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Anti-antibodies
Anti-antibodies PHILIP G.
H. GEL1 AND ANDREW S. KELUS
Department of Experimental Pathology, University o f Birmingham, Birmingham, England
I. Introduction . . . . . . . . . . . . . 11. Anti-antibodies in the Immunization Process and “Subcomplementarity” 111. Immunogenicity of “Altered” IgG. . . . . . . . . IV. Rheumatoid Factor and Rheumatoid Factor-like Antibodies . . V. Changes in the IgG Molecule Leading to the Production of Rheumatoid Factor-like Antibodies: the Fc Piece . . . . . . . VI. Distortion in the Fab Piece of IgG: Anticomplex Antibodies . . VII. “Natural” Anti-antibodies: Agglutinators . . . . . . VIII. Experimental Anticomplex Antibodies . . . . . . . IX. Molecular Location of Revealed Determinants . . . . . X. “Anticlone” Antibodies . . . . . . . . . . XI. Anticlone Antibodies by Heteroimrnunization . . . . . XII. Isoimmune Anticlone Antibodies (Idiotypes) . . . . . . XIII. Biological Significance of Anti-antibodies . . . . . . References . . . . . . . . . . . . .
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1. Introduction
It is remarkable how comparatively recently the proteins of the plasma have been differentiated as physicochemical entities, and antibody activity has been shown to be the property of well-defined proteins rather than of the “serum” as a whole. Only for the last decade have the various classes of immunoglobulins and the possibility of interspecific differences as well as intraspecific differences between immunoglobulins of a single class been recognized. At the same time the concept that an animal may react immunologically against its own autogenous antigens has been generally accepted. An anti-antibody in the sense used here means an antibody which will react as such with an Ig molecule1 because that molecule is an antibody, not just because that molecule is a y-globulin. This does not imply that the antibody-combining site of that molecule is necessarily the locus of interaction, which has never been definitively shown. The word ‘We employ the terminology recently proposed (Nomenclature for Human Immunoglobulins, 1964 ) of immunoglobulins or their components, using the blanket symbol Ig when no particular class of immunoglobulin is referred to; though most of the work discussed in detail refers to “ordinary 7 S y-globulin,” IgG. 461
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“becausc,”in fact, covers three quite diffcrent situiiti~l~s. The first is
when anti-antibodies are elicitable because the antibody has reacted with antigen beforehand (in a way characteristic, as far as one can judge, only of antibodies) so as to expose, or to form de nouo, structures which, though potentially present in any Ig molecule. are not immunogenic in the intact molecule. The second is when anti-antibodies are elicitable because the antibody-combining site itself, in the intact molecule, is immunogenic and is involved in the interaction. The third is when there is an absolute correlation between the presence of a particular specific cornbining site and an immunogenic site elsewhere on the molecule. A fourth situation, strictly excluded by this definition, is when the antibodies all come from a highly restricted subclass of, say, IgG, but where there are molecules in this subclass capable of reacting with the presumed antiantibody yet which do not appear to possess antibody activity themselves. These may, of course, be antibodies of low or negligible affinity for the antigen but which are, in fact, present because of and produced by the original induction stimulus; or they may be quite unrelated to it. The earlier speculations and experiments on anti-antibodies were along the lines (1) whether heterologous antisera could be raised against antibodies as such (as opposed to the “normal” proteins of the serum) and ( 2 ) whether the change in immunoglobulin thought to be “induced” by antigen might itself be recognized as foreign by the animal in which it occurred and whether this might play a part in the immunization process. Pioneer work along these lines is discussed in that off-beat but stimulating book “Immuno-catalysis” by Sevag ( 1945). The earliest studies by Ehrlich, Bordet, Moreschi, and others were influenced by the idea that antibody was a substance arising de novo, unrelated in any way to the pre-existing proteins of the serum. Thus antisera that neutralized thc activities of antibodies ( Iysins, agglutinins) were termed “anti-antibodies.” By the time of the paper by Eagle (1930), antibodies were recognized as members of a class of serum protein. Smith and Marrack (1930) were the first to state clearly that, since antitoxin, when precipitated by a precipitin, still combines with toxin, different groups on the antitoxin molecules must be involved in the two reactions-a point confirmed by the studies of Eagle (1936). Treffers and Heidelberger (1941) in a study of horse antisera concluded that “the groupings responsible for the antibody function constitute either a small part of the total protein molecule or else are non-antigenic.” Other work on these lines (e.g., Ando et al., 1938; Northrop, 1942) is difficult to interpret, presumably owing to lack of knowledge at the time of the macroglobulin
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(IgM) nature of some horse antibodies and of their possible nonreactivity or cross-reactivity with antisera to IgG. A simple-minded attempt to demonstrate anti-antibodies is, therefore, likely to encounter a number of difficulties. If heteroimmunization is used, ( 1 ) the bulk of the antibody will be species-specific and ( 2 ) antibodies specific to a class or even a subclass of Ig molecules may be confused with specific anti-antibodies. If isoimmunization is used, antibodies may be produced specific for genetic differences on IgG (or other proteins) not common to donor and recipient, i.e., to allotypic determinants. Since the bulk of our discussion here will be concerned with rabbit antibodies, and since the genetic situation with rabbit IgG allotypes (Dray et al., 1962) is fairly simple and well worked out, it is advantageous to outline at this stage the relevant data on this-not because antiallotypic antibodies are anti-antibodies in any sense used here, but because it is necessary to be aware of the allotypic situation whenever intraspecies cross-immunizations are used ( see review, Kelus and Gell, 1967). At present there are two well-defined loci, a and b, giving rise to allotypic determinants. The a-locus determinants are located on the heavy chain (Asl, 2, 3); the b-locus determinants (As4, 5, 6 ), on the light chain, and possibly also in the heavy chain (Feinstein et al., 1963). There is evidence for at least one other locus for IgG, and one for the heavy chain of IgM (Kelus and Gell, 1965). Quite extensive breeding data from both Europe and America for laboratory rabbits have not given any indication of further alleles at either the a or b locus. The blocus determinants are quite strongly immunogenic, although the use of some kind of adjuvant is usually needed to raise strongly precipitating antisera; the a-locus determinants are much less so, and adjuvants are always needed. A proportion of IgG molecules (10-20%) seems to lack determinants from one or the other locus. II. Anti-antibodies in the Immunization Process and “Subcomplementarity”
Before we consider more recent experiments on the direct elicitation of anti-antibodies, the work of Najjar (1963) and his colleagues may be considered. To this group must go the credit of having persistently drawn attention to the possible role of molecular distortions in the immune process, although the rather elaborate theoretical structure which they have erected upon their experiments is not acceptable to many workers today. Najjar considers that “when the first antibody” [to whose process of formation we shall return in a moment] “reacts in the antigenic site, both antibody and antigen are shown to be altered simul-
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taneousl!,. Antibodies are then formed against these sites of altered configuration. This process continues as further sites appear on the antigen and reacting antibody” (Najjar and Robinson, 1959). Thus the antibodies formed in the later stages of immunization would be to novel determinants not present in the “native” antigen, a proportion of them, indeed, to determinants not on antigen but on antibody. “After the third injection [of a native protein antigen] over 80% of antibody protein reacted with [an antigen-antibody complex made at equivalence] . . . leaving behind the remainder which reacted with antigen only” (Najjar and Fisher, 1955). The authors go on to advance as supporting evidence the work discussed below of Milgrom and Dubiski and others on rheumatoid factor-like anti-antibodies. Although it is difficult to accept either the authors’ experiments, or their supporting evidence, as being only, or even most readily, explainable in terms of their theory, this does not imply that the theory, first put forward at a time when autoantibodies were still hardly respectable, does not contain much truth. It is not quite clear whether the distortion of the antibody is thought of as being antigen-specific, which is hard to envisage in physicochemical terms, or a nonspecific result of antigen-antibody interaction, in which case the later stages of immunization should lack all specificity, since antibodies developed against one complex would react with all other complexes. Such antibodies are, indeed, sometimes demonstrable ( see below) but are produced only in special circumstances and not apparently as a general rule. Failure to eliminate allotypic differences between rabbits may make some experiments hard to interpret (Najjar and Fisher, 1955), though not those in which autostimulation is thought to have occurred (Harshman et aZ., 1963); however, application of newer knowledge of Ig structure and the use of the digerential power of gel-diffusion techniques to demonstrate antibodies of different specificities can bring a greater precision to such experiments. Najjar and co-workers go on to postulate that precipitation, in fact, occurs only when the “fit” of antigen and antibody is not exact (“subcomplementary”), so leading to some distortion. “Qualitatively there could then be three types of immune globulin synthesized in response to an antigen stimulus. The first type would seem to be those that are sufEciently sub-complementary to the antigen site to undergo configurational alterations, i.e. antibodies detectable by the usual immunochemical techniques.” [These are considered to arise because antibody is synthesized in relation to antigenic fragments, as discussed by Campbell (1957) and others, as well as to native antigen with its normal tertiary structure.] “To the second type belong the globulins that are comple-
