- Email: [email protected]
[10] Antiidiotypic antibodies
174
SERUMIMMUNOGLOBULINS
[10]
[10] A n t i i d i o t y p i c A n t i b o d i e s B y DAVID E. BRILES a n d JOHN F. KEARNEY
Introduction In the broad sense, idiotypy is the variation among the variable regions of different immunoglobulins.l Such differences can be detected by amino acid sequence analysis, peptide mapping, isoelectric focusing, differences in antibody specificity, or reactivity with antisera directed against different antibody variable region determinants. Although all of these procedures provide information about variations in variable region structure, the term idiotypy is generally used to refer to variable region characterizations carried out with antibodies to variable region determinants. Antigenic determinants associated with the variable regions of different antibodies are referred to as idiotypic determinants 2 and antibodies that recognize them are referred to as antiidiotypic antibodies. An idiotypic determinant detected by a single hybridoma antiidiotypic antibody (or one of the many antibodies in a heterologous antiidiotypic sera) is said to be an idiotope. 3 The constellation of idiotopes detected by the various antiidiotypic antibodies of a single idiotypic serum, by a group of different antiidiotypic sera, or by a group of hybridoma antiidiotypic antibodies is the idiotype of the antibody under study. This chapter is divided into three parts. The first describes the preparation of antiidiotypic sera. The second describes the immunoassays developed for using antiidiotypic sera to characterize the idiotypes of myeloma proteins, hybridoma antibodies, and antibodies in immune sera. The third section describes the methods that have been developed for use with hybridoma antiidiotypic antibodies.
P r e p a r a t i o n of A n t i i d i o t y p i c Sera
The early idiotypic studies of human and mouse myeloma antibodies were conducted using antiidiotypic sera raised in rabbits, goats, or guinea pigs. The immunization procedures used generally involved the injection
10. M/ikel/i and K. Karjalainen, Cold Spring Harbor Symp. Quant. Biol. 41, 735 (1976). z j. Oudin, Proc. R. Soc. London, Ser. B 166, 207 (1966b). 3 M. Reth, T. Imanishi-Kari, and K. Rajewsky, Eur. J. Immunol. 9, 1004 (1979).
METHODS IN ENZYMOLOGY, VOL. 116
Copyright © 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.
[10]
ANTI1DIOTYPIC ANTIBODIES
175
of partially purified antibodies in complete Freund's adjuvant (CFA). 3a In this chapter, we will refer to any immunoglobulin used to elicit an antiidiotypic antibody as a "proband" antibody. Appropriate immunization procedures are no different from those that would be used to prepare antibodies to any macromolecular protein antigen. 4,5 Although a number of different immunization procedures have been used to elicit antiidiotypic antibodies, we have found that a relatively simple method provides adequate results. Rabbits are immunized intraperitoneally with 100-1000 tzg of the proband antibody emulsified in 1 ml of C.FA. 4-6 The more highly purified the antibodies used for immunization are, the higher will be the percentage of the elicited antibodies that recognize idiotypic determinants on the proband molecule. In practice, however, complete purity is not required. In fact, when making antiidiotypic sera to myeloma proteins, purification by three sequential ammonium sulfate precipitations generally works quite well. For mouse antibodies, precipitation from a 47% saturated ammonium sulfate solution is usually satisfactory. Whenever the antigenic specificity of a proband antibody is known, it is desirable to purify it by affinity chromatography from an antigen column. 