragments, we do observe that as the fragments become smaller, approaching 20-amino acid peptides, at least some of them take on new formats with the emergence o f new determinants and the loss of others. Then, as the fragments become even smaller, approaching 8-12 amino acid haptenic-sized peptides, there is a loss of even these new determinants, the emergence of yet others, and the recovery o f some original determinants. In the continuing catabolic process, of course, immunologic identity eventually becomes lost altogether. The process may be quite rapid, as it is in rabbits (1), or it may be sluggish, accounting for the eventual build-up and retention of fragment activity in the blood (24, 35, 38). The association of fragments with serum components (a-globulin, bilirubin, /3-1ipoprotein, endogenous autoantibody) is likely to slow the catabolic process even further (2). The heterogeneity o f the fragments with respect to size (2), affinity (22, 38), and SDS-PAGE analysis (9) is evidence for the general breakdown o f MBP beyond the three large segments A, B, and C of Table 1.
Binding Affinities and RIAs There is one more parameter of the MBP-anti-MBP system that has led to seemingly disparate results from laboratory to laboratory--the matter o f binding affinity. As Reichlin (40) has pointed out in his review, different nonrepeating determinants of a given protein in many cases give rise to different binding
55
Table ! Amino Acid Sequence of the Three Segments of Myelin Basic Protein (MBP) of Bovine* and Human1 Origins:~ Segment
A
Species
Sequence
bovine human
0 0 0 0 0 0 0 0 l 1 2 2 3 3 5 0 5 0 5 0 5 ASAQKRPSQR - - SKYLA SASTMDHARHGFLPRHRDTGILDSLGRF ASQKR'PSQRHGSKYLATASTMDHARHGFLPRHRDTGILDS
0 4 5 bovine human
bovine human
0 5 5
0 6 0
0 6 5
0 7 0
0 7 5
IGRF
0 8 0
0 8 5
FG SDRGAPKRGSGKDGHHAARTTHYGSLPQKAQGHRPQDENPVVHF FGGDR,GAPKRGSGKD SHH PARTAHYGSLPQK SHG -RTQDQDPVVHF
0 9 0 C
0 5 0
0 4 0
0 9 5
I 0 0
I 0 5
1 ! 0
i I 5
I 2 0
1 2 5
1 3 0
l 3 5
i 4 0
1 4 5
FKNIVTPRTPPPSQGKGRGLSLSRFSWGAEGQKPGFGYGGRASDYKSAHKGLKGHDAQGT FKNIVTPRTPPPSQGKGRGLSLSRFSWGAEGQRPGFGYGGRASDYKSAHKGFKGVDAQGT
1
5 0 LSK LSK
1
I
!
5 6 6 5 0 5 IFKLGGRDSRSGSPMARR IFK LGGRDSRSGSPMARR
I
7 0
*As given by Eylar et al. (26). A variant in the bovine sequence has also been found (5) in which the serine at residue 2 is deleted. 1As given by Carnegie and Dunkley (7), A genetic variant in the h u m a n sequence has also been found (10) in which one of the two glycines at residues 45-46 has been interchanged with serine. :[:The N-terminal residue #1 is blocked by an acetyl group. The arginine at 108 is methylated on occasion. Glutamine 104 m a y be deamidated in some isolated products. O n e molecule out o f five may be phosphorylated at residues such as serine-55 (endogenous) or serine-110 (exogenous). The C-terminal arginine-170 is extremely labile in isolated MBP. Further details and references may be found in the review by Carnegie and Moore (8).
affinities in an antiserum; moreover, the range of affinities for any one determinant may be considerably more restricted than the overall range of binding affinities for the total protein. MBP has these characteristics (42), with some determinants raising antibodies with affinities much higher than others and with even the lower affinity antibodies (as determined in liquidphase RIAs) having relatively high association constants. When MBP is fragmented into a heterogeneous collection of smaller peptides but still remains reactive with the antiserum against the whole molecule,
36
the various antibody populations in that antiserum will react with the various peptides--one population with one fragment with a very high affinity, another population with another fragment of only a moderate affinity, and so on. Thus, the fragments can be described in terms of the affinity with which they react. For purposes of measurement, the higher affinities become more dominant in antibody RIAs at the higher dilutions of antibody and antigen (42); thus, when inhibition assays are carried out at the higher dilutions, higher affinity fragments can be detected (21). When this
method (called dual-dilution RIAs) was applied to antisera from Lewis rats undergoing EAE (22) or to sera from MS patients (38), heterogeneity of fragments (MBP-serum factors) with respect to affinity was readily seen, with a shifting profile occurring during the time course. Palfreyman et al. (36) had been among the first to recognize the small size of MBP-like material in the CSFs of patients with head injuries and had adopted a method of inhibition RIA that depended on matching inhibition curves of test samples with those of standard MBP. The inhibition curves of the
Clinical Immunology Newsletter
CSF samples were not parallel, suggesting the existence of crossreacting material o f smaller molecular size and, probably, degraded MBP. In the sera o f patients with cerebrovascular accidents (CVAs) (35), relatively high levels o f MBP-like material were also found, and again a nonparallel effect was obtained "indicating differences between the antigenic nature of MBP and the immunoreactivity found in the test serum." A shift to a more restricted population of binding affinities is also likely. Palfreyman et al. (35) also found evidence o f anti-MBP antibodybinding activity in the sera o f five o f the 58 patients who had a history of previous stroke, classifying the antibodies as having low avidity. In the sera o f some o f our clinically well subjects, we also found antiMBP binding activity o f relatively low or moderate affinity (38), while at the same time we detected MBP fragments of relatively high affinity. In one of four patients with clinically active MS, we found the reverse situation: anti-MBP antibody of relatively high affinity and MBP fragments o f relatively low affinity. The B Segment of MBP Segment B, more than A or C, appears to accumulate in the CSFs o f patients undergoing episodes of severe demyelination (45, 50, 51). In 93 specimens from patients with various neurologic diseases, Whitaker et al. (50) found crossreactive peptides in active-MS CSF, in myelopathic CSF, and in CSFs o f acute-stage CVA patients, but "virtually no material" in the same samples reactive with antisera to peptides o f the A and C segments. In collaboration with other groups, Whitaker and his colleagues were able to extend their findings to 582 samples (51) with no change in the overall result. Cohen et ai. (11) were very careful to point out that " n o t all antisera to MBP will react with the CSF myelin basic protein" in the 697 samples they h~/d measured.
CoDvrizht © 1982 by G. K. Hall & Co.