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mentary to the antigen site and, therefore, not detectable under ordinary conditions. The third type falls between these two and includes a spectrum of molecules with varying degrees of sub-complementarity.” Though this theory can be used to explain certain types of cross-reactivity, the basic supposition that immunological precipitation is due, not to “complementary” antigen-antibody interaction leading to lattice formation, but solely to the uncovering of hydrophobic groups as a result of subcomplementary molecular distortion, would seem to ignore the whole history of immunochemistry from Landsteiner and Marrack onward. More recent studies of what conditions do, in fact, lead to molecular distortion, and in what parts of the molecule, have made the situation much clearer. Ill. lmmunogenicity of “Altered” IgG
This work entails five different kinds of experiments: ( a ) experiments with red cells or bacteria coated with antibody and injected into other animals of the same species (isoimmunization) or of a different species (heteroimmunization ) ; ( b) experiments in which antigen-antibody complexes are made in various ratios and used for immunizing other animals of the same species; ( c ) experiments in which IgG is altered in uitro and reinjected into the donor animal; ( d ) the demonstration in normal rabbit sera of IgM antibodies against rabbit IgG antibody which has reacted with antigen; and ( e ) strong immunization with any antigen may be found to give rise to antibodies reacting with Ig of one sort or another. By such experiments, four evidently different kinds of antibodies have been demonstrated, singly or in combination. 1. Antiallotypic antibodies, when isoimmunization by methods ( a ) or ( b ) has been used-such methods led, in fact, to the original demonstrations of IgG allotypes by Oudin (1956). Even where the known allotypes are identical in donor and recipient, a new allotype may be uncovered, the characteristic of this situation being that the preimmunization serum of the donor and of certain other rabbits will react with the antibody elicited. These antiallotypic antibodies need not be considered further here. 2. Rheumatoid factor-like antibodies [methods ( a ) , ( b ) , ( c ) , and ( e ) ]-these characteristically cross-react with Ig of species other than the donor. 3. Anticomplex antibodies [methods ( a ) and ( b ) ; allotypic differences being eliminated]- these can be shown to react with any antigenantibody complexes made with the Ig of the same species or with antibody-coated cells, but not with unreacted antibody. It is an open question
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PHILIP G . H. GELL AND Ah’DREW S. KELUS
whether “antigen-specific” anticomplex antibodies also occur. The ‘hatural” antibodies ( d ) fall also into this group. 4. “Anticlone” antibody [methods ( a ) and (b)]-these will react only with the actual antibody used as immunogen, not with preimmunization serum from the same animal-the antigen need not be in the form of a complex. Immunoconglutinin ( Coombs and Coombs, 1953)-although not an anti-antibody under our definition is an additional type of antibody, which is likely to be detectable if looked for with an appropriate test system and which, if unsuspected, may lead to misleading conclusions. However, it has not been recorded as precipitating antibody. It is evidently directed against activated autogenous complement, and occurs regularly as an accompaniment of any fairly intense immunization. Its properties have recently been fully reviewed (Coombs et al., 1961). IV. Rheumatoid Factor and Rheumatoid Factor-like Antibodies
Whether one considers rheumatoid factor ( RF) , as arising in rheumatoid arthritis and some related diseases in man, to be relevant to this discussion depends upon one’s opinion as to its origin. There seems little doubt that it is an IgM antibody induced by “damaged IgG, and it is possible, but not proved, that this damage arises because the IgG has reacted, as antibody, with some extrinsic, e.g., bacterial, antigen, and therefore the RF is brought within our definition of an anti-antibody. This hypothesis was put forward by Dubiski (1958). The extensive chemical and experimental work on human RF has been reviewed elsewhere (e.g., Glynn, 1963). Williams and Kunkel (1965) in a discussion of antiantibodies refer to a number of antibodies of this nature occurring in rheumatoid arthritis sera. Similarly the identification of the Gm and InV allotypes of man by means of normal ( SNagg) sera and rheumatoid (RF, Ragg) sera, is outside the compass of this review. We shall, therefore, not consider further the spectrum of antibodies in human RF sera except when showing analogies with those experimentally produced. Milgrom and Witebsky ( 19sO) showed that autoimmunization of rabbits with their own IgG precipitated with ammonium sulfate or alum (and presumably mildly denatured), in Freunds complete adjuvant, gave rise to precipitating and complement-fixing antibodies which reacted very much better with human than with rabbit IgG. McCluskey et al. (1%2), in a controlled study of various methods of denaturation, recorded the appearance of antibodies of this type. These results can be taken as possible models for the production of anti-antibody.
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The rheumatoid factor-like substance (RFLS) of Abruzzo and Christian (1961), arising from long immunization of rabbits with formalin-killed bacteria, showed somewhat different properties. This was a macroglobulin which reacted with immune complexes, made both with human and rabbit antisera, and with cells coated with heat-aggregated rabbit and human IgG. Cross-absorbtion showed that the rabbit IgG was the homologous, and human IgG the cross-reacting antigen; in these properties it shows an analogy with human RF. Aho and Wager (1961) and Williams and Kunkel (1963) described similar antibodies in rabbits both of 19 and 7s type, after immunization with various protein antigens. In man, RF-like antibodies may be demonstrable in many chronic diseases (as quoted by Abruzzo and Christian, 1961) and even transiently after prophylactic immunization with tetanus or diphtheria toxoid ( Svec and Dingle, 1965). V. Changes in the IgG Molecule leading to the Production of Rheumatoid Factor-like Antibodies: the Fc Piece
There is evidence that the precipitation of RF with heated IgG is the result of changes in the Fc piece of the latter (Henney and Stanworth, 1965b), involving S-S bond rupture and some aggregation. The Fc part of the molecule is also involved in complement fixation and in the irritant properties of antigen-antibody complexes ( Ishizaka and Ishizaka, 1964 ) . The RF-like antibodies induced by Milgrom and Witebsky (1960) and McCluskey et al. (1962) were evidently produced in response to Fc piece changes. This sort of change evidently gives rise to a determinant which is both autoimmunogenic and widely cross-reacting-as with other types of cross-reacting system, heated IgG from some species, e.g., rabbit, reacts very much better with human R F than does that from others, e.g., horse ( Henney and Stanworth, 1965a). Antigen-antibody combination may also give rise to similar Fc piece changes: it is as a result of some such process that the RF-like antibodies of Abruzzo and Christian are likely to have been provoked. Antigen-antibody complexes and reacted antibody on the surface of cells (as in the Rose-Waaler test) will for the same reason react with human RF. The changes induced by antigen-antibody union, however, are more complex than those induced by heat “denaturation.” (An unfortunate word to use in this connection, in that it often implies an “all-or-none” irreversible reaction, whereas the changes which we are considering are progressive, and, in the early stages at least, reversible.) In the first place, those changes occurring when antibody reacts with a soluble antigen are
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PHILIP G. H. GELL AND ASDREW S. KELUS
critically dependent upon the antigen (Ag)-antibody ( Ab) ratio of the complex formed, being maximal in moderate antigen excess, where the ratio is Ags Ab,. and probably minimal both at the gross antigen excess ratio ( Ag, Ab, Ag,,) and at equivalence. Reaction of antibody with a cell, e.g. ;in erythrocyte, will produce some distortional changes in antibody apparently independently of the ratio of the reactants, and even with soluble antigens there is some evidence that the larger the antigen the wider the range of ratios at which distortion occurs (Henney and Stanworth, 1966). The distortion of antibody on union at a critical ratio is reflected in increase in free -SH (Robert and Grabar, 1957) and in optical rotation (Ishizaka and Campbell, 1959). That the latter was due to changes in antibody rather than antigen was indicated by the fact that the increase in optical rotation was still demonstrable when an optically inactive antigen was used. Distortion of antigen does, however, sometimes occur, most elegantly shown in studies with enzyme-antienzyme systems, e.g., those of Pollock ( 1964) with antipenicillinases where clear-cut changes in substrate specificity and kinetics were demonstrable, as well as those investigated by Najjar (1963).
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VI.