7-~° A typical preparation of adjuvant for the injection of 3 rabbits would require 1200/zg of purified antibody suspended in 1.6 ml of phosphatebuffered saline (PBS) (0.056 M Na2HPO4, 0.016 M KHzPO4, 0.077 M NaC1, pH 7.2). The PBS-antibody mixture is then added to a 15-ml round-bottom glass centrifuge tube containing 2.4 ml of commercially prepared (Difco) mycobacteria-oil suspension. If a stable emulsion is not obtained, it may be due to the effects of salts or proteins present in the antigen preparation. To obtain a stable emulsion if this occurs, the ratio of oil:aqueous phase can be varied slightly, usually by adding slightly more oil. 3a Abbreviations: ALL, acute lymphocytic leukemia; AP, alkaline phosphatase; BDB, bisdiazotized benzidine; BSA, bovine serum albumin; CFA, complete Freund's adjuvant; ELISA, enzyme-linked immunoadsorbant assay; IdI, individual idiotypic determinants; IdX, cross-reacting idiotypic determinant; PBS, phosphate-buffered saline; SPRIA, solid phase radioimmunoassay; VH, heavy chain variable region; VL, light chain variable region. 4 M. W. Chase, Methods lmmunol. Immunochem. 1, 209 (1967). 5 B. A. L. Hurn and S. M. Chantler, this series, Vol. 70, p. 104. 6 D. E. Briles and R. M. Krause, J. lmmunol. 109, 1311 (1972). 7 j. C. Jaton, D. C. Brandt, and P. Vassalli, in "Immunological Methods" (I. Lefkovits and B. Pernis, eds.), p. 43. Academic Press, New York, 1979. 8 j. S. Garvey, N. E. Cremer, and D. H. Sussdorf, in "Methods in Immunology," 3rd ed., p. 245. Benjamin, Reading, Massachusetts, 1977. 9 D. C. Parker, R. M. Krause, and L. Wofsy, Immunochemistry 10, 727 (1973). i0 B. Chesebro and H. Metzger, Biochemistry 11, 766 (1972).
176
SERUMIMMUNOGLOBULINS
[10]
A pilot bleeding should be made at one month after the primary injection. To determine if antibody has been produced, a small amount of antiserum is mixed with an equal amount of antigen solution (about 1 mg/ ml) in a capillary tube.l~ If no precipitate, or only a weak precipitate, forms (after several hours), the animal should be boosted with a second injection identical to the first. Rabbits are usually bled 7 to 10 days after the second injection. Animals that fail to show detectable responses after two injections are discarded. Animals showing only weak responses after the second injection are given a third injection about 3-4 weeks after the second. High titer antisera will give a positive precipitin reaction within 5-10 min of being mixed with the proband protein in a capillary tube. Since the titer following CFA immunizations generally stays high for several weeks, we generally pool several weekly bleedings from each rabbit before the animal is bled out. When antiidiotype antibodies are made in heterologous species, the recipient animal responds not only to the idiotypic determinants, but also to other determinants on the immunizing immunoglobulin molecule, as well as any contaminating molecules from the species that produced the immunizing immunoglobulin. Thus, the presence of a precipitin reaction between the antiidiotypic sera and the immunizing antigen preparation does not guarantee that the rabbit has begun to make antiidiotypic antibody. In practice, however, whenever a good precipitating serum is obtained, it is almost certain to contain a significant amount of antiidiotypic antibody. Antibodies to constant region determinants and those to contaminating serum molecules can be effectively removed by absorption with pooled normal serum from the species that provided the immunizing antigen. If the immunizing antibody was of a high enough purity so that no antibodies binding other serum components were elicited, then pooled 3'globulin would be the ideal absorbing material. If the proband antibody was of mouse origin, then pooled normal mouse serum from the same inbred strain that provided the proband antibody is used so that any antiimmunoglobulin allotype antibodies will be removed. Originally, antiidiotypic sera were absorbed by simply mixing the antiserum with an excess of normal serum or pooled immunoglobulin and then centrifuging out any precipitate that might occur. T2,~3This procedure proved adequate for studies using precipitation, passive hemagglutination, passive hemagglutination inhibition, direct binding radioimmunoas11 p. H. Maurer, Methods Immunol. Immunochem. 3, 36 (1971). i2 H. G. Kunkel, Fed. Proc., Fed. Am. Soc. Exp. Biol. 29, 55 (1970). 13 H. G. Kunkel, M. Mannik, and R. C. Williams, Science 140, 1218 (1963).