They had found that the best way to raise an effective antiserum for their assays was to immunize rabbits with 250-mg guinea pig spinal cord in complete Freund's adjuvant and boost with MBP. The Whitaker reagents were raised to segment B itself or to human MBP that " c o n tained intact BP as well as an assortment o f BP peptides, which is a common occurrence in the preparation o f human B P " (50). Careful selection among a number of antisera was required to obtain effective ones since most anti-MBP antisera in previous studies had failed to react with segment B (48). Quite obviously, the peptides measured by both the Cohen and the Whitaker groups represent only a select portion o f the MBP molecule, so there could easily be free antibodies against some MBP specificities in solution with free peptides of other specificities. That Cohen and Gutstein (12) did not find anti-MBP antibodies in CSFs o f MS patients whereas Panitch et al. (37) did is not surprising since the Cohen group used a liquid-phase type assay whereas the Panitch group used a solid-phase type assay. As Fritz et al. (27) have shown, an anti-MBP antibody may be refractory to a peptide in liquidphase but fully reactive with the same peptide adsorbed to glass surfaces. The Present Dilemma In conclusion, it should be pointed out that the number of potential determinants in an MBP molecule that will give rise to a humoral response appears to exceed by several fold the actual number involved in raising any one given antiserum; consequently, the effective valence of MBP for any given antiserum may be no more than 3 to 6, obviously a mere sampling o f the number of specificities available. A relatively large library of reagent antisera to MBP woula therefore be required to cover the number of possibilities. Since no accepted standards exist even to identify and capitalize on the major MBP deter-
minants or their respective " t y p i n g " antisera, there is little agreement among the laboratories o f the world as to what constitutes a suitable reagent for an RIA. The problem is compounded by the fact that MBP tends to fragment in vivo as well as in vitro and that the resulting degradation products tend to clear from the circulation at different rates. This leaves behind in the body fluids an accumulation o f only a part of the MBP molecule with its own set of intact determinants. Only certain o f the reagent antisera that happen to have antibodies specific for this set can be used effectively in competitive inhibition RIAs. The problem is further compounded by the fact that certain o f the MBP fragments tend to change their conformation, resulting in the loss o f determinants of the parent molecule and the gain o f new ones. Such fragments remain "silent" during the search for inhibitory material in biologic fluids using antibodies to the parent proteins, unless solid-phase RIA techniques are adopted. The problem with the solid-phase approach, however, is that very low affinity nonspecific binding is difficult to control, and high affinity binding (which is usually low titered) is hard to distinguish. Particularly in the evaluation of binding parameters and in the quantitative analysis o f inhibitory materials, the liquid-phase approach is far superior and far less complicatedi however, it can provide only part of the answer. The dilemma of the moment, therefore, is that there is not even one satisfactory method for the quantitative inhibition analysis of MBP fragments that is certain to capture the full complement of all parts of this biologically important molecule. Until such a method has been devised and placed in service in clinical laboratories around the world, the evaluation of MBP and its antibodies in clinical material will have to remain a relatively haphazard exercise.
57
References I. Bashlr, R. M., and J. N. Whitaker. 1980. Metabolism of a peptide of human myelin basic protein in the rabbit. Neurology (N.Y.) 30:1184-1192. 2. Bashir, R. M., and J. N. Whitaker. 1980. Molecular features of immunoreactive myelin basic protein in cerebrospinal fluid of persons with multiple sclerosis. Ann. Neurol. 7:50-57. 3. Bernard, C. C. A., el al. 1981. Antibody to myelin basic protein in extracts of multiple sclerosis brain. Immunology 43:447-457. 4. Braun, P. E., and S. W. Brostoff. 1977. Proteins of myelin, pp. 201-231. In P. Morell (ed.), Myelin. Plenum Press, New York. 5. Brostoff, S. W., et ai. 1974. Specific cleavage of the AI protein from myelin with cathepsin D. J. Biol. Chem. 249:559-567. 6. Brostoff, S. W., and E. I1. Eylar. 1971. Localization of methylated arginine in the AI protein from myelin. Proc. Natl. Aead. Sci. U.S.A. 68:765-769. 7. Carnegie, P. R., and P. R. Dtmkley. 1975. Basic proteins of central and peripheral nervous system myelin. Adv. Neurochem. 1:95-135. 8. Carnegie, P. R., and W. J. Moore. 1980. Myelin basic protein, pp. 119-143. In R. A. Bradshaw and D. M. Schneider (eds.), Proteins of the nervous system. Raven Press, New York. 9. Carson, J. H., et al. 1978. Components in multiple sclerosis cerebrospinal fluid that are detected by radioimmunoassay for myelin basic protein. Proc. Natl. Acad. Sci. U.S.A. 75:1976-1978. 10. Chou, C.-H. J., et al. 1978. Identity of myelin basic protein from multiple sclerosis and human control brain. J. Neurochem. 30:745-750. 11. Cohen, S. R., et al. 1978. Cerebrospinal fluid myelin basic protein and multiple sclerosis, pp. 513-519. In J. Palo (ed.), Myelination and demyelination. Plenum Press, New York. 12. Cohen, S. R., and H. S. Gulstein. 1978. Spinal fluid differences in experimental allergic encephalomyelitis and multiple sclerosis. Science 199:30t -303. 13. Cohen, S. R., R. M. llerndon, and G. M. McKhann. 1976. Radioimmunoassay of myelin basic protein in spinal fluid. An index of active demyelination. N. Engl. J. Med. 295:1455-1457. 14. Cohen, S. R., G. M. MeKhann, and M. Guarnleri. 1975. A radioimmunoassay for myelin basic protein
15. 16.
17.
18.
19.
20.
21.
22.
23.
and its use for quantitative measurements. J. Neurochem. 25:371-376. Day, E. D. 1981. Myelin basic protein. Contemp. Top. Mol. Immunol. 8:1-39. Day, E. D., et al. 1981. Affinity purification of an acylated and radiolabelled synthetic derivative of residues 75-83 of bovine myelin basic protein, 'z*I-$79: A model for the purification of picomole quantities of specific peptide fragments of myelin basic protein and antibodies against them. J. Neuroimmunol. I:311-324. Day, E. D., el al. 1981. Equilibrium and non-equilibrium competitive inhibitions of anti-peptide antibody binding by parent myelin basic protein and 18 related peptide sequences. Neurochem. Res. 6(5):577-593. Day, E. D., el al. 1981. Immunogenicity of synthetic peptide sequences $81 and $82 (residues 68-83 and 65-83) of bovine myelin basic protein: Time-course of antibody responses in rats and rabbits. J. Neuroimmunol. 1:205-216. Day, E. D., et ai. 1981. Synthetic peptides from region 65-84 of bovine myelin basic protein: Radioimmunoassays and equilibrium competitive inhibition studies with antibodies prepared against myelin basic protein. Neurochem. Res. 6(8):913-929. Day, E. D., and O. M. Pitts. 1974. Radioimmunoassay of myelin basic protein in sodium sulfate, lmmunochemistry 11:652-659. Day, E. D., and V. A. Varitek. 1981. Radioimmunoassays of antibodies to myelin basic protein (MBP), dual-dilution radioimmunoassays, and competitive binding radioimmunoassays to measure MBP and its fragments, pp. 380-385. In N. R. Rose and H. Friedman (eds.), Manual of clinical immunology, 2nd ed. American Society for Microbiology, Washington, D.C. Day, E. D., V. A. Vafitek, and P. Y. Paterson. 1981. Endogenous myelin basic protein-serum factors (MBP-SFs) in Lewis rats: Evidence for their heterogeneity and reactivity with anti-MBP antibodies of different affinities. J. Neurol. Sci. 49:1-17. Dayhoff, M. O., and W. C. Barker. 1972. Mechanisms in molecular evolution: Examples, pp. 41-45. In M. O. Dayhoff (ed.), Atlas of protein sequence and structure, vol. 5. National Biomedical Research Foundation, Washington, D.C.