Distortion in the Fob Piece of IgG: Anticomplex Antibodies
Thus both physical treatment, and union with antigen, will produce alterations of the IgG (antibody) molecule in its Fc piece. In the latter situation there is evidence, however, that other and, perhaps, mere significant changes occur at the Fab end of the molecule. These changes are recognizable by altered immunogenicity, but antisera elicited by the altered molecules still react more strongly with homologous than with heterologous IgG. This is in contrast with the cross-reactivity of the Fc piece changes. The anti-antibodies so produced have the property of reacting with homologous antibody as it exists in a complex at antigen excess or attached to a cell, but not with heated IgG or with heterospecific IgG complexes. They are demonstrable, as discussed below, in a proportion of normal rabbit sera under special conditions, or as a result of experimental immunization with complexes formed in uitro. No unequivocal demonstration that this kind of anti-antibody arises as a result of true spontaneous autostimulation has apparently appeared, though the production of immunoconglutinin (Coombs and Coombs, 1953) provides an analogy. One might expect a course of repeated large injections of antigen, as in the experimental production of “serum sickness nephritis,” to provoke
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their appearance, but Boyns (1966) during experiments of this type has hitherto failed to find any antibodies of this nature. VII. “Natural” Anti-antibodies: Agglutinators
The “anti-antibodies” described by Milgrom and Dubiski ( 1957) were later identified by Dubiski et al. (1958) as probably antiallotypic antibodies. However, Milgrom ( 1962) described antibodies ( “agglutinators”) in a proportion of normal rabbits, which in a subsequent communication (Fudenberg et al., 1963) were found to be macroglobulins capable of reacting with antigen-antibody complexes made from antibody digested with papain and therefore lacking the Fc piece, as well as with rabbit red cells sensitized with rabbit isoagglutinin. Cohen and Tissot ( 1965) analyzing the latter situation, showed that only certain isoagglutinins could, on reacting with the red cells, reveal a site capable of combining with the agglutinator, and there was some evidence from family data that the “agglutinator trait” was under genetic control. Although it was not possible to decide whether the agglutinator resulted from specific stimulation or not, it would seem reasonable to refer to it as a “natural” antibody, by analogy with other “natural” isoagglutinins. VIII. Experimental Anticomplex Antibodies
Antisera were raised by isoimmunization against preformed soluble complexes made in vitro by Leskowitz (1960), but he concluded that the antibodies demonstrated were probably against allotypic determinants rather than against complexes as such. Henney et al. (1965) immunized rabbits with preformed bovine serum albumin ( BSA )-anti-B.S.A. complexes, the known allotypes of donor and recipient being identical, and analyzed the resulting antisera by gel diffusion. In this study no RF-like antibodies were produced, though antibodies were produced against the antigen contained in the complexes used as immunogen. There was no evidence of the production of antiallotypic or other antibodies reacting with the free antibody used to make the immunogenic complex, but true anticomplex (anti-cpx) antibodies could be demonstrated. By this we mean antibodies that will react on agar diffusion with complexes made between other rabbit antibodies and their respective antigens. The reaction of anti-cpx antisera with the immunizing complex ( BSA-anti-BSA) and with BSA alone was more complicated (see Fig. l ) , since the anti-cpx antiserum contained in addition much anti-BSA antibody-as one would expect when immunization was carried out with BSA-antibody complexes in antigen excess. Therefore, when the anti-cpx antiserum was diffused in agar against BSA,
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complexes were formed which then reacted in situ with the anti-cpx antibodies present in the same serum. However, the expected strong spur was formed when complexes made between another antigen and its antiserum were reacted in an adjacent cup. Further information was obtained as to the location in the IgG molecule of the new “determinant,” produced on complexing, in that, though no reaction was obtained between the anti-cpx serum and samples of fresh IgG nor with several samples of heated IgG capable of reacting with R F (via changes in the Fc piece), a definite reaction occurred
FIG.1. Diagram to illustrate the reactions of anticomplex antiserum. Note spur between homologous and heterologous systems; both anti-BSA and anticomplex antibodies pile up in this line. (BSA = bovine serum albumin.)
with the separated Fab pieces obtained by papain digestion and with a (heavy; 7 ) A-chain preparation made by the method of Fleischman et al. ( 1962). It was tentatively concluded that the determinants revealed by this study were in the Fab region of the heavy chain, contiguous to the antibody-combining site, revealed and made immunogenic as a result of the distortion produced by reaction with antigen. Two samples out of thirty of stored IgG, in which some aggregation had occurred, also showed the presence of this same determinant reacting with the anti-cpx serum. Thus though attempts to expose this Fab determinant experimentally by physical means were unsuccessful, one may suppose its appearance not to be absolutely confined to antigen-antibody reactions, but producihlc also 11y other forms of degradation, of unknown nature. A notable characteristic of the anti-cpx serum u7as that it reacted most strongly on gel diffusion with complexes in that ratio at which
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maximal distortion of the molecule occurred as judged by optical rotation and -SH group increases. Similar studies with ferritin (Henney and Stanworth, 1966) showed an essentially similar picture, except that in this case much RF-like antibody was elicited, and complexes made with this larger antigen molecule reacted over a larger range of AgAb ratios. (Here also an additional precipitation line appeared in the anti-cpx-ferritin diffusion pattern, which could possibly be due to antibodies against distortions on the antigen. ) Since, however, even antibody-BSA complexes are reactive with RF sera, one may assume that changes in the Fc piece normally occur coincidentally with changes in the Fab piece, though the former may not always elicit specific antibodies. IX. Molecular Location of Revealed Determinants
From the foregoing experiments it is clear that the distortion of the IgG molecule, either by physical means or as a result of its reaction with antigen, can lead to the appearance of immunogenic determinants, as postulated by Najjar. Different situations arise, however, depending upon whether these determinants are in the Fc or the Fab part of the molecule. Broadly we may say that the antibodies we have described as RF-like are directed against Fc determinants, whereas those described as anticomplex are against Fab determinants. If this general interpretation is correct, it suggests that the determinants on complexes are not created by the process of distortion of the chains, but merely revealed. If, as suggested by electron microscopy (Feinstein and Rowe, 1965), the two halves of the IgG molecule, each containing one antibody-active site, may be forced apart on reacting with antigen, it would not be surprising that areas on the H ( 7 ) chain, normally concealed within the molecule, should be exposed. That these should be potentially immunogenic implies that the normal self-tolerance to autogenous IgG extends to the whole molecule but not to its fragments. X. “Anticlone” Antibodies
The last type of anti-antibody is of a kind quite different from those which we have discussed hitherto, and its lucid description is a matter of some difficulty, owing to the complexity of the system, though in essence it is simple enough. Some preliminary discussion of the use of the word “clone” is, therefore, justified. It is now a commonplace that each myeloma protein, of man or of mouse, possesses an individual specificity which distinguishes it from all other myeloma proteins, and from the “normal” Ig of the same animal
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(Kunkel, 1965). This specificity is quite independent of the specificity that determines light or heavy chain class, but resides in some other part of the H chain. These proteins are often referred to as “monoclonal”-an assumption based, perhaps, upon Burnet’s original postulate of a marked tendency to somatic mutation of Ig-producing cells, and hence on the possibility that any IgG molecule in the serum may be different in detail from all other IgG molecules. Burnet postulated that such differences arise by somatic mutation. The very word, therefore, implies a quite complicated and still controversial theory of Ig biosynthesis, which was devised by Burnet primarily to explain heterogeneity at the antibodycombining site, though without excluding it at other points on the molecule. Nevertheless the concept of a “clone” of cells is a helpful one to indicate the exceptionally limited specificity of certain determinants both on myeloma cells and on the types of antibody pinpointed by the anti-antibodies which we shall describe. As long as one is aware of the danger of allowing a word to direct the track of one’s thinking, and continuously alert to prevent its doing so, it is perhaps safe to use it. By an anticlone antibody w7e mean, therefore, an anti-antibody that will react with another antibody of defined specificity only, and not with normal IgG or other antibodies or complexes from that animal. There has been a gradation both in specificity and in geographical site of action in the types of antibody we have been describing, from the Fc end of the molecule to the Fab end in the region of the combining site- “anticlone” antibodies appear to be directed either to the combining site itself or to a part of the molecule specifically codetermined with it, though not necessarily determined by it. XI. Anticlone Antibodies by Heteroirnmunization
One would expect that immunization of one species with the antigenantibody complexes of another would produce antispecies IgG antibodies, perhaps some RF-like and anti-cpx antibodies, and possibly some antibodies to a limited class of heavy-chain IgG, and stop at that. The situation following the injection of human Ab-Ag complexes into rabbits was analyzed by McDuffie et al. (1958) and they showed that some antibody was only removable by the specific immunogen. Subsequently Kunkel et al. (1963) described rabbit antisera against isolated human antibodies which after full absorption with normal IgG reacted specifically with the immunogen, i.e., the human antibodies had individual specificity. The Fab piece of the molecule contained the reacting site.