[10]
ANTIIDIOTYPICANTIBODIES
177
says and inhibition radioimmunoassays, and should be satisfactory for direct ELISA and inhibition ELISA assays. However, the presence of complexes in the sera completely prevents the application of such sera to immunofluorescence studies or to studies using antiidiotypic antibodies for inhibition in the Jerne plaque assay. By pasing the immune sera over Sepharose columns bearing the proteins to be used for absorption, the sera can be absorbed without the formation of complexes. 7,8 The absorption is carried out at room temperature and the volume of absorbant required is dependent on the amount of the absorbing immunoglobulin that is attached to the Sepharose, the amount of unwanted antibody in the antiserum, and the affinity of the interaction between the unwanted antibody and the immunoglobulin on the column. The antiserum to be absorbed is generally passed slowly, at room temperature, over a column with a volume similar to the volume of the serum being absorbed. If unwanted antibody remains the absorption is repeated. Care must be taken not to over absorb the antiserum since even "idiotypically specific" antibody may weakly react with some of the non-idiotype-bearing antibodies in normal serum. When anti-idiotypic sera are properly absorbed with normal immunoglobulin, all of the antibodies are removed from the sera except those to the most unique idiotypic determinants of the immunizing molecule. If it is desirable to obtain an antiserum that would detect the more common idiotopes, such as those associated with particular variable gene families, ~4,15 then it is best to absorb with only one, or at most a few, different antibody variable regions. This can be done by absorbing the antiserum with a single myeloma protein of the same heavy and light chain isotypes as the proband antibody. If the absorbing protein was prepared by ammonium sulfate precipitation, it will probably have enough contaminating serum proteins to remove any antibodies to nonimmunoglobulin molecules. When antiidiotypic antibodies are made to rabbit antibodies, it is customary to inject the proband antibodies in adjuvant into allotypically matched rabbits 2 or into guinea pigs tolerized by prior injection with 5 mg of deaggregated (25,000 g for 30 min) normal rabbit immunoglobulin in saline. 16In either technique the only antibodies produced are antiidiotypic antibodies and no absorption is required. Claflin and Davie 17developed a procedure that allowed them to purify 14 M. J. Bosma, C. DeWitt, S. J. Hausman, R. Marks, and M. Potter, J. Exp. Med. 146, 1041 (1977). l~ p. Basta, H. Kubagawa, J. F. Kearney, and D. E. Briles, J. lmmunol. 130, 2423 (1983). i6 K. Eichmann and T. J. Kindt, J. Exp. Med. 134, 532 (1971). t7 j. L. Claflin and J. M. Davie, J. Immunol. 114, 70 (1975).
178
SERUM IMMUNOGLOBULINS
[10]
idiotypic antibodies reactive with determinants in the antigen binding site of the proband antibody. The procedure is carried out as follows: Antiidiotypic antibodies are made against an immunoglobulin specific for a hapten, such as phosphocholine. The absorbed antiidiotypic serum is then passed over a Sepharose column bearing the proband antibody. The column is washed witha 0.2 M pH 8.3 borate buffer containing 0.15 M NaCI to elute unbound serum proteins. The antibinding site antibodies are eluted from the column by passing the hapten, in this case 10 3 M phosphorylcholine in the same borate buffer over the column. These antibodies are then dialyzed against the borate buffer to remove the hapten. ~7 Antiidiotypic antibodies to mouse myeloma and hybridoma antibodies have also been made in mice. In this case, it has been found that if mice are used that express the same allotype as the injected antibody, the antiidiotypic response is very weak. Better responses are usually obtained if the mouse antibodies are injected into allotypically mismatched mice. The small amount of antiallotype antibody produced is generally absorbed out with a Sepharose column bearing the normal serum obtained from the same mouse strain that provided the proband immunoglobulin.~8
Assay Procedures Double Diffusion in Agar The earliest idiotypic assay in routine use was double diffusion in agar. 2,~2,19This assay has the advantage of not requiring prior absorption of the antiidiotypic sera, since precipitin patterns with idiotype-bearing sera will form a spur behind patterns with sera lacking the aprpopriate idiotype. 2,19,2° In fact, it was this property of the assay that led to the original discovery ofidiotypy. 2,j9 The assay is seldom used now because it is difficult to quantitate, requires large amounts of the antiidiotypic serum and test antibody, and is only applicable to antiidiotypic sera that contain antibodies to a number of distinct idiotopes on the same proband molecule. The assay has an additional disadvantage of giving false negatives, since a precipitin band is often not obtained unless the concentrations of antisera and antigen loaded into a double diffusion pattern are close to equivalence. The assay is usually inappropriate for studies using hybridoma antiidiotypic antibodies because of the frequent failure of monois R. Lieberman, M. Potter, E. B. Mushinski, W. Humphrey, Jr., and S. Rudikoff, J. Exp. Med. 139, 983 (1974). z9 R. J. Slater, S. M. Ward, and H. G. Kunkel, J. Exp. Med. 101, 85 (1955). 20 j. Munoz, Methods IrnmunoL Immunochem. 3, 146 (1971).