24. Delassalle, A., et al. 1980. Radioimmunoassay of the myelin basic protein in biological fluids, conditions improving sensitivity and specificity. Biochimie 62:159-165. 25. Driseol|, B. F., A. J. Kramer, and M. W. Kies. 1974. Myelin basic protein: Location of multiple independent antigenic regions. Science 184:73-75. 26. Eylar, E. It., et al. 1971. Basic A protein of the myelin membrane: The complete amino acid sequence. J. Biol. Chem. 246:5770-5784. 27. Fritz, R. B., et al. 1979. The immune response of Lewis rats to peptide 68-88 o f guinea pig myelin basic protein. 1I. B cell determinants. J. lmmunol. 123:1544-1547. 28. Hashim,,G. 1978. Myelin basic protein: Structure, function, and antigenic determinants, lmmunol. Rev. 39:60-107. 29. Jemmerson, R., and E. Margoliash. 1979. Specificity of the antibody response o f rabbits to a self antigen. Nature 282:468-471. 30. Linthlcum, D. S., et al. 1981. Detection of antibodies to myelin basic protein by solid-phase radioimmunoassay with ['~q] protein A. J. Neuroimmunol. 1:17-26. 31. Martenson, R. E. 1981. Prediction of the secondary structure of myelin basic protein. J. Neurochem. 36:1543-1560. 32. Marlenson, R. E., and G. E. Deibler. 1975. Partial characterization of basic proteins o f chicken, turtle, and frog central nervous system myelin. J. Neurocbem. 24:79-88. 33. Martenson, R. E., G. E. Deibler, and M. W. Kies. 1971. The occurrence of two myelin basic proteins in the central nervous system of rodents in the suborders Myomorpha and Sciuromorpha. J. Neurochem. 18:2427-2433. 34. Moscarello, M. A. 1976. Chemical and physical properties of myelin proteins. Current Topics in Membranes and Transport 8:1-28. 35. Palfreyman, J. W., et al. 1979. Radioimmunoassay of serum myelin basic protein and its application to patients with cerebrovascular accident. Clin. Chim. Acta 92:403-409. 36. Palfreyman, J. W., D. G. T. Thomas, and J. G. Ratcliffe. 1978. Radioimmunoassay of human myelin basic protein in tissue extract, cerebrospinal fluid, and serum znd its clinical application to patients with head injury. Clin. Chim. Acta 82:259-270. 37. Paniteh, H. S., C. J. Hooper, and K. P. Johnson. 1980. CSF antibody to myelin basic protein. Measure-
=ll
58
Clin~ca!ImmunologyNewsletter
38.
39.
40.
41. 42.
ment in patients with multiple sclerosis and subacute sclerosing panencephalitis. Arch. Neurol. 37:206-209. Paterson, P. Y., el al. 1981. Endogenous myelin basic protein-serum factors (MBP-SFs) and anti-MBP antibodies in humans: Occurrence in sera of clinically well subjects and patients with multiple sclerosis. J. Neurol. Sci. 52:37-51. Pills, O. M., V. A. Varitek, and E. D. Day. 1976. The extensive crossreaction of several syngeneic rat,anti-BP antiserums with myelin basic proteins (BP) of other species, lmmunochemistry 13:307-312. Reichlin, M. 1975. Amino acid substitution and the antigenicity of globular proteins. Adv. lmmunol. 20:71-123. Rose, N. 1981. International symposium on thyroid autoirl)munity. Clin. Immunol. Newsl. 2."167-168. Varitek, V. A., and E. D. Day.
43.
44.
45.
46.
1979. Relative affinity of antisera for myelin basic protein (MBP) and degree of affinity heterogeneity. Mol. Immunol. 16:163-172. Wallace, A. D., R. Shapira, and R. B. Fritz. 1978. Isolation and characterization of rabbit antibodies to bovine myelin basic protein. Immunochemistry 15:47-54. Webb, C., et al. 1973. In vivo and in vitro immunological crossreactions between basic encephalitogen and synthetic basic polypeptides capable of suppressing experimental allergic encephalomyelitis. Eur. J. lmmunol. 3:279-286. Whitaker, J. N. 1977. Myelin encephalitogenic protein fragments in cerebrospinal fluid of persons with multiple sclerosis. Neurology (N.Y.) 27:919-920. Whitaker, J. N. 1978. Immunochemieal comparisons among myelin basic proteins. Comp.
Biochem. Physiol. [B] 59:299-306. 47. Whitaker, J. N., el al. 1975. Antigenic determinants of bovine myelin encephalitogenic protein recognized by rabbit antibody to myelin encephalitogenic protein. J. Biol. Chem. 250:9106-9111. 48. Whitaker, J. N., et al. 1977. Molecular internalization of a region of myelin basic protein. J. Exp. Med. 146:317-331. 49. Whitaker, J. N., el al. 1979. Antigenic regions for the humoral response to myelin basic protein. Mol. Immunol. 16:495-501. 50. Whitaker, J. N., et al. 1980. Antigenic features of myelin basic protein-like material in cerebrospinal fluid. J. Immunol. 124:1148-1153. 51. Whitaker, J. N., el al. 1980. lmmunoreactive myelin basic protein in the cerebrospinal fluid in neurological disorders. Ann. Neurol. 7:58-64.
Guest Editorial Radioimmunoassays for Myelin Basic Protein: Clinical Applications Helene C. Rauch Department of Immunology and Microbiology Wayne State University School of Medicine Detroit, Michigan 48201 Dr. Eugene Day brings to our attention in this issue the apparently contradictory findings that antibody to myelin basic protein (MBP), the m a j o r structural component o f myelin, has been detected by some investigators using radioimmunoassay in extracts of brains or in cerebrospinal fluids (CSFs) of multiple sclerosis (MS) patients, while other investigators not only did not detect antibody in similar cases but instead found immunologically reactive MBP (or fragments thereof) present. Moreover, the presence of both antigen and specific antibody in the circulation simultaneously is not unique to MS but has been reported in several autoimmune diseases. Now, Dr. Day offers an interesting resolution to this apparent paradox by pointing out some in-
Copyright © 1982 by G. K. Hall & Co.