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Under the conditions of the experiment it was not possible to demonstrate the absence of the reactant from preimmunization serum. XII. Isoimrnune Anticlone Antibodies (Idiotypes)
An anticlone antibody arising from isoimmunization was described independently by Oudin and Michel (1963) and Gell and Kelus ( 1964a), which arose out of routine attempts to identify new allotypes in rabbits. This kind of antibody has been called “idiotypic” by Oudin (1966). The normal way to raise antiallotypic sera is to take a donor animal of say As1/4 (genotype l,la/4,4b), and immunize it with a bacterial suspension (e.g., Proteus vulgaris). The antibody so raised is coated on a suspension of the bacterium, the coated suspension washed, injected into the recipient animal of allotype say AslJ5 (or any animal having As1 but not As4). Antiallotypic antibodies are then elicited in the recipient specsc for As4. The process of coating onto a bacterium both gives a painless way of separating the immunoglobulin of the donor, and adds (when Profeus vulgaris is used) a marked adjuvant effect presumably due to the endotoxin of Proteus vulgaris intimately associated with the antigen. Much the same effect can be achieved by making equivalence precipitates with antibody from a donor animal with any antigen, say BSA, and injecting the precipitate in Freund’s complete adjuvant (FCA ) (Oudin, 1956). The results to be discussed arose from using this process of immunization between animals of identical allotypes. We shall use for descriptive purposes our own notation and experiments, though the general methods and conclusions are similar to those of Oudin and Michel. Should there be undetected differences between say As4 determinants in various strains of rabbits, or “new” allotypes on other Ig classes, such methods should demonstrate them-indeed, in one such experiment a new allotype on macroglobulin (Msl) was identified (Kelus and Gell, 1965). But a large proportion of the immunized animals produced antibodies (R/a) which would react with the donor antiserum (D/a) only, and not at all with (1) preimmunization donor serum (D/o), ( 2 ) normal serum from progenitors or progeny of the donor, ( 3 ) antibody subsequently or simultaneously raised in the donor to other antigens, and ( 4 ) sera from over fifty other rabbits immunized against the same antigen as that used to immunize the donor (see Fig. 2). On the other hand, anti-antibodies of this type were elicited against the antibody of one donor (D/a) in several other animals; these all gave a reaction of identity on gel diffusion against D/a indicating that a single substance was being identified. Evidence was presented that this
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substance was reactive as antibody against an antigen of Proteus vulgaris and that all this antibody and only this antibody was the “antigen”
reacting with R/a. Its electrophoretic mobility was shown to be restricted in the slow region of the IgG range. In a subsequent communication, serum from another donor was analyzed (Gell and Kelus, 196413) which contained a number of antibodies to various constituents of Proteus vulgaris-independent anti-antibodies corresponding to two and possibly three of these multiple components were seen. In the work in progress we have ourselves raised twelve D/a-R/a systems in pairs of homo-
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FIG. 2. Diagram to illustrate the reactions of anticlone antibodies. Pr-Proteus extract; RJa-antiserum of recipient 1 immunized against Dda; &/a-antiserum of recipient 2 immunized against Dda; Dda-donor 1 : anti-Proteus uulgoris antibody; D d o d o n o r 1: preimmunization serum; Ddb-donor 1: long postimmunization 2: anti-Proteus serum (after immunization with another antigen); Dda-donor antibody. (Note: In this diagram R,/a is shown as absorbed with Proteus bacilli so that no reaction occurs against Proteus extract.
allotypic rabbits: none of these D/a determinants were cross-reacting (Kelus and Gell, 1966). If the interpretation of these experiments is correct it would appear that rabbits can recognize individual determinants correlated with and wholly specific to particular antibodies in individual antisera. These determinants are not characteristic of antibodies from different rabbits against the same immunogen, as one might expect if the combining site to, say, a particular Proterls uulgaris component had just one mandatory amino acid composition and arrangement. On this ground we were hesitant in regarding the anti-antibody as being directed against the combining site itself, since one would suppose that all structures, defined
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by a pattern ot atomic radii and potential H bonds, which when functioning as antibody could be complementary to the combining site of a Proteus antigen, would when functioning as antigens provoke the same or at least a cross-reacting antibody. The simplest explanation would seem to be that all the cells producing the D/a antibody are derived from a single progenitor cell which has undergone at least two somatic mutations, one, on the Burnet hypothesis, to shape the antibody-combining site, another, quite independent one to shape the determinant recognized by the anti-antibody. The antibodycombining site may still be nonimmunogenic either from its position on the molecule or because it is entirely dependent on a labile tertiary structure of the Fab piece, which is not preserved in a macrophageprocessed fragment. The correlation of the immunogenic D/a determinants with a particular antibody specificity would then be a purely coincidental effect of random mutations in a single progenitor cell. If this is the case a particular type of D/a determinant will not be under genetic control. Clearly the decision as to whether the determinant on D/a recognized by this kind of anti-antibody is determined by or merely randomly correlated with the combining site will depend upon further chemical analysis. In the noninbred rabbits which we have used, there does not appear to be any simple genetic law controlling the production of D/a determinant in antibody: since among thirty progeny of 2 bucks producing D/a determinants (recognizable by R/a anti-antibodies), no reacting D/a determinants were demonstrable in the progeny sera before or after successful immunization with Proteus vulgaris ( Kelus and Gell, 1966). Nevertheless, the interpretation in terms of random somatic mutation and cloning may be oversimplified or wrong. An alternative hypothesis, in terms of multiple genes and their alleles capable of contributing elements in the structure of Ig (in particular of the Fd and possibly light chains ), among which antigen would “select” those put together in just the right way, is possible and easier to reconcile with the clear demonstration of genetic control of the capacity to respond to certain synthetic antigens ( Levine and Benacerraf, 1964; McDevitt and Sela, 1965). XIII. Biological Significance of Anti-antibodies
Autoantibodies to changes in the reacted antibody molecule, such as those of Abruzzo and Christian, may well play a part in potentiating immune elimination of antigen-especially when the antigen is weakly immunogenic and antibody production against it is poor, so that small
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antigen-excess complexes occur and are cleared rather inefficiently. A similar role has been postulated for immunoconglutinin (Coombs et uZ. 1961) . Whether autoanticlone antibodies can be formed is a much more complex problem. In our own recent work two animals were injected with their own antibody under conditions regularly effective in raising antiantibodies by isoimmunization, without any autoanti-antibodies being produced; but much more experimentation would be necessary to prove that this would never occur. Much speculation has been devoted to the question as to whether and how the body protects itself against (somatic) mutant cells in order to preserve its identity. It is hard to believe that it can regularly react to “private” determinants on its own antibodies, not so much because the process would be self-destructive as that it would lead to an infinite regress of anti-antibody production. If self-tolerance is to be invoked to explain this, then it must exist or be induced specifically to the possible private determinants on any single one of its possible antibodies-sinee the determinants are potentially immunogenic as shown by isoimmunization. Burnet’s clonal elimination theory could give a mechanism for this, but it would entail the elimination of a number of clones equal to the number of possible antibodies. If an antibody site is uniquely associated with a recognizable mutant sequence, then this number would be equal to the number of possible mutations associated with and determining the antibody character of the IgG molecule. Since all possible mutant sequences in this part of the molecule might crop up as antibodies they would all have to be rendered nonimmunogenic. UnIess the amino acid composition at or near this Combining site is very similar in all possible antibodies-which would contradict the demand for a wide range of heterogeneity there-one would expect this wholesale elimination of clones to eliminate response to a wide range of potential antigens. Clonal elimination, that is to say, would have to cope not merely with “autologous I g G but with all possible somatic mutants of IgG associated with the combining site. Alternatively one may admit that antibodies are potentially immunogenic in the host, but that their immunogenicity is very low. Hence thc infinite regress is cut short at an early stage-except, perhaps, in systemic lupus erythematosus, in which such high levels of IgG occur-and one is approaching a position very close to that taken by Najjar, as discussed above. In general it may be said that work on anticlone antibodies, with their uniquely limited specificity, could throw light on many unsolved problems in the analysis of the genesis and mechanism of the immune response.
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REFERENCES Abruzzo, J. L., and Christian, C. L. (1961). J. Ex&. Med. 114, 791. Aho, K., and Wager, 0. (1961). Ann. Med. Exptl. Fenniae (Helsinki) 39, 79. Ando, K., Takeda, S., and Ilamano, M. (1938). J. Immunol. 34, 303. Boyns, A. R. (1966). Pb.D. Thesis, Birmingham University. Campbell, D. H. (1957). Blood 12, 589. Cohen, C., and Tissot, R. G. (1965). J. Immunol. 95, 273. Coombs, A. M., and Coombs, R. R. A. (1953). J. H y g . 51, 509. Coomhs, R. R. A., Coombs, A. M., and Ingram, D. G. (1961). “The Serdogy of Conglutination and its Relation to Disease.” Blackwell, Oxford. Dray, S., Dubiski, S., Kelus, A., Lennox, E. S., and Oudin, J. (1962). Nature 195, 785. Dubiski, S. (1958). Folia Biol. (Cracow) 6, 47. Dubiski, S., Dudziak, Z., and Skalba, D. ( 1958). Proc. 7th Intern. Congr. Microbial., 1958, Almquist & Wiksell, Uppsala, p. 201. Eagle, H. (1930). J. Immunol. 18, 393. Eagle, H. (1936). J . Immunol. 30, 339. Feinstein, A., and Rowe, A. J. (1965). Nature 205, 147. Feinstein, A., Gell, P. G. H., and Kelus, A. S. (1963). Nature 200, 853. Fleischman, J. B., Pain, R. H., and Porter, R. R. (1962). Arch. Biochem. Biophys. Suppl. 1, 174. Fudenberg, H. H., Goodman, J., and Milgrom, F. (1963). Transfusion 3, 422. Gell, P. G. H., and Kdus, A. S. (1964a). Nature 201, 687. Gell, P. G. H., and Kelus, A. S. (1964b). “Molecular and Cellular Basis of Antibody Formation” (J. Sterzl et aZ. eds.), p. 201. Publishing House of Czech. Acad. Sci., Prague (distr., Academic Press, New York). Glynn, L. E. (1963). In “Clinical Aspects of Immunology” (P. G . H. Cell and R. R. A. Coombs, eds.), p. 593. Blackwdl, Oxford. Harshman, S., Robinson, J. P., and Najjar, V. A. (1963). Ann. N.Y. Acad. Sci. 103, 688. Henney, C. S., and Stanworth, D. R. (1965a). In “Protides of Biological Fluids,” (H. Peeters, ed.), p. 155. Elsevier, Amsterdam. Henney, C. S., and Stanworth, D. R. (1965b). Immunology 9, 139. Henney, C. S., and Stanworth, D. R. (1966). Nature 210, 1071. Henney, C. S., Stanworth, D. R., and Gell, P. G. H. (1965). Nature 205, 1079. Ishizaka, K., and Campbell, D. H. (1959). J . Immunol. 83, 116. Ishizaka, K., and Ishizaka, T. (1964). J. Immunol. 93, 59. Kelus, A. S., and Gell, P. G. H. (1965). Nature 206, 313. Kelus, A. S., and Gell, P. G. H. (1966). In preparation. Kelus, A. S., and Gell, P. G. H. (1967). Progr. Allergy 11 (in press). Kunkel, H. G., (1965). Harvey Lect. Ser. 59, 219. Kunkel, H. G., Mannik, M., and Williams, R. C. (1963). Science 140, 1218. Leskowitz, S. (1960). J. Immunol. 85, 56. Levine, B. B., and Benacerraf, B. (1964). J . Erptl. Aled. 120, 955. i’vIcCluskey, R. T., Miller, F., and Benacerraf, B. (1962). J. Erptl. Med. 115, 253. McDevitt, H. 0. and Seln, W., (1965). J. Exptl. Med. 122, 517. McDuffie, F. C., Kabat, E. A., Allen, P. Z., and Williams, C. A., Jr. (1958). J. Immunol. 81, 48.