[10]
ANTIID1OTYPIC ANTIBODIES
179
clonal antibodies to give precipitation reactions with nonpolymeric antigens.
Passive Hemagglutination and Passive Hemagglutination Inhibition Passive hemagglutination and hemagglutination inhibition provided a major advance in the detection of idiotypy. ~2 Proband or other test immunoglobulins are coupled to red cells, which can then be agglutinated by antiidiotypic antibodies. This procedure is easier to quantitate than double diffusion reactions, antibody-antigen equivalence is not a major concern and the antiserum need not contain antibodies to multiple idiotopes on the same test molecule. Thus, an immune serum, or hybridoma antibody, that recognized only a single idiotope on an immunoglobulin molecule could cause the agglutination of red cells coated with that antibody. One of three reagents is generally used to coat sheep red cells with immunoglobulin: carbodiimide, zl bis-diazotized benzidine (BDB) 22 or the chromic chloride. 23 All three procedures have the potential of making antibody-antibody and red cell-red cell bonds as well as making antibody-red cell bonds. Carbodiimide and BDB make covalent bonds. Since both of these reactions are relatively slow they allow for the production of very reproducible batches of sensitized red cells. The carbodiimide and BDB procedures have the disadvantage that mg amounts of the test antigens are required. The chromic chloride procedure requires much smaller amounts of protein but the reaction is very fast. This can make it difficult to produce similar degrees of aggregation of the antibody and the red cells. This can result in coated cell preparations that vary in their ability to be agglutinated. Details of the passive hemagglutination technique have been given in another chapter [9] in this volume and will not be duplicated here. In the hemagglutination inhibition assay, red cells are coated with the proband immunoglobulin, as for passive hemagglutination, and agglutinated with the smallest amount of the absorbed antiidiotypic serum that yields a strongly positive pattern. Test antibodies suspected of expressing idiotopes shared by the proband antibody are then used to attempt inhibition of agglutination. In general the inhibitors are titrated out with 2- or 3fold dilutions in the microtitration wells prior to the addition of a constant amount of antiidiotypic antibody to each well. Finally the proband-coated red cells are added to each well as for the passive hemagglutination as2~ H. M. Johnson, K. Brenner, and H. E. Hall, J. lmmunol. 97, 791 (1966). 22 C. A. Williams and M. W. Chase, eds., "Methods in Immunology and Immunochemistry," Vol. 1, p. 164. Academic Press, New York, 1967. z3 E. R. Gold and H. H. Fudenberg, J. lrnmunol. 99, 859 (1967).