herent challenges in the design of a completely satisfactory test to detect the presence o f intact MBP or its fragments in a patient's CSF in order to indicate the occurrence o f an acute demyelinative event such as stroke, head trauma or MS exacerbation. In 1975, Cohen, McKhann, and Guarnieri introduced the use o f a competitive radioimmunoassay (RIA) to detect MBP in CSF as a diagnostic indicator of myelin breakdown in the central nervous system (CNS). It was, on the surface, a simple R1A in which antibody, produced in rabbits using CNS tissue or MBP, reacted with MBP present in CSF. Some discrepancies a m o n g reports using RIA systems have caused questions to be raised concerning, in particular, some negative observations. For example, when MBP-reactive material was not observed by one investigator under fluid-phase RIA conditions using his rabbit antiserum but was obsqrved by another using a solid-phase RIA system and different rabbit antisera, there seemed to be a serious contradiction. Not really. The answer is seen
by Dr. Day to depend in part upon the various schemes for immunizing rabbits (of various genetic background) whereby antibodies with different specificities are raised that recognize different determinants such as sequence regions or secondary structural conformation, the latter being more prominent. Variatio~ "n antigenic specificities a m o n g i m m t r a b b i t sera coupled with two bas,d differences in RIA techniques has led to the present dilemma. In the solid-phase RIA the MBP is rigidly affixed to a surface, thus exposing some determinants while others may be simultaneously obscured, while in a liquid-phase RIA the MBP is "flexible" and only some sequences as well as only some structural determinants may be exposed. Such an analysis of the various assays readily explains the apparent conflict among reports from different laboratories, as well as the concomitant circulation of MBP-reactive material and antiMBP antibodies. How to solve the dilemma (i.e., how to develop a uniform testing procedure that detects all or most of the MBPreactive material in CSF) remains to
59
Vol. 3, No. 9
Radioimmunoassays for Myelin Basic Protein Eugene D. Day, Ph.D. Departments of Microbiol~gy&nmunology and Surger3, Duke University~Medical Center Durhanl, North Carolina 27710
The C o n f l i c t By means of a solid-phase radioimmunoassay (RIA) based upon '2q-protein-A detection of adsorbed immunoglobulin in myelin basic protein (MBP)-coated wells o f plastic microtiter plates (30), Bernard et al. (3) have been able to detect anti-MBP antibody in extracts o f multiple sclerosis (MS) brain. The authors were therefore able to explain and support the findings of Panitch et al. (37) that the cerebrospinal fluids (CSFs) o f progressive MS patients often contain anti-MBP antibodies measurable by solid-phase techniques. These findings would appear to be in direct conflict with other lines of evidence. For example, Cohen and Gutstein (12) had been able to show that in the spinal fluids of MS patients there were no detectable anti-MBP antibodies even though, by their liquid-phase RIA technique, they were easily able to measure antiMBP activity in the CSFs of sheep afflicted with experimental allergic encephalomyelitis (EAE). Cohen and his colleagues, after developing an effective liquid-phase RIA for measuring MBP in biologic fluids (14), had been able to show in a blind study of 303 CSFs from patients with a variety of neurologic
disorders that those patients with active demyelinating disease not only did not contain detectable antibody but actually registered relatively high levels of MBP (13). To punctuate this fact Cohen et al. (I 1) had been able to demonstrate similar findings in an additional 394 patients. I m m u n o c h e m i c a l F e a t u r e s o f the MBP Molecule The findings of Panitch et al. (37) and Cohen et al. (l l) are not in conflict, however; the data from both groups may be taken as correct and definitive, and the issue can easily be resolved. The reasons why free anti-MBP antibody may be found by one group while free antigen may be found by another stem from some highly interesting features o f the MBP molecule. Therefore, before discussing any further the clinical findings of MBP a n d / o r its fragments in CSFs, it would be well to review some of the known immunochemical features o f the MBP molecule as it appears in liquid and solid phases. Since a number o f reviews concerning the immunologic, biologic, and chemical aspects of MBP have recently appeared (4, 7, 8, 15, 28, 34), it will not be necessary to present more than a capsular view of the molecule. When the two complete amino acid sequences for bovine and human myelin are compared (Table I), using the original sequence o f Eylar et al. (26) as a frame of
reference to establish the numbering system,* the molecule falls naturally into three segments that are joined by two phenylalanine doublets: A, residues 1-43; B, residues 44-89; and C, residues 90-170. Segments A and C, the N- and C-terminal portions, respectively, have a higher degree of homology'than segment B, one of the many facts that contrast the evolutionary aspects o f MBP with most other proteins. The regions where gap events and interchanges disrupt homologies more frequently are the N-terminal and C-terminal ends (23). At residues 99-t01 in segment C, there is a highly conserved knot of three prolines that was once thought to form a U-type p bend in the molecule, thus simulating a *A different sequence for bovine MBP has also appeared (5) in ~vhichthe serine at position #2 is deleted. In some immunologic studies (e.g., 27, 50) this Brostoff sequence is used, causing segments A, B, and C to be numbered 1-42, 43-88, and 89-169, respectively, and in one review (15) a numbering system based on kno~vnmaximum homology among the species is presented, causing segments A, B, and C to be numbered 1-44, 45-90, and 91-171, respectively.
In This Issue Radioimmunoassays for Myelin Basic Protein . . . . . . . . . . . . . . . . . . . . . . . 53 The present state of the art and a dilemma Guest Editorial . . . . . . . . . . . . . . . . . 59 Clinical applications o f radioimmunoassays for myelin basic protein
hairpin (5), but Martenson (31) disagrees. In the highly conserved C-terminal leg, an internal deletion o f ,.,40 residues, 118-158, occurs in two-thirds of the MBP molecules in the rat. Other Myomorpha (mouse, hamster) as well as Scittromorpha (prairie dog, woodchuck, squirrel) also bear this deletion (presumably the same sequence) to produce in those mammals a mixture o f small and large MBP (33). The hystricomorphs (guinea pig, chinchilla) as well as other vertebrates (rabbit, sheep, chicken, frog) retain the full framework of the C segment of the bovine and human sequences. Microheterogeneity (see Table 1, third footnote) undoubtedly' blays at least a small role in immunologic responses and reactivities, but it is believed that by far the greatest contributor to immunologic differences among MBP molecules of different species is conformational, thus causing differences even in homologous portions o f the MBP molecule.
MBP Cross-Reactivity A m o n g the Species The differences among MBP sequences of the species do not engage the attention o f immunologists as much as the similarities-leading to a high degree of cross-reactivity and self-reactivity. This is not to say that there are not important differences that arise because o f nonhomologous determinants. Whitaker (46), for example, has measured such differences among basic proteins from human, monkey, dog, sheep, pig, cow, guinea pig, rat, rabbit, chicken, and turtle using the Wasserman-Levine method of quantitative microcomplement fixation. In a typical response, even though rabbit-anti-human MBP did not seem to be able to differentiate between human and monkey MBP, it reacted better with dog, sheep, pig, and guinea pig MBPs than with cow, rat (large), or rabbit, and reacted least well with rat (small), chicken or turtle. Even so, the reaction with rabbit MBP was still threequarters of the maximum, and the reaction with chicken MBP still over
54
a quarter. And even in the case of a rabbit-anti-chicken MBP antiserum (32), double immunodiffusion reactivity was obtained with a high degree of identity among precipitin lines and complete fusion among human, rabbit, guinea pig, and cow MBPs or among frog, turtle, chicken, and cow MBPs. In our laboratory (39) the question asked was "whether syngeneic rat antisera raised against Lewis rat MBP would be directed primarily against those parts o f the MBP molecule that were private to the species or whether they would cross-react fully with MBPs from other species, indicating an indifference to species in the Witebskian sense of organ specificity and autoimmunity." A panel of different MBPs coupled to Sepharose 4B was used to absorb several individual syngeneic antiMBP antisera from Lewis rats, and then the absorbed sera were compared with the unabsorbed sera with respect to their antigenic binding capacity for different '2Sl-labeled MBPs. More than 80% of the antibodies in each serum were found to be cross-reactive with the different MBPs, and less than 20% were specific only for the homologous '2q-MBP rat (large). Coupled with the fact that such syngeneic antibodies were also found to display relatively high affinities in their reactions with MBP (42), these findings were taken by Roset to "exclude the JemmersonMargoliash hypothesis (29) that the late evolving, species-restricted determinants are the ones most likely to be involved in autoimmune responses." In the world of clinical immunology, scientists may be used to thinking in terms of a thyroglobulin model (41), in which cross-reactions among the species play little part, but, as Roset has pointed out, "Thyroglobulin represents one end of the spectrum o f organ-specific antigens since there is relatively little cross-reaction among thyroglobulins of different tN. R. Rose. 1981. Personal communication.