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hlilgrom, F. ( 1962). Vox Sanguinis 7, 545. Milgrom, F., and Dubiski, S. (1957). Nuture 179, 1351. Milgrom, F., and Witebsky, E. (1960). J. Am. Med. Assuc. 174, 56. Najjar, V. A. (1963). Physiol. Rev. 43, 243. Najjar, V. A., and Fisher, J. (1955). Science 122, 1272. Najjar, V, A,, and Robinson, J. P. (1959). in “Immunity and Virus Infection” (V. A. Najjar, ed.), p. 71. Wiley, New York. Nomenclature for Human Immunoglobulins. ( 1964). Bull. Wmld Health Organ. 30, 447. Northrop, J. H. (1942). J . Gen. Physiol. 25, 465. Oudin, J. (19513). Compt. Rend. Acud. Sci. 242, 2489, 2606. Oudin, J. (1966). Proc. Roy. SOC. B (in press). Oudin, J., and hfichel, M. (1963). Compt. Rend. Acad. Sci. 257, 805. Pollock, 51. R. (1964). Immunology 7, 707. Robert, B., and Grabar, P. (1957). Ann. Inst. Pasteur 92, 56. Sevag, hl. C. ( 1945). “Immuno-Catalysis.” Thomas, Springfield, Illinois. Smith, F. C., and blarrack, J. R. (1930). Brit. J . Exptl. Puthol. 11, 494. Svec, K. H., and Dingle, J. H. (1965). Arthritis Rheumut. 8, 524. Treffers, H. P., and Heidelberger, M. (1941). J. Erptl. Med. 73, 125, 293. Williams, R. C., Jr., and Kunkel, H. G. (1963). Proc. Soc. Exptl. Biol. 112, 554. Williams, R C., Jr., and Kunkel, H. G. (1965). Ann. N.Y. Acad. Sci. 124, 860.
H. GEL1 AND ANDREW S. KELUS
Department of Experimental Pathology, University o f Birmingham, Birmingham, England
I. Introduction . . . . . . . . . . . . . 11. Anti-antibodies in the Immunization Process and “Subcomplementarity” 111. Immunogenicity of “Altered” IgG. . . . . . . . . IV. Rheumatoid Factor and Rheumatoid Factor-like Antibodies . . V. Changes in the IgG Molecule Leading to the Production of Rheumatoid Factor-like Antibodies: the Fc Piece . . . . . . . VI. Distortion in the Fab Piece of IgG: Anticomplex Antibodies . . VII. “Natural” Anti-antibodies: Agglutinators . . . . . . VIII. Experimental Anticomplex Antibodies . . . . . . . IX. Molecular Location of Revealed Determinants . . . . . X. “Anticlone” Antibodies . . . . . . . . . . XI. Anticlone Antibodies by Heteroimrnunization . . . . . XII. Isoimmune Anticlone Antibodies (Idiotypes) . . . . . . XIII. Biological Significance of Anti-antibodies . . . . . . References . . . . . . . . . . . . .
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1. Introduction
It is remarkable how comparatively recently the proteins of the plasma have been differentiated as physicochemical entities, and antibody activity has been shown to be the property of well-defined proteins rather than of the “serum” as a whole. Only for the last decade have the various classes of immunoglobulins and the possibility of interspecific differences as well as intraspecific differences between immunoglobulins of a single class been recognized. At the same time the concept that an animal may react immunologically against its own autogenous antigens has been generally accepted. An anti-antibody in the sense used here means an antibody which will react as such with an Ig molecule1 because that molecule is an antibody, not just because that molecule is a y-globulin. This does not imply that the antibody-combining site of that molecule is necessarily the locus of interaction, which has never been definitively shown. The word ‘We employ the terminology recently proposed (Nomenclature for Human Immunoglobulins, 1964 ) of immunoglobulins or their components, using the blanket symbol Ig when no particular class of immunoglobulin is referred to; though most of the work discussed in detail refers to “ordinary 7 S y-globulin,” IgG. 461
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“becausc,”in fact, covers three quite diffcrent situiiti~l~s. The first is
when anti-antibodies are elicitable because the antibody has reacted with antigen beforehand (in a way characteristic, as far as one can judge, only of antibodies) so as to expose, or to form de nouo, structures which, though potentially present in any Ig molecule. are not immunogenic in the intact molecule. The second is when anti-antibodies are elicitable because the antibody-combining site itself, in the intact molecule, is immunogenic and is involved in the interaction. The third is when there is an absolute correlation between the presence of a particular specific cornbining site and an immunogenic site elsewhere on the molecule. A fourth situation, strictly excluded by this definition, is when the antibodies all come from a highly restricted subclass of, say, IgG, but where there are molecules in this subclass capable of reacting with the presumed antiantibody yet which do not appear to possess antibody activity themselves. These may, of course, be antibodies of low or negligible affinity for the antigen but which are, in fact, present because of and produced by the original induction stimulus; or they may be quite unrelated to it. The earlier speculations and experiments on anti-antibodies were along the lines (1) whether heterologous antisera could be raised against antibodies as such (as opposed to the “normal” proteins of the serum) and ( 2 ) whether the change in immunoglobulin thought to be “induced” by antigen might itself be recognized as foreign by the animal in which it occurred and whether this might play a part in the immunization process. Pioneer work along these lines is discussed in that off-beat but stimulating book “Immuno-catalysis” by Sevag ( 1945). The earliest studies by Ehrlich, Bordet, Moreschi, and others were influenced by the idea that antibody was a substance arising de novo, unrelated in any way to the pre-existing proteins of the serum. Thus antisera that neutralized thc activities of antibodies ( Iysins, agglutinins) were termed “anti-antibodies.” By the time of the paper by Eagle (1930), antibodies were recognized as members of a class of serum protein. Smith and Marrack (1930) were the first to state clearly that, since antitoxin, when precipitated by a precipitin, still combines with toxin, different groups on the antitoxin molecules must be involved in the two reactions-a point confirmed by the studies of Eagle (1936). Treffers and Heidelberger (1941) in a study of horse antisera concluded that “the groupings responsible for the antibody function constitute either a small part of the total protein molecule or else are non-antigenic.” Other work on these lines (e.g., Ando et al., 1938; Northrop, 1942) is difficult to interpret, presumably owing to lack of knowledge at the time of the macroglobulin
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(IgM) nature of some horse antibodies and of their possible nonreactivity or cross-reactivity with antisera to IgG. A simple-minded attempt to demonstrate anti-antibodies is, therefore, likely to encounter a number of difficulties. If heteroimmunization is used, ( 1 ) the bulk of the antibody will be species-specific and ( 2 ) antibodies specific to a class or even a subclass of Ig molecules may be confused with specific anti-antibodies. If isoimmunization is used, antibodies may be produced specific for genetic differences on IgG (or other proteins) not common to donor and recipient, i.e., to allotypic determinants. Since the bulk of our discussion here will be concerned with rabbit antibodies, and since the genetic situation with rabbit IgG allotypes (Dray et al., 1962) is fairly simple and well worked out, it is advantageous to outline at this stage the relevant data on this-not because antiallotypic antibodies are anti-antibodies in any sense used here, but because it is necessary to be aware of the allotypic situation whenever intraspecies cross-immunizations are used ( see review, Kelus and Gell, 1967). At present there are two well-defined loci, a and b, giving rise to allotypic determinants. The a-locus determinants are located on the heavy chain (Asl, 2, 3); the b-locus determinants (As4, 5, 6 ), on the light chain, and possibly also in the heavy chain (Feinstein et al., 1963). There is evidence for at least one other locus for IgG, and one for the heavy chain of IgM (Kelus and Gell, 1965). Quite extensive breeding data from both Europe and America for laboratory rabbits have not given any indication of further alleles at either the a or b locus. The blocus determinants are quite strongly immunogenic, although the use of some kind of adjuvant is usually needed to raise strongly precipitating antisera; the a-locus determinants are much less so, and adjuvants are always needed. A proportion of IgG molecules (10-20%) seems to lack determinants from one or the other locus. II. Anti-antibodies in the Immunization Process and “Subcomplementarity”
Before we consider more recent experiments on the direct elicitation of anti-antibodies, the work of Najjar (1963) and his colleagues may be considered. To this group must go the credit of having persistently drawn attention to the possible role of molecular distortions in the immune process, although the rather elaborate theoretical structure which they have erected upon their experiments is not acceptable to many workers today. Najjar considers that “when the first antibody” [to whose process of formation we shall return in a moment] “reacts in the antigenic site, both antibody and antigen are shown to be altered simul-
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taneousl!,. Antibodies are then formed against these sites of altered configuration. This process continues as further sites appear on the antigen and reacting antibody” (Najjar and Robinson, 1959). Thus the antibodies formed in the later stages of immunization would be to novel determinants not present in the “native” antigen, a proportion of them, indeed, to determinants not on antigen but on antibody. “After the third injection [of a native protein antigen] over 80% of antibody protein reacted with [an antigen-antibody complex made at equivalence] . . . leaving behind the remainder which reacted with antigen only” (Najjar and Fisher, 1955). The authors go on to advance as supporting evidence the work discussed below of Milgrom and Dubiski and others on rheumatoid factor-like anti-antibodies. Although it is difficult to accept either the authors’ experiments, or their supporting evidence, as being only, or even most readily, explainable in terms of their theory, this does not imply that the theory, first put forward at a time when autoantibodies were still hardly respectable, does not contain much truth. It is not quite clear whether the distortion of the antibody is thought of as being antigen-specific, which is hard to envisage in physicochemical terms, or a nonspecific result of antigen-antibody interaction, in which case the later stages of immunization should lack all specificity, since antibodies developed against one complex would react with all other complexes. Such antibodies are, indeed, sometimes demonstrable ( see below) but are produced only in special circumstances and not apparently as a general rule. Failure to eliminate allotypic differences between rabbits may make some experiments hard to interpret (Najjar and Fisher, 1955), though not those in which autostimulation is thought to have occurred (Harshman et aZ., 1963); however, application of newer knowledge of Ig structure and the use of the digerential power of gel-diffusion techniques to demonstrate antibodies of different specificities can bring a greater precision to such experiments. Najjar and co-workers go on to postulate that precipitation, in fact, occurs only when the “fit” of antigen and antibody is not exact (“subcomplementary”), so leading to some distortion. “Qualitatively there could then be three types of immune globulin synthesized in response to an antigen stimulus. The first type would seem to be those that are sufEciently sub-complementary to the antigen site to undergo configurational alterations, i.e. antibodies detectable by the usual immunochemical techniques.” [These are considered to arise because antibody is synthesized in relation to antigenic fragments, as discussed by Campbell (1957) and others, as well as to native antigen with its normal tertiary structure.] “To the second type belong the globulins that are comple-