180
SERUM IMMUNOGLOBULINS
[10]
says. One advantage of this asay over direct agglutination is that only a single preparation of red cells is needed to examine the idiotype of a number of test molecules. This eliminates complications in the direct assay that arise from differences in the efficiency of coating the red cells with the different antigen preparations. Another major advantage of the inhibition assay is that it is well suited to studies attempting to detect complete idiotypic identity, or a lack of complete identity, between a proband immunoglobulin and any test immunoglobulin. ~2 In order for a test antibody to completely inhibit the agglutination of proband-conjugated red blood cells by the antiidiotypic sera, it will have to have all of the idiotopes recognized by the antisera on the proband molecule. If the same minimal amount of the test protein and the proband antibody were required for complete inhibition of agglutination, then the molecules would be said to be idiotypically identical as determined by this assay. Kunkel originally used this assay to demonstrate that idiotypically identical myeloma antibodies were seldom, if ever, observed, and that even in normal human sera, individual idiotypic determinants (IdI) of any particular proband antibody were extremely r a r e . 12
Kunkel et al. 24 also demonstrated that if an idiotypically cross-reactive antibody (which we will refer to as the reference antibody), rather than the proband antibody, was used to coat the red cells, the agglutination could be inhibited by any antibody that carried the idiotope(s) that were shared between the proband and the reference antibody (and detected by the idiotypic antiserum). Using this type of assay, it is possible to readily detect idiotypic similarities among different antibodies with the same specificity or among antibodies using the same VH or VL region genes. Idiotypic determinants that are shared by idiotypically nonidentical antibodies are referred to as IdX determinants.
Radioimmuno-Idiotypic Assays Two major types of inhibition radioimmunoassays have been used. In both, the ability of various test antibodies to inhibit the reactivity of the antiidiotypic antisera with a radioactively labeled proband antibody or cross-reactive reference antibody is measured. In an indirect precipitation system, z5 a rabbit antiidiotypic serum and a radioactively labeled mouse proband antibody are mixed together and allowed to incubate for a short time. Next a goat anti-rabbit immunoglobulin serum, that has been ab24 H, G. Kunkel, V. Agnello, F. G. Joslin, R. J. Winchester, and J. D. Capra, J. Exp. Med. 137, 331 (1973). z~ M. G. Kuettner, A. Wang, and A. Nisonoff, J. Exp. Med. 135, 579 (1972),
ll0]
ANTIIDIOTYPICANTIBODIES
181
sorbed so it does not react with mouse immunoglobulin, is added at a concentration that has been previously determined to precipitate all of the rabbit immunoglobulin and any ~25I-labeled antigen bound to it. By counting the amount of radioactivity in either the supernatant or the pellet, it is possible to determine the percentage of the radioactivity bound to the antiidiotypic antibody. When used as an inhibition assay, test antigens are added to the mixture to see if they can block the binding of the labeled proband antibody to the antiidiotypic antibody. The major advantages of this system is that test antibodies that share some, but not all, of the idiotopes of the proband antibody give a plateau of partial inhibition regardless of the amount of inhibitor used. This is usually not a property of the solid phase radioimmunoassay (SPRIA) described below. However this assay has not been widely adopted by other laboratories, in part, because large amounts of anti-rabbit immunoglobulin antibody are required and the fact that it is not as easy to perform as the SPRIA. S PRI A
The major radioimmuno-idiotypic assay currently in use is the SPRIA, 2 in which a 7-globulin fraction of the antiidiotypic serum is allowed to absorb, generally overnight at 4 °, to the surface of vinyl microtitration 26,27plates (disposable polyvinyl Flex " V " Plates, Dynatec Laboratories). The plates are then rinsed with PBS and blocked for at least 1 hr with 1% BSA-PBS to prevent any further nonspecific adsorption, washed with PBS, and the wells filled with 100 txl of 1% BSA-PBS containing 30,000 cpm of either the 125I-labeled28 proband or an idiotypically crossreactive immunoglobulin. Inhibitors can be included in the label mixture to determine their ability to inhibit the binding of the labeled molecule to the antiidiotypic antibodies. After 4-12 hr of reaction at 37 °, the plates are washed with buffer and dried. The dry plates are placed in a special jig (Fig. 1) and the wells are cut from the plates with a hack saw. The radioactivity associated with the wells is then determined in a gamma counter. In practice, the inhibitors are diluted out in the antibody-coated plate after it has been rinsed with PBS, blocked with BSA, and its wells filled with 100 tzl of 1% BSA. To the first well in each row of 8, 100/xl of an appropriate dilution of a test inhibitor is added. Fifty microliter microtitration wands (Dynatech Laboratories) are then used to make 3-fold dilu26 D. Carson and M. Weigert, Proc. Natl. Acad. Sci. U.S.A. 70, 235 (1973). 27 T. N. Marion, A. R. Lawton, III, J. F. Kearney, and D. E. Briles, J. Immunol. 128, 668 (1982). 28 F. C. Greenwood, W. M. Hunter, and J. S. Glover, J. Biochem. 89, 114, (1963).