species, whereas myelin basic protein typifies the other extreme, showing extensive cross-reactions. Obviously, the evolutionary concepts based on one antigen would not apply to the other." T h e A n t i g e n i c Valence o f M B P The first question might well be, " W h e r e are the antigenic determinants located in the MBP molecule that react and cross-react with anti-MBP antibodies? And how many determinants are there?" A valence of 3 is suggested by a Heidelberger-type treatment (20) of the quantitative precipitin reaction o f a published individual rabbit antiserum to bovine M B P , , but a valence of 4 or more is suggested by Wallace et al. (43), who carried out a liquid-phase Heidelberger-type analysis of one of their rabbit antisera to bovine MBP. Wallace et al. also measured the distribution of solid-phase adsorption activity among the three segments of MBP referred to in Table 1 and found a fairly uniform concentration of antibodies against each of the three parts with a tendency to react somewhat more with the C segment, somewhat less with the A segment, and less uniformly with segment B. Driscoll et al. (25) analyzed seven syngeneic guinea pig (strain 13) antiMBP antisera with respect to the distribution o f activity among six fragments, 1-19, 1-89, 44-88, 1-I 16, 90-170, and 117-169, as compared to whole-molecule MBP, finding that none reacted with 1-19, two with all five of the active fragments (a valence of at least 5 with " b r o a d specificity"), and five with only some o f the active fragments ("narrow specificity"). Whitaker et al. (49) compared rabbit and guinea pig antisera for their reactivities with peptides 90-170, 1-39, and 44-89, finding that all guinea pig antisera preferred 90-170, whereas rabbit antisera reacted with 1-39 as well. Low and variable reactivity with 44-89 was obtained with either type of antiserum. ~:J. Boggs. 1981. Personal communication.
Clinical ImmunologyNewsletter
Disparate RIAs At this juncture in our discussion, we should now focus upon two distinct observations that have emerged from RIA data in the MBP system: a) liquid-phase RIAs capture a smaller and possibly different spectrum o f anti-MBP antibodies than do solid-phase methods; b) small peptide fragments of MBP carry determinants that often do not cross-react with whole-molecule MBP and vice versa, even though the amino acid sequences of the fragments may be homologous to the parent whole molecule. The problems are, perhaps, best illustrated in the work of Fritz et al. (27), who were studying~the B-cell immune responses of Lewis rats to peptide 69-89 (referred to by them as 68-88) homologous to the sequence of guinea pig MBP for that region. In a liquid-phase sodium sulfate RIA with '~q-labeled guinea pig MBP, the rat antipeptide antiserum had no capacity to bind, yet in a solid-phase assay with guinea pig MBP adsorbed to glass, antibodies from the antipeptide antiserum did effectively bind. As possible explanations for this "unexpected finding," the authors suggested either "confotmational changes associated with adsorption o f the protein to a solid matrix" or "dissociation [of relatively low affinity complexes] in the presence of high concentrations of sodium sulfate." It had been noted previously that rabbit antisera to monkey or bovine MBP failed to react with the 44-89 peptide (47), but that if antisera were raised in rabbits to the 44-89 peptide linked to rabbit serum albumin (48) good reactions in a liquid R1A could be obtained with the peptide, even though these antipeptide antisera would not react with whole-molecule MBP. A difference in conformation between the peptide and the parent whole molecule ("molecular internalization" o f part or all o f the 44-89 region in whole MBP) was taken as the explanation. In our laboratory (19), it was shown that synthetic peptides from region 65-84
Copyright © 1982 by G. K. Hall & Co.
of bovine MBP would not react with antibodies in any of nine pools of syngeneic rat anti-MBP antisera, and only peptide $82 (bovine MBP residues 65-83 plus glycine) would react even to a Iimited extent with 14/15 rabbit antisera to MBPs from several species. At the same time (18), whole-molecule, parent bovine MBP would not react with any of 26 rat and rabbit antisera to peptides $82 and S81 (residues 68-83 plus glycine), even though the antipeptide antisera were highly reactive in liquid-phase RIAs with the labeled peptides. Dr. Joan Boggs of the Hospital for Sick Children in Toronto, Ontario, has informed us~ that an anti-S82 antiserum that does not interact with '2SI-MBP in solution (thus confirming that fact) does, however, precipitate lipid vesicles containing MBP in a sequestered form. Meanwhile, we have determined that the same anti-S82 antiserum has a major population o f antibodies that reacts with a conformational determinant of $82 but not with small overlapping sequences o f the same $82 region (17), and that a minor population of antibodies in that antiserum, reacting with a sequential determinant (16), is not involved in MBP cross-reaction. Recently, in our laboratory, K. J. Lazarus also found populations of antibodies in some rabbit antisera against whole-molecule MBP that react with synthetic peptide $24, which is half as large as $82 and representative o f its N-terminal half (residues 65-74 plus glycine), yet do not interact with intermediate $82. These results suggest that the 20-amino acid $82 peptide does indeed have a conformation all its own in solution that is not found in either whole-molecule MBP or the shorter I l-amino acid $24 peptide. The Consequences of MBP Catabolism Where does this leave us with respect to the interpretation of existing reports of immunologically identified MBP molecules and their :l:J. Boggs. 1981. Personal communication.
fragments in CSFs, sera, and other biologic fluids? We observe that MBP is a rather labile protein even in vivo and that it does tend to fragment, yet we find that much of the immunologic reactivity o f antibodies to the whole molecule is retained by reactivity with the large pieces. Consequently, it is sometimes impossible to determine whether a whole molecule or its pieces or both are involved in any one particular immunologic reaction. Although we know little about the conformation o f the large fragments, we do observe that as the fragments become smaller, approaching 20-amino acid peptides, at least some of them take on new formats with the emergence o f new determinants and the loss of others. Then, as the fragments become even smaller, approaching 8-12 amino acid haptenic-sized peptides, there is a loss of even these new determinants, the emergence of yet others, and the recovery o f some original determinants. In the continuing catabolic process, of course, immunologic identity eventually becomes lost altogether. The process may be quite rapid, as it is in rabbits (1), or it may be sluggish, accounting for the eventual build-up and retention of fragment activity in the blood (24, 35, 38). The association of fragments with serum components (a-globulin, bilirubin, /3-1ipoprotein, endogenous autoantibody) is likely to slow the catabolic process even further (2). The heterogeneity o f the fragments with respect to size (2), affinity (22, 38), and SDS-PAGE analysis (9) is evidence for the general breakdown o f MBP beyond the three large segments A, B, and C of Table 1.