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mentary to the antigen site and, therefore, not detectable under ordinary conditions. The third type falls between these two and includes a spectrum of molecules with varying degrees of sub-complementarity.” Though this theory can be used to explain certain types of cross-reactivity, the basic supposition that immunological precipitation is due, not to “complementary” antigen-antibody interaction leading to lattice formation, but solely to the uncovering of hydrophobic groups as a result of subcomplementary molecular distortion, would seem to ignore the whole history of immunochemistry from Landsteiner and Marrack onward. More recent studies of what conditions do, in fact, lead to molecular distortion, and in what parts of the molecule, have made the situation much clearer. Ill. lmmunogenicity of “Altered” IgG
This work entails five different kinds of experiments: ( a ) experiments with red cells or bacteria coated with antibody and injected into other animals of the same species (isoimmunization) or of a different species (heteroimmunization ) ; ( b) experiments in which antigen-antibody complexes are made in various ratios and used for immunizing other animals of the same species; ( c ) experiments in which IgG is altered in uitro and reinjected into the donor animal; ( d ) the demonstration in normal rabbit sera of IgM antibodies against rabbit IgG antibody which has reacted with antigen; and ( e ) strong immunization with any antigen may be found to give rise to antibodies reacting with Ig of one sort or another. By such experiments, four evidently different kinds of antibodies have been demonstrated, singly or in combination. 1. Antiallotypic antibodies, when isoimmunization by methods ( a ) or ( b ) has been used-such methods led, in fact, to the original demonstrations of IgG allotypes by Oudin (1956). Even where the known allotypes are identical in donor and recipient, a new allotype may be uncovered, the characteristic of this situation being that the preimmunization serum of the donor and of certain other rabbits will react with the antibody elicited. These antiallotypic antibodies need not be considered further here. 2. Rheumatoid factor-like antibodies [methods ( a ) , ( b ) , ( c ) , and ( e ) ]-these characteristically cross-react with Ig of species other than the donor. 3. Anticomplex antibodies [methods ( a ) and ( b ) ; allotypic differences being eliminated]- these can be shown to react with any antigenantibody complexes made with the Ig of the same species or with antibody-coated cells, but not with unreacted antibody. It is an open question
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whether “antigen-specific” anticomplex antibodies also occur. The ‘hatural” antibodies ( d ) fall also into this group. 4. “Anticlone” antibody [methods ( a ) and (b)]-these will react only with the actual antibody used as immunogen, not with preimmunization serum from the same animal-the antigen need not be in the form of a complex. Immunoconglutinin ( Coombs and Coombs, 1953)-although not an anti-antibody under our definition is an additional type of antibody, which is likely to be detectable if looked for with an appropriate test system and which, if unsuspected, may lead to misleading conclusions. However, it has not been recorded as precipitating antibody. It is evidently directed against activated autogenous complement, and occurs regularly as an accompaniment of any fairly intense immunization. Its properties have recently been fully reviewed (Coombs et al., 1961). IV. Rheumatoid Factor and Rheumatoid Factor-like Antibodies
Whether one considers rheumatoid factor ( RF) , as arising in rheumatoid arthritis and some related diseases in man, to be relevant to this discussion depends upon one’s opinion as to its origin. There seems little doubt that it is an IgM antibody induced by “damaged IgG, and it is possible, but not proved, that this damage arises because the IgG has reacted, as antibody, with some extrinsic, e.g., bacterial, antigen, and therefore the RF is brought within our definition of an anti-antibody. This hypothesis was put forward by Dubiski (1958). The extensive chemical and experimental work on human RF has been reviewed elsewhere (e.g., Glynn, 1963). Williams and Kunkel (1965) in a discussion of antiantibodies refer to a number of antibodies of this nature occurring in rheumatoid arthritis sera. Similarly the identification of the Gm and InV allotypes of man by means of normal ( SNagg) sera and rheumatoid (RF, Ragg) sera, is outside the compass of this review. We shall, therefore, not consider further the spectrum of antibodies in human RF sera except when showing analogies with those experimentally produced. Milgrom and Witebsky ( 19sO) showed that autoimmunization of rabbits with their own IgG precipitated with ammonium sulfate or alum (and presumably mildly denatured), in Freunds complete adjuvant, gave rise to precipitating and complement-fixing antibodies which reacted very much better with human than with rabbit IgG. McCluskey et al. (1%2), in a controlled study of various methods of denaturation, recorded the appearance of antibodies of this type. These results can be taken as possible models for the production of anti-antibody.
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The rheumatoid factor-like substance (RFLS) of Abruzzo and Christian (1961), arising from long immunization of rabbits with formalin-killed bacteria, showed somewhat different properties. This was a macroglobulin which reacted with immune complexes, made both with human and rabbit antisera, and with cells coated with heat-aggregated rabbit and human IgG. Cross-absorbtion showed that the rabbit IgG was the homologous, and human IgG the cross-reacting antigen; in these properties it shows an analogy with human RF. Aho and Wager (1961) and Williams and Kunkel (1963) described similar antibodies in rabbits both of 19 and 7s type, after immunization with various protein antigens. In man, RF-like antibodies may be demonstrable in many chronic diseases (as quoted by Abruzzo and Christian, 1961) and even transiently after prophylactic immunization with tetanus or diphtheria toxoid ( Svec and Dingle, 1965). V. Changes in the IgG Molecule leading to the Production of Rheumatoid Factor-like Antibodies: the Fc Piece
There is evidence that the precipitation of RF with heated IgG is the result of changes in the Fc piece of the latter (Henney and Stanworth, 1965b), involving S-S bond rupture and some aggregation. The Fc part of the molecule is also involved in complement fixation and in the irritant properties of antigen-antibody complexes ( Ishizaka and Ishizaka, 1964 ) . The RF-like antibodies induced by Milgrom and Witebsky (1960) and McCluskey et al. (1962) were evidently produced in response to Fc piece changes. This sort of change evidently gives rise to a determinant which is both autoimmunogenic and widely cross-reacting-as with other types of cross-reacting system, heated IgG from some species, e.g., rabbit, reacts very much better with human R F than does that from others, e.g., horse ( Henney and Stanworth, 1965a). Antigen-antibody combination may also give rise to similar Fc piece changes: it is as a result of some such process that the RF-like antibodies of Abruzzo and Christian are likely to have been provoked. Antigen-antibody complexes and reacted antibody on the surface of cells (as in the Rose-Waaler test) will for the same reason react with human RF. The changes induced by antigen-antibody union, however, are more complex than those induced by heat “denaturation.” (An unfortunate word to use in this connection, in that it often implies an “all-or-none” irreversible reaction, whereas the changes which we are considering are progressive, and, in the early stages at least, reversible.) In the first place, those changes occurring when antibody reacts with a soluble antigen are
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PHILIP G. H. GELL AND ASDREW S. KELUS
critically dependent upon the antigen (Ag)-antibody ( Ab) ratio of the complex formed, being maximal in moderate antigen excess, where the ratio is Ags Ab,. and probably minimal both at the gross antigen excess ratio ( Ag, Ab, Ag,,) and at equivalence. Reaction of antibody with a cell, e.g. ;in erythrocyte, will produce some distortional changes in antibody apparently independently of the ratio of the reactants, and even with soluble antigens there is some evidence that the larger the antigen the wider the range of ratios at which distortion occurs (Henney and Stanworth, 1966). The distortion of antibody on union at a critical ratio is reflected in increase in free -SH (Robert and Grabar, 1957) and in optical rotation (Ishizaka and Campbell, 1959). That the latter was due to changes in antibody rather than antigen was indicated by the fact that the increase in optical rotation was still demonstrable when an optically inactive antigen was used. Distortion of antigen does, however, sometimes occur, most elegantly shown in studies with enzyme-antienzyme systems, e.g., those of Pollock ( 1964) with antipenicillinases where clear-cut changes in substrate specificity and kinetics were demonstrable, as well as those investigated by Najjar (1963).