182
SERUM IMMUNOGLOBULINS MICROTITER
JL_ t
PLATE'-~
-~
÷ TEMPLATE A N D M/CROTITER PLATE ~SSEMBLY
T ~
"t7~'~°ol~l
I
cm
I ! ~
/
~ :~IIT-~I I
('/IT #/A#/. . . . "~.~ !¢/"
[
~
M/CROTITER
I (
i
~ l "
II~ II I I .3 ,,2 III_ t ' - ~
,,
I L
/
~
E--~
' 4"
TEMP'ATE'
1~51/8" hi
/ [ I
~:~
PLADT, - .JsrZ I" RLATE
7 iIII---"
k
( Z 7 4 re, Is)
/ +i ~--8~m i
I)
Il mm
t_t_ ~
T~MPLATE ~ 1 1< 11
~
[10]
r Ii 0 J" #-~i ~ ~T ~3
CUTTING PLAN p
TE .P. TE AFTER c u r r l N G O F F
TOP OF WELLS
/
~
~1/8 ~J
,~
BOTTOMS REMAIN
~ SNUG IN TEMPLATE UNTIL PUSHED OUT AND INTO ~,ALS OR TEST
TUBES--
__
!"-.
I I I
\ SUPPORT
~1~11/6" ~ 1/16 I GUIDE SLOT F O R S A W
METAL X CUTTING GUIOE
FINGER NO TCBES TO AlL) IN REMOVING
TEMP, ATE ANO
MI
ASSE
'!
OL
FIG. 1. Apparatus for cutting microtitration wells from microtitration plates. The wells of a microtitration plate are first pressed into a template with 96 holes drilled in an identical pattern to those on a vinyl 96-well plate. The diameter of the holes in the template is small enough so that about 1/8 in. of each well protrudes from the top of the template thus allowing it to be cut offin a later step. The vinyl plate-template sandwich is then placed into a 1/2 in. slot between two stationary blocks. The slot between these two blocks has metal guides down each side that will position the saw blade 1/16 in. from one on the blocks. The vinyl plate-template sandwich is inserted so that the vinyl plate is on the same side of the slot as the path for the saw blade. A hacksaw is then used to cut the wells from the vinyl plate. Once all of the wells are cut the saw blade is disconnected from the saw and pulled out horizontally so as not to dislodge any of the cut wells from the template. The template is then lifted out the top of the slot and the wells punched into plastic tubes compatible with the gamma counter being used. t i o n s t h r o u g h 7 w e l l s . N o i n h i b i t o r is a d d e d to t h e e i g h t h well in e a c h r o w , t h u s p r o v i d i n g a set o f m a x i m u m b i n d i n g c o n t r o l s . T h e p l a t e s h o u l d also c o n t a i n at l e a s t t w o w e l l s that h a v e n o t b e e n c o a t e d w i t h a n t i i d i o t y p i c a n t i b o d y . T h e s e w e l l s will s e r v e as m i n i m u m , o r b a c k g r o u n d , b i n d i n g c o n t r o l s . B e f o r e t h e a s s a y is c a r r i e d o u t , a t e s t t i t r at i o n is p e r f o r m e d to d e t e r m i n e t h e o p t i m a l d i l u t i o n o f t h e a n t i i d i o t y p i c a n t i b o d y to be u sed . I n this t y p e o f a s s a y , it is r a r e f o r o v e r 50% o f t he a d d e d p r o b a n d 125I-counts to b i n d t h e a n t i b o d y o n t h e p l a s t i c s u r f a c e , r e g a r d l e s s o f t h e a n t i b o d y
[10]
ANTIIDIOTYPICANTIBODIES
183
concentration used to coat the plates. For an inhibition assay, the antibody concentration used to coat the plates is one that will bind 2/3 of the number of counts bound by an optimal concentration of antiidiotypic antibody. This will ensure that the amount of antibody adsorbed to the wells is limiting. The antibody used to coat the wells need not be highly purified but should be largely free of contaminating serum albumin, which will block the antibody's adsorption to the plastic microtiter wells. Immunoglobulin