Binding Affinities and RIAs There is one more parameter of the MBP-anti-MBP system that has led to seemingly disparate results from laboratory to laboratory--the matter o f binding affinity. As Reichlin (40) has pointed out in his review, different nonrepeating determinants of a given protein in many cases give rise to different binding
55
Table ! Amino Acid Sequence of the Three Segments of Myelin Basic Protein (MBP) of Bovine* and Human1 Origins:~ Segment
A
Species
Sequence
bovine human
0 0 0 0 0 0 0 0 l 1 2 2 3 3 5 0 5 0 5 0 5 ASAQKRPSQR - - SKYLA SASTMDHARHGFLPRHRDTGILDSLGRF ASQKR'PSQRHGSKYLATASTMDHARHGFLPRHRDTGILDS
0 4 5 bovine human
bovine human
0 5 5
0 6 0
0 6 5
0 7 0
0 7 5
IGRF
0 8 0
0 8 5
FG SDRGAPKRGSGKDGHHAARTTHYGSLPQKAQGHRPQDENPVVHF FGGDR,GAPKRGSGKD SHH PARTAHYGSLPQK SHG -RTQDQDPVVHF
0 9 0 C
0 5 0
0 4 0
0 9 5
I 0 0
I 0 5
1 ! 0
i I 5
I 2 0
1 2 5
1 3 0
l 3 5
i 4 0
1 4 5
FKNIVTPRTPPPSQGKGRGLSLSRFSWGAEGQKPGFGYGGRASDYKSAHKGLKGHDAQGT FKNIVTPRTPPPSQGKGRGLSLSRFSWGAEGQRPGFGYGGRASDYKSAHKGFKGVDAQGT
1
5 0 LSK LSK
1
I
!
5 6 6 5 0 5 IFKLGGRDSRSGSPMARR IFK LGGRDSRSGSPMARR
I
7 0
*As given by Eylar et al. (26). A variant in the bovine sequence has also been found (5) in which the serine at residue 2 is deleted. 1As given by Carnegie and Dunkley (7), A genetic variant in the h u m a n sequence has also been found (10) in which one of the two glycines at residues 45-46 has been interchanged with serine. :[:The N-terminal residue #1 is blocked by an acetyl group. The arginine at 108 is methylated on occasion. Glutamine 104 m a y be deamidated in some isolated products. O n e molecule out o f five may be phosphorylated at residues such as serine-55 (endogenous) or serine-110 (exogenous). The C-terminal arginine-170 is extremely labile in isolated MBP. Further details and references may be found in the review by Carnegie and Moore (8).
affinities in an antiserum; moreover, the range of affinities for any one determinant may be considerably more restricted than the overall range of binding affinities for the total protein. MBP has these characteristics (42), with some determinants raising antibodies with affinities much higher than others and with even the lower affinity antibodies (as determined in liquidphase RIAs) having relatively high association constants. When MBP is fragmented into a heterogeneous collection of smaller peptides but still remains reactive with the antiserum against the whole molecule,
36
the various antibody populations in that antiserum will react with the various peptides--one population with one fragment with a very high affinity, another population with another fragment of only a moderate affinity, and so on. Thus, the fragments can be described in terms of the affinity with which they react. For purposes of measurement, the higher affinities become more dominant in antibody RIAs at the higher dilutions of antibody and antigen (42); thus, when inhibition assays are carried out at the higher dilutions, higher affinity fragments can be detected (21). When this
method (called dual-dilution RIAs) was applied to antisera from Lewis rats undergoing EAE (22) or to sera from MS patients (38), heterogeneity of fragments (MBP-serum factors) with respect to affinity was readily seen, with a shifting profile occurring during the time course. Palfreyman et al. (36) had been among the first to recognize the small size of MBP-like material in the CSFs of patients with head injuries and had adopted a method of inhibition RIA that depended on matching inhibition curves of test samples with those of standard MBP. The inhibition curves of the
Clinical Immunology Newsletter
CSF samples were not parallel, suggesting the existence of crossreacting material o f smaller molecular size and, probably, degraded MBP. In the sera o f patients with cerebrovascular accidents (CVAs) (35), relatively high levels o f MBP-like material were also found, and again a nonparallel effect was obtained "indicating differences between the antigenic nature of MBP and the immunoreactivity found in the test serum." A shift to a more restricted population of binding affinities is also likely. Palfreyman et al. (35) also found evidence o f anti-MBP antibodybinding activity in the sera o f five o f the 58 patients who had a history of previous stroke, classifying the antibodies as having low avidity. In the sera o f some o f our clinically well subjects, we also found antiMBP binding activity o f relatively low or moderate affinity (38), while at the same time we detected MBP fragments of relatively high affinity. In one of four patients with clinically active MS, we found the reverse situation: anti-MBP antibody of relatively high affinity and MBP fragments o f relatively low affinity. The B Segment of MBP Segment B, more than A or C, appears to accumulate in the CSFs o f patients undergoing episodes of severe demyelination (45, 50, 51). In 93 specimens from patients with various neurologic diseases, Whitaker et al. (50) found crossreactive peptides in active-MS CSF, in myelopathic CSF, and in CSFs o f acute-stage CVA patients, but "virtually no material" in the same samples reactive with antisera to peptides o f the A and C segments. In collaboration with other groups, Whitaker and his colleagues were able to extend their findings to 582 samples (51) with no change in the overall result. Cohen et ai. (11) were very careful to point out that " n o t all antisera to MBP will react with the CSF myelin basic protein" in the 697 samples they h~/d measured.
CoDvrizht © 1982 by G. K. Hall & Co.
They had found that the best way to raise an effective antiserum for their assays was to immunize rabbits with 250-mg guinea pig spinal cord in complete Freund's adjuvant and boost with MBP. The Whitaker reagents were raised to segment B itself or to human MBP that " c o n tained intact BP as well as an assortment o f BP peptides, which is a common occurrence in the preparation o f human B P " (50). Careful selection among a number of antisera was required to obtain effective ones since most anti-MBP antisera in previous studies had failed to react with segment B (48). Quite obviously, the peptides measured by both the Cohen and the Whitaker groups represent only a select portion o f the MBP molecule, so there could easily be free antibodies against some MBP specificities in solution with free peptides of other specificities. That Cohen and Gutstein (12) did not find anti-MBP antibodies in CSFs o f MS patients whereas Panitch et al. (37) did is not surprising since the Cohen group used a liquid-phase type assay whereas the Panitch group used a solid-phase type assay. As Fritz et al. (27) have shown, an anti-MBP antibody may be refractory to a peptide in liquidphase but fully reactive with the same peptide adsorbed to glass surfaces. The Present Dilemma In conclusion, it should be pointed out that the number of potential determinants in an MBP molecule that will give rise to a humoral response appears to exceed by several fold the actual number involved in raising any one given antiserum; consequently, the effective valence of MBP for any given antiserum may be no more than 3 to 6, obviously a mere sampling o f the number of specificities available. A relatively large library of reagent antisera to MBP woula therefore be required to cover the number of possibilities. Since no accepted standards exist even to identify and capitalize on the major MBP deter-
minants or their respective " t y p i n g " antisera, there is little agreement among the laboratories o f the world as to what constitutes a suitable reagent for an RIA. The problem is compounded by the fact that MBP tends to fragment in vivo as well as in vitro and that the resulting degradation products tend to clear from the circulation at different rates. This leaves behind in the body fluids an accumulation o f only a part of the MBP molecule with its own set of intact determinants. Only certain o f the reagent antisera that happen to have antibodies specific for this set can be used effectively in competitive inhibition RIAs. The problem is further compounded by the fact that certain o f the MBP fragments tend to change their conformation, resulting in the loss o f determinants of the parent molecule and the gain o f new ones. Such fragments remain "silent" during the search for inhibitory material in biologic fluids using antibodies to the parent proteins, unless solid-phase RIA techniques are adopted. The problem with the solid-phase approach, however, is that very low affinity nonspecific binding is difficult to control, and high affinity binding (which is usually low titered) is hard to distinguish. Particularly in the evaluation of binding parameters and in the quantitative analysis o f inhibitory materials, the liquid-phase approach is far superior and far less complicatedi however, it can provide only part of the answer. The dilemma of the moment, therefore, is that there is not even one satisfactory method for the quantitative inhibition analysis of MBP fragments that is certain to capture the full complement of all parts of this biologically important molecule. Until such a method has been devised and placed in service in clinical laboratories around the world, the evaluation of MBP and its antibodies in clinical material will have to remain a relatively haphazard exercise.