+
VI.
Distortion in the Fob Piece of IgG: Anticomplex Antibodies
Thus both physical treatment, and union with antigen, will produce alterations of the IgG (antibody) molecule in its Fc piece. In the latter situation there is evidence, however, that other and, perhaps, mere significant changes occur at the Fab end of the molecule. These changes are recognizable by altered immunogenicity, but antisera elicited by the altered molecules still react more strongly with homologous than with heterologous IgG. This is in contrast with the cross-reactivity of the Fc piece changes. The anti-antibodies so produced have the property of reacting with homologous antibody as it exists in a complex at antigen excess or attached to a cell, but not with heated IgG or with heterospecific IgG complexes. They are demonstrable, as discussed below, in a proportion of normal rabbit sera under special conditions, or as a result of experimental immunization with complexes formed in uitro. No unequivocal demonstration that this kind of anti-antibody arises as a result of true spontaneous autostimulation has apparently appeared, though the production of immunoconglutinin (Coombs and Coombs, 1953) provides an analogy. One might expect a course of repeated large injections of antigen, as in the experimental production of “serum sickness nephritis,” to provoke
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their appearance, but Boyns (1966) during experiments of this type has hitherto failed to find any antibodies of this nature. VII. “Natural” Anti-antibodies: Agglutinators
The “anti-antibodies” described by Milgrom and Dubiski ( 1957) were later identified by Dubiski et al. (1958) as probably antiallotypic antibodies. However, Milgrom ( 1962) described antibodies ( “agglutinators”) in a proportion of normal rabbits, which in a subsequent communication (Fudenberg et al., 1963) were found to be macroglobulins capable of reacting with antigen-antibody complexes made from antibody digested with papain and therefore lacking the Fc piece, as well as with rabbit red cells sensitized with rabbit isoagglutinin. Cohen and Tissot ( 1965) analyzing the latter situation, showed that only certain isoagglutinins could, on reacting with the red cells, reveal a site capable of combining with the agglutinator, and there was some evidence from family data that the “agglutinator trait” was under genetic control. Although it was not possible to decide whether the agglutinator resulted from specific stimulation or not, it would seem reasonable to refer to it as a “natural” antibody, by analogy with other “natural” isoagglutinins. VIII. Experimental Anticomplex Antibodies
Antisera were raised by isoimmunization against preformed soluble complexes made in vitro by Leskowitz (1960), but he concluded that the antibodies demonstrated were probably against allotypic determinants rather than against complexes as such. Henney et al. (1965) immunized rabbits with preformed bovine serum albumin ( BSA )-anti-B.S.A. complexes, the known allotypes of donor and recipient being identical, and analyzed the resulting antisera by gel diffusion. In this study no RF-like antibodies were produced, though antibodies were produced against the antigen contained in the complexes used as immunogen. There was no evidence of the production of antiallotypic or other antibodies reacting with the free antibody used to make the immunogenic complex, but true anticomplex (anti-cpx) antibodies could be demonstrated. By this we mean antibodies that will react on agar diffusion with complexes made between other rabbit antibodies and their respective antigens. The reaction of anti-cpx antisera with the immunizing complex ( BSA-anti-BSA) and with BSA alone was more complicated (see Fig. l ) , since the anti-cpx antiserum contained in addition much anti-BSA antibody-as one would expect when immunization was carried out with BSA-antibody complexes in antigen excess. Therefore, when the anti-cpx antiserum was diffused in agar against BSA,
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complexes were formed which then reacted in situ with the anti-cpx antibodies present in the same serum. However, the expected strong spur was formed when complexes made between another antigen and its antiserum were reacted in an adjacent cup. Further information was obtained as to the location in the IgG molecule of the new “determinant,” produced on complexing, in that, though no reaction was obtained between the anti-cpx serum and samples of fresh IgG nor with several samples of heated IgG capable of reacting with R F (via changes in the Fc piece), a definite reaction occurred
FIG.1. Diagram to illustrate the reactions of anticomplex antiserum. Note spur between homologous and heterologous systems; both anti-BSA and anticomplex antibodies pile up in this line. (BSA = bovine serum albumin.)
with the separated Fab pieces obtained by papain digestion and with a (heavy; 7 ) A-chain preparation made by the method of Fleischman et al. ( 1962). It was tentatively concluded that the determinants revealed by this study were in the Fab region of the heavy chain, contiguous to the antibody-combining site, revealed and made immunogenic as a result of the distortion produced by reaction with antigen. Two samples out of thirty of stored IgG, in which some aggregation had occurred, also showed the presence of this same determinant reacting with the anti-cpx serum. Thus though attempts to expose this Fab determinant experimentally by physical means were unsuccessful, one may suppose its appearance not to be absolutely confined to antigen-antibody reactions, but producihlc also 11y other forms of degradation, of unknown nature. A notable characteristic of the anti-cpx serum u7as that it reacted most strongly on gel diffusion with complexes in that ratio at which
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maximal distortion of the molecule occurred as judged by optical rotation and -SH group increases. Similar studies with ferritin (Henney and Stanworth, 1966) showed an essentially similar picture, except that in this case much RF-like antibody was elicited, and complexes made with this larger antigen molecule reacted over a larger range of AgAb ratios. (Here also an additional precipitation line appeared in the anti-cpx-ferritin diffusion pattern, which could possibly be due to antibodies against distortions on the antigen. ) Since, however, even antibody-BSA complexes are reactive with RF sera, one may assume that changes in the Fc piece normally occur coincidentally with changes in the Fab piece, though the former may not always elicit specific antibodies. IX. Molecular Location of Revealed Determinants
From the foregoing experiments it is clear that the distortion of the IgG molecule, either by physical means or as a result of its reaction with antigen, can lead to the appearance of immunogenic determinants, as postulated by Najjar. Different situations arise, however, depending upon whether these determinants are in the Fc or the Fab part of the molecule. Broadly we may say that the antibodies we have described as RF-like are directed against Fc determinants, whereas those described as anticomplex are against Fab determinants. If this general interpretation is correct, it suggests that the determinants on complexes are not created by the process of distortion of the chains, but merely revealed. If, as suggested by electron microscopy (Feinstein and Rowe, 1965), the two halves of the IgG molecule, each containing one antibody-active site, may be forced apart on reacting with antigen, it would not be surprising that areas on the H ( 7 ) chain, normally concealed within the molecule, should be exposed. That these should be potentially immunogenic implies that the normal self-tolerance to autogenous IgG extends to the whole molecule but not to its fragments. X. “Anticlone” Antibodies
The last type of anti-antibody is of a kind quite different from those which we have discussed hitherto, and its lucid description is a matter of some difficulty, owing to the complexity of the system, though in essence it is simple enough. Some preliminary discussion of the use of the word “clone” is, therefore, justified. It is now a commonplace that each myeloma protein, of man or of mouse, possesses an individual specificity which distinguishes it from all other myeloma proteins, and from the “normal” Ig of the same animal
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(Kunkel, 1965). This specificity is quite independent of the specificity that determines light or heavy chain class, but resides in some other part of the H chain. These proteins are often referred to as “monoclonal”-an assumption based, perhaps, upon Burnet’s original postulate of a marked tendency to somatic mutation of Ig-producing cells, and hence on the possibility that any IgG molecule in the serum may be different in detail from all other IgG molecules. Burnet postulated that such differences arise by somatic mutation. The very word, therefore, implies a quite complicated and still controversial theory of Ig biosynthesis, which was devised by Burnet primarily to explain heterogeneity at the antibodycombining site, though without excluding it at other points on the molecule. Nevertheless the concept of a “clone” of cells is a helpful one to indicate the exceptionally limited specificity of certain determinants both on myeloma cells and on the types of antibody pinpointed by the anti-antibodies which we shall describe. As long as one is aware of the danger of allowing a word to direct the track of one’s thinking, and continuously alert to prevent its doing so, it is perhaps safe to use it. By an anticlone antibody w7e mean, therefore, an anti-antibody that will react with another antibody of defined specificity only, and not with normal IgG or other antibodies or complexes from that animal. There has been a gradation both in specificity and in geographical site of action in the types of antibody we have been describing, from the Fc end of the molecule to the Fab end in the region of the combining site- “anticlone” antibodies appear to be directed either to the combining site itself or to a part of the molecule specifically codetermined with it, though not necessarily determined by it. XI. Anticlone Antibodies by Heteroirnmunization
One would expect that immunization of one species with the antigenantibody complexes of another would produce antispecies IgG antibodies, perhaps some RF-like and anti-cpx antibodies, and possibly some antibodies to a limited class of heavy-chain IgG, and stop at that. The situation following the injection of human Ab-Ag complexes into rabbits was analyzed by McDuffie et al. (1958) and they showed that some antibody was only removable by the specific immunogen. Subsequently Kunkel et al. (1963) described rabbit antisera against isolated human antibodies which after full absorption with normal IgG reacted specifically with the immunogen, i.e., the human antibodies had individual specificity. The Fab piece of the molecule contained the reacting site.