isolated from the rabbit anti-idiotypic serum by three sequential precipitations from 50% saturated ammonium sulfate is generally sufficiently free of albumin for use in this assay. The assay is quantitated by determining the amount of each test antigen required to provide 50% inhibition of the uninhibited binding of the labeled proband to the antiidiotypic serum-coated microtiter wells. As a basis of comparison, the binding of the labeled proband antibody is also inhibited with unlabeled proband antibody and the values for each test antigen are presented as the quotient of the number of/xg proband required for 50% inhibition divided by the number of/xg of test antigen required for 50% inhibition. Thus, a test antigen with antibody activity equal to that of the proband would have a value of one and could be said to show idiotypic identity (IdI) by this assay. Any antigen that cross reacts more weakly with the antiidiotypic antiserum from the proband would have a lower value. With the assay set up as described, antibodies that share only a few idiotopes with the proband antibody provide minimal inhibition. To examine the idiotypy of shared determinants, a cross-reactive protein can be used as the 125I-labeled reference protein. 29,3° When this is done, the binding of the reference protein will be able to be completely inhibited by any test protein that carries the idiotopes of the reference protein that are recognized by antiproband antibodies in the idiotypic antiserum. As in the hemagglutination inhibition assay, when the SPRIA is set up in this way, it is said to detect IdX determinants. This assay has one major drawback. When inhibitors are used that share some but not all of idiotopes detected on the reference (or proband) antibody by the antiidiotypic antibodies, they will sometimes give complete inhibition at high concentrations even though they do not contain all of the idiotopes detected by the antiserum. Although this is difficult to appreciate conceptually, it is a real property of the assay. 29 D. E. Briles and R. M. Krause, J. lmmunol. 113, 522 (1974). 30 D. Hansburg, D. E. Briles, and J. M. Davie, J. lmmunol. 119, 1406 (1977).
184
SERUM IMMUNOGLOBULINS
[10]
Testing for Idiotypes o f Antibodies in Immune Serum One of the frequent uses of most of these assays is to examine the expression of idiotypes in the immune serum of animals immunized with particular antigens. The examinations of antibodies in immune serum presents certain ambiguities. When the amount of specific antibody in one serum required to give 50% idiotypic inhibition is more than the required amount of the specific antibody of another serum, it is never clear if the difference is due to the fact that the antibodies in the less inhibitory serum cross-react only weakly with the antiidiotypic serum, or if the less inhibitory serum simply has very few molecules possessing the idiotype in question. Another problem in interpreting data from immune serum studies is that the inhibition observed can be the combined effect of several different serum antibodies, each of which shares different idiotopes with the proband. Both of these problems can be overcome to some degree by using monoclonal antiidiotypic antibodies and by examining the expression of idiotypes expressed by individual B cells and plasma cells (see below).