57
References I. Bashlr, R. M., and J. N. Whitaker. 1980. Metabolism of a peptide of human myelin basic protein in the rabbit. Neurology (N.Y.) 30:1184-1192. 2. Bashir, R. M., and J. N. Whitaker. 1980. Molecular features of immunoreactive myelin basic protein in cerebrospinal fluid of persons with multiple sclerosis. Ann. Neurol. 7:50-57. 3. Bernard, C. C. A., el al. 1981. Antibody to myelin basic protein in extracts of multiple sclerosis brain. Immunology 43:447-457. 4. Braun, P. E., and S. W. Brostoff. 1977. Proteins of myelin, pp. 201-231. In P. Morell (ed.), Myelin. Plenum Press, New York. 5. Brostoff, S. W., et ai. 1974. Specific cleavage of the AI protein from myelin with cathepsin D. J. Biol. Chem. 249:559-567. 6. Brostoff, S. W., and E. I1. Eylar. 1971. Localization of methylated arginine in the AI protein from myelin. Proc. Natl. Aead. Sci. U.S.A. 68:765-769. 7. Carnegie, P. R., and P. R. Dtmkley. 1975. Basic proteins of central and peripheral nervous system myelin. Adv. Neurochem. 1:95-135. 8. Carnegie, P. R., and W. J. Moore. 1980. Myelin basic protein, pp. 119-143. In R. A. Bradshaw and D. M. Schneider (eds.), Proteins of the nervous system. Raven Press, New York. 9. Carson, J. H., et al. 1978. Components in multiple sclerosis cerebrospinal fluid that are detected by radioimmunoassay for myelin basic protein. Proc. Natl. Acad. Sci. U.S.A. 75:1976-1978. 10. Chou, C.-H. J., et al. 1978. Identity of myelin basic protein from multiple sclerosis and human control brain. J. Neurochem. 30:745-750. 11. Cohen, S. R., et al. 1978. Cerebrospinal fluid myelin basic protein and multiple sclerosis, pp. 513-519. In J. Palo (ed.), Myelination and demyelination. Plenum Press, New York. 12. Cohen, S. R., and H. S. Gulstein. 1978. Spinal fluid differences in experimental allergic encephalomyelitis and multiple sclerosis. Science 199:30t -303. 13. Cohen, S. R., R. M. llerndon, and G. M. McKhann. 1976. Radioimmunoassay of myelin basic protein in spinal fluid. An index of active demyelination. N. Engl. J. Med. 295:1455-1457. 14. Cohen, S. R., G. M. MeKhann, and M. Guarnleri. 1975. A radioimmunoassay for myelin basic protein
15. 16.
17.
18.
19.
20.
21.
22.
23.
and its use for quantitative measurements. J. Neurochem. 25:371-376. Day, E. D. 1981. Myelin basic protein. Contemp. Top. Mol. Immunol. 8:1-39. Day, E. D., et al. 1981. Affinity purification of an acylated and radiolabelled synthetic derivative of residues 75-83 of bovine myelin basic protein, 'z*I-$79: A model for the purification of picomole quantities of specific peptide fragments of myelin basic protein and antibodies against them. J. Neuroimmunol. I:311-324. Day, E. D., el al. 1981. Equilibrium and non-equilibrium competitive inhibitions of anti-peptide antibody binding by parent myelin basic protein and 18 related peptide sequences. Neurochem. Res. 6(5):577-593. Day, E. D., el al. 1981. Immunogenicity of synthetic peptide sequences $81 and $82 (residues 68-83 and 65-83) of bovine myelin basic protein: Time-course of antibody responses in rats and rabbits. J. Neuroimmunol. 1:205-216. Day, E. D., et ai. 1981. Synthetic peptides from region 65-84 of bovine myelin basic protein: Radioimmunoassays and equilibrium competitive inhibition studies with antibodies prepared against myelin basic protein. Neurochem. Res. 6(8):913-929. Day, E. D., and O. M. Pitts. 1974. Radioimmunoassay of myelin basic protein in sodium sulfate, lmmunochemistry 11:652-659. Day, E. D., and V. A. Varitek. 1981. Radioimmunoassays of antibodies to myelin basic protein (MBP), dual-dilution radioimmunoassays, and competitive binding radioimmunoassays to measure MBP and its fragments, pp. 380-385. In N. R. Rose and H. Friedman (eds.), Manual of clinical immunology, 2nd ed. American Society for Microbiology, Washington, D.C. Day, E. D., V. A. Vafitek, and P. Y. Paterson. 1981. Endogenous myelin basic protein-serum factors (MBP-SFs) in Lewis rats: Evidence for their heterogeneity and reactivity with anti-MBP antibodies of different affinities. J. Neurol. Sci. 49:1-17. Dayhoff, M. O., and W. C. Barker. 1972. Mechanisms in molecular evolution: Examples, pp. 41-45. In M. O. Dayhoff (ed.), Atlas of protein sequence and structure, vol. 5. National Biomedical Research Foundation, Washington, D.C.