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Under the conditions of the experiment it was not possible to demonstrate the absence of the reactant from preimmunization serum. XII. Isoimrnune Anticlone Antibodies (Idiotypes)
An anticlone antibody arising from isoimmunization was described independently by Oudin and Michel (1963) and Gell and Kelus ( 1964a), which arose out of routine attempts to identify new allotypes in rabbits. This kind of antibody has been called “idiotypic” by Oudin (1966). The normal way to raise antiallotypic sera is to take a donor animal of say As1/4 (genotype l,la/4,4b), and immunize it with a bacterial suspension (e.g., Proteus vulgaris). The antibody so raised is coated on a suspension of the bacterium, the coated suspension washed, injected into the recipient animal of allotype say AslJ5 (or any animal having As1 but not As4). Antiallotypic antibodies are then elicited in the recipient specsc for As4. The process of coating onto a bacterium both gives a painless way of separating the immunoglobulin of the donor, and adds (when Profeus vulgaris is used) a marked adjuvant effect presumably due to the endotoxin of Proteus vulgaris intimately associated with the antigen. Much the same effect can be achieved by making equivalence precipitates with antibody from a donor animal with any antigen, say BSA, and injecting the precipitate in Freund’s complete adjuvant (FCA ) (Oudin, 1956). The results to be discussed arose from using this process of immunization between animals of identical allotypes. We shall use for descriptive purposes our own notation and experiments, though the general methods and conclusions are similar to those of Oudin and Michel. Should there be undetected differences between say As4 determinants in various strains of rabbits, or “new” allotypes on other Ig classes, such methods should demonstrate them-indeed, in one such experiment a new allotype on macroglobulin (Msl) was identified (Kelus and Gell, 1965). But a large proportion of the immunized animals produced antibodies (R/a) which would react with the donor antiserum (D/a) only, and not at all with (1) preimmunization donor serum (D/o), ( 2 ) normal serum from progenitors or progeny of the donor, ( 3 ) antibody subsequently or simultaneously raised in the donor to other antigens, and ( 4 ) sera from over fifty other rabbits immunized against the same antigen as that used to immunize the donor (see Fig. 2). On the other hand, anti-antibodies of this type were elicited against the antibody of one donor (D/a) in several other animals; these all gave a reaction of identity on gel diffusion against D/a indicating that a single substance was being identified. Evidence was presented that this
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substance was reactive as antibody against an antigen of Proteus vulgaris and that all this antibody and only this antibody was the “antigen”
reacting with R/a. Its electrophoretic mobility was shown to be restricted in the slow region of the IgG range. In a subsequent communication, serum from another donor was analyzed (Gell and Kelus, 196413) which contained a number of antibodies to various constituents of Proteus vulgaris-independent anti-antibodies corresponding to two and possibly three of these multiple components were seen. In the work in progress we have ourselves raised twelve D/a-R/a systems in pairs of homo-
-
FIG. 2. Diagram to illustrate the reactions of anticlone antibodies. Pr-Proteus extract; RJa-antiserum of recipient 1 immunized against Dda; &/a-antiserum of recipient 2 immunized against Dda; Dda-donor 1 : anti-Proteus uulgoris antibody; D d o d o n o r 1: preimmunization serum; Ddb-donor 1: long postimmunization 2: anti-Proteus serum (after immunization with another antigen); Dda-donor antibody. (Note: In this diagram R,/a is shown as absorbed with Proteus bacilli so that no reaction occurs against Proteus extract.
allotypic rabbits: none of these D/a determinants were cross-reacting (Kelus and Gell, 1966). If the interpretation of these experiments is correct it would appear that rabbits can recognize individual determinants correlated with and wholly specific to particular antibodies in individual antisera. These determinants are not characteristic of antibodies from different rabbits against the same immunogen, as one might expect if the combining site to, say, a particular Proterls uulgaris component had just one mandatory amino acid composition and arrangement. On this ground we were hesitant in regarding the anti-antibody as being directed against the combining site itself, since one would suppose that all structures, defined
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by a pattern ot atomic radii and potential H bonds, which when functioning as antibody could be complementary to the combining site of a Proteus antigen, would when functioning as antigens provoke the same or at least a cross-reacting antibody. The simplest explanation would seem to be that all the cells producing the D/a antibody are derived from a single progenitor cell which has undergone at least two somatic mutations, one, on the Burnet hypothesis, to shape the antibody-combining site, another, quite independent one to shape the determinant recognized by the anti-antibody. The antibodycombining site may still be nonimmunogenic either from its position on the molecule or because it is entirely dependent on a labile tertiary structure of the Fab piece, which is not preserved in a macrophageprocessed fragment. The correlation of the immunogenic D/a determinants with a particular antibody specificity would then be a purely coincidental effect of random mutations in a single progenitor cell. If this is the case a particular type of D/a determinant will not be under genetic control. Clearly the decision as to whether the determinant on D/a recognized by this kind of anti-antibody is determined by or merely randomly correlated with the combining site will depend upon further chemical analysis. In the noninbred rabbits which we have used, there does not appear to be any simple genetic law controlling the production of D/a determinant in antibody: since among thirty progeny of 2 bucks producing D/a determinants (recognizable by R/a anti-antibodies), no reacting D/a determinants were demonstrable in the progeny sera before or after successful immunization with Proteus vulgaris ( Kelus and Gell, 1966). Nevertheless, the interpretation in terms of random somatic mutation and cloning may be oversimplified or wrong. An alternative hypothesis, in terms of multiple genes and their alleles capable of contributing elements in the structure of Ig (in particular of the Fd and possibly light chains ), among which antigen would “select” those put together in just the right way, is possible and easier to reconcile with the clear demonstration of genetic control of the capacity to respond to certain synthetic antigens ( Levine and Benacerraf, 1964; McDevitt and Sela, 1965). XIII. Biological Significance of Anti-antibodies
Autoantibodies to changes in the reacted antibody molecule, such as those of Abruzzo and Christian, may well play a part in potentiating immune elimination of antigen-especially when the antigen is weakly immunogenic and antibody production against it is poor, so that small
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antigen-excess complexes occur and are cleared rather inefficiently. A similar role has been postulated for immunoconglutinin (Coombs et uZ. 1961) . Whether autoanticlone antibodies can be formed is a much more complex problem. In our own recent work two animals were injected with their own antibody under conditions regularly effective in raising antiantibodies by isoimmunization, without any autoanti-antibodies being produced; but much more experimentation would be necessary to prove that this would never occur. Much speculation has been devoted to the question as to whether and how the body protects itself against (somatic) mutant cells in order to preserve its identity. It is hard to believe that it can regularly react to “private” determinants on its own antibodies, not so much because the process would be self-destructive as that it would lead to an infinite regress of anti-antibody production. If self-tolerance is to be invoked to explain this, then it must exist or be induced specifically to the possible private determinants on any single one of its possible antibodies-sinee the determinants are potentially immunogenic as shown by isoimmunization. Burnet’s clonal elimination theory could give a mechanism for this, but it would entail the elimination of a number of clones equal to the number of possible antibodies. If an antibody site is uniquely associated with a recognizable mutant sequence, then this number would be equal to the number of possible mutations associated with and determining the antibody character of the IgG molecule. Since all possible mutant sequences in this part of the molecule might crop up as antibodies they would all have to be rendered nonimmunogenic. UnIess the amino acid composition at or near this Combining site is very similar in all possible antibodies-which would contradict the demand for a wide range of heterogeneity there-one would expect this wholesale elimination of clones to eliminate response to a wide range of potential antigens. Clonal elimination, that is to say, would have to cope not merely with “autologous I g G but with all possible somatic mutants of IgG associated with the combining site. Alternatively one may admit that antibodies are potentially immunogenic in the host, but that their immunogenicity is very low. Hence thc infinite regress is cut short at an early stage-except, perhaps, in systemic lupus erythematosus, in which such high levels of IgG occur-and one is approaching a position very close to that taken by Najjar, as discussed above. In general it may be said that work on anticlone antibodies, with their uniquely limited specificity, could throw light on many unsolved problems in the analysis of the genesis and mechanism of the immune response.
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