24. Delassalle, A., et al. 1980. Radioimmunoassay of the myelin basic protein in biological fluids, conditions improving sensitivity and specificity. Biochimie 62:159-165. 25. Driseol|, B. F., A. J. Kramer, and M. W. Kies. 1974. Myelin basic protein: Location of multiple independent antigenic regions. Science 184:73-75. 26. Eylar, E. It., et al. 1971. Basic A protein of the myelin membrane: The complete amino acid sequence. J. Biol. Chem. 246:5770-5784. 27. Fritz, R. B., et al. 1979. The immune response of Lewis rats to peptide 68-88 o f guinea pig myelin basic protein. 1I. B cell determinants. J. lmmunol. 123:1544-1547. 28. Hashim,,G. 1978. Myelin basic protein: Structure, function, and antigenic determinants, lmmunol. Rev. 39:60-107. 29. Jemmerson, R., and E. Margoliash. 1979. Specificity of the antibody response o f rabbits to a self antigen. Nature 282:468-471. 30. Linthlcum, D. S., et al. 1981. Detection of antibodies to myelin basic protein by solid-phase radioimmunoassay with ['~q] protein A. J. Neuroimmunol. 1:17-26. 31. Martenson, R. E. 1981. Prediction of the secondary structure of myelin basic protein. J. Neurochem. 36:1543-1560. 32. Marlenson, R. E., and G. E. Deibler. 1975. Partial characterization of basic proteins o f chicken, turtle, and frog central nervous system myelin. J. Neurocbem. 24:79-88. 33. Martenson, R. E., G. E. Deibler, and M. W. Kies. 1971. The occurrence of two myelin basic proteins in the central nervous system of rodents in the suborders Myomorpha and Sciuromorpha. J. Neurochem. 18:2427-2433. 34. Moscarello, M. A. 1976. Chemical and physical properties of myelin proteins. Current Topics in Membranes and Transport 8:1-28. 35. Palfreyman, J. W., et al. 1979. Radioimmunoassay of serum myelin basic protein and its application to patients with cerebrovascular accident. Clin. Chim. Acta 92:403-409. 36. Palfreyman, J. W., D. G. T. Thomas, and J. G. Ratcliffe. 1978. Radioimmunoassay of human myelin basic protein in tissue extract, cerebrospinal fluid, and serum znd its clinical application to patients with head injury. Clin. Chim. Acta 82:259-270. 37. Paniteh, H. S., C. J. Hooper, and K. P. Johnson. 1980. CSF antibody to myelin basic protein. Measure-
=ll
58
Clin~ca!ImmunologyNewsletter
38.
39.
40.
41. 42.
ment in patients with multiple sclerosis and subacute sclerosing panencephalitis. Arch. Neurol. 37:206-209. Paterson, P. Y., el al. 1981. Endogenous myelin basic protein-serum factors (MBP-SFs) and anti-MBP antibodies in humans: Occurrence in sera of clinically well subjects and patients with multiple sclerosis. J. Neurol. Sci. 52:37-51. Pills, O. M., V. A. Varitek, and E. D. Day. 1976. The extensive crossreaction of several syngeneic rat,anti-BP antiserums with myelin basic proteins (BP) of other species, lmmunochemistry 13:307-312. Reichlin, M. 1975. Amino acid substitution and the antigenicity of globular proteins. Adv. lmmunol. 20:71-123. Rose, N. 1981. International symposium on thyroid autoirl)munity. Clin. Immunol. Newsl. 2."167-168. Varitek, V. A., and E. D. Day.
43.
44.
45.
46.
1979. Relative affinity of antisera for myelin basic protein (MBP) and degree of affinity heterogeneity. Mol. Immunol. 16:163-172. Wallace, A. D., R. Shapira, and R. B. Fritz. 1978. Isolation and characterization of rabbit antibodies to bovine myelin basic protein. Immunochemistry 15:47-54. Webb, C., et al. 1973. In vivo and in vitro immunological crossreactions between basic encephalitogen and synthetic basic polypeptides capable of suppressing experimental allergic encephalomyelitis. Eur. J. lmmunol. 3:279-286. Whitaker, J. N. 1977. Myelin encephalitogenic protein fragments in cerebrospinal fluid of persons with multiple sclerosis. Neurology (N.Y.) 27:919-920. Whitaker, J. N. 1978. Immunochemieal comparisons among myelin basic proteins. Comp.
Biochem. Physiol. [B] 59:299-306. 47. Whitaker, J. N., el al. 1975. Antigenic determinants of bovine myelin encephalitogenic protein recognized by rabbit antibody to myelin encephalitogenic protein. J. Biol. Chem. 250:9106-9111. 48. Whitaker, J. N., et al. 1977. Molecular internalization of a region of myelin basic protein. J. Exp. Med. 146:317-331. 49. Whitaker, J. N., el al. 1979. Antigenic regions for the humoral response to myelin basic protein. Mol. Immunol. 16:495-501. 50. Whitaker, J. N., et al. 1980. Antigenic features of myelin basic protein-like material in cerebrospinal fluid. J. Immunol. 124:1148-1153. 51. Whitaker, J. N., el al. 1980. lmmunoreactive myelin basic protein in the cerebrospinal fluid in neurological disorders. Ann. Neurol. 7:58-64.
Guest Editorial Radioimmunoassays for Myelin Basic Protein: Clinical Applications Helene C. Rauch Department of Immunology and Microbiology Wayne State University School of Medicine Detroit, Michigan 48201 Dr. Eugene Day brings to our attention in this issue the apparently contradictory findings that antibody to myelin basic protein (MBP), the m a j o r structural component o f myelin, has been detected by some investigators using radioimmunoassay in extracts of brains or in cerebrospinal fluids (CSFs) of multiple sclerosis (MS) patients, while other investigators not only did not detect antibody in similar cases but instead found immunologically reactive MBP (or fragments thereof) present. Moreover, the presence of both antigen and specific antibody in the circulation simultaneously is not unique to MS but has been reported in several autoimmune diseases. Now, Dr. Day offers an interesting resolution to this apparent paradox by pointing out some in-
Copyright © 1982 by G. K. Hall & Co.
herent challenges in the design of a completely satisfactory test to detect the presence o f intact MBP or its fragments in a patient's CSF in order to indicate the occurrence o f an acute demyelinative event such as stroke, head trauma or MS exacerbation. In 1975, Cohen, McKhann, and Guarnieri introduced the use o f a competitive radioimmunoassay (RIA) to detect MBP in CSF as a diagnostic indicator of myelin breakdown in the central nervous system (CNS). It was, on the surface, a simple R1A in which antibody, produced in rabbits using CNS tissue or MBP, reacted with MBP present in CSF. Some discrepancies a m o n g reports using RIA systems have caused questions to be raised concerning, in particular, some negative observations. For example, when MBP-reactive material was not observed by one investigator under fluid-phase RIA conditions using his rabbit antiserum but was obsqrved by another using a solid-phase RIA system and different rabbit antisera, there seemed to be a serious contradiction. Not really. The answer is seen
by Dr. Day to depend in part upon the various schemes for immunizing rabbits (of various genetic background) whereby antibodies with different specificities are raised that recognize different determinants such as sequence regions or secondary structural conformation, the latter being more prominent. Variatio~ "n antigenic specificities a m o n g i m m t r a b b i t sera coupled with two bas,d differences in RIA techniques has led to the present dilemma. In the solid-phase RIA the MBP is rigidly affixed to a surface, thus exposing some determinants while others may be simultaneously obscured, while in a liquid-phase RIA the MBP is "flexible" and only some sequences as well as only some structural determinants may be exposed. Such an analysis of the various assays readily explains the apparent conflict among reports from different laboratories, as well as the concomitant circulation of MBP-reactive material and antiMBP antibodies. How to solve the dilemma (i.e., how to develop a uniform testing procedure that detects all or most of the MBPreactive material in CSF) remains to
59