Production and Specificity Testing of Monoclonal Antiidiotype Antibodies
Production of Monoclonal Antiidiotypic Antibodies We have successfully used an immunization protocol originally described by Lieberman et al. 18for the production of allogeneic antiidiotype antibodies in mice. This procedure has been satisfactory not only for the production of antiidiotype antibodies against mouse and human idiotypes, but also as a general method for production of monoclonal antibodies to a variety of foreign proteins and cells. The procedure involves local subcutaneous immunizations, followed by fusion of the lymph node cells obtained from nodes draining the sites of immunization. The protocol for immunization consists of 4-5 50 t~l injections at 3 day intervals. Generally, 1-100/~g of purified antibody is injected per mouse, distributed in six subcutaneous sites: rear foot pads, inguinal regions and subaxillary regions. The first immunization consists of the appropriate amount of antibody in PBS emulsified with an equal volume of CFA (Difco). The next 3-4 immunizations consisting of the same amount of antigen in 50 ~1 of physiological saline administered into the same sites. One day after the last immunization the draining lymph nodes are removed. Single cell suspensions are made by teasing the lymph nodes with fine forceps, and the cells obtained are fused with a
[10]
ANTUDIOTYP1C ANTIBODIES
185
non-secreting myeloma such as Ag8.653 (obtainable from American Type Culture Collection) by methods previously described. 31,32 This protocol has worked successfully for constructing hybridomas to both mouse and human immunoglobulin idiotypes. 33 Hybridoma antibodies have also been prepared to idiotypes expressed by surface antibody of human B cell neoplasms. This has been done by injection of the B lymphoma cells themselves or by injecting antibody produced by cell lines obtained by fusing lymphomas with nonsecreting plasmacytoma lines. 34,35
Screening for Monoclonal Antiidiotypic Antibodies Enzyme-Linked Immunoabsorbent Assay (ELISA). The details of the ELISA method have been presented in another chapter [8] in this volume and will not be duplicated here. The advantages of an ELISA assay over a SPRIA are (1) the stability of the conjugated protein (some have been used for 5-6 years without significant loss of stability). (2) The lack of necessity to take special safety precautions because of the non-radioactive nature of the marker used. (3) The possibility of using lower cost equipment for accurate quantitation of the assay. Isologous or Allologous Antiidiotype Antibodies. Isologous or allologous antiidiotypic antibodies are made by immunizing mice with mouse antibodies obtained, respectively, from the same or a different inbred line as the injected mouse. In the particular modification developed to screen for culture wells containing isologous or allologous (mouse anti-mouse) antiidiotypic antibodies, the microtitration plate is first coated with the proband antibody, and then blocked with 1% BSA, prior to incubation with tissue culture supernatants suspected of containing antiidiotypic antibody. The plate is then washed in borate buffered saline and incubated with a mixture of several antiisotype antibodies that detect all mouse isotypes except the light and heavy chain isotype of the proband antibody. Finally, the plate is incubated with an appropriate substrate. The assay system will detect any antibody in the tissue culture supernatant that reacts with the proband antibody whether it reacts with constant 31 J. F. Kearney, A. Radbruch, B. Liesegang, and K. Rajewsky, J. lmmunol. 123, 1548 (1979). 32 R. Stohrer, M. C. Lee, and J. F. Kearney, J. lrnmunol. 131, 1375 (1983). 33 H. Kubagawa, M. Mayumi, J. F. Kearney, and M. D. Cooper, J. Exp. Med. 156, 1010 (1982). 34 M. Mayumi, H. Kubagawa, G. A. Omura, W. E. Gathings, J. F. Kearney, and M. D. Cooper, J. Immunol. 129, 904 (1982). 35 K. Thielemans, D. G. Maloney, T. Meeker, J. Fujimoto, C. Doss, R. A. Warnke, J. Bindl, J. Gralow, R. A. Miller, and R. Levy, J. lmmunol. 133, 495 (1984).
186
SERUMIMMUNOGLOBULINS
[10]
region, or with idiotypic determinants. 36 To determine whether or not the antibody in the supernatant is reacting with constant or variable region determinants, the same supernatant is also added to microtitration wells coated with different members of a panel of mouse monoclonal antibodies that have a different specificity than the proband antibody but show the same light and heavy chain isotypes as the proband antibodies. Those monoclonal antibodies that react only with the proband antibody are idiotypically specific for the V region. Once a putative antiidiotypic antibody is detected by this procedure it is recloned and retested by the same assay. Xenogeneic Antiidiotype Antibodies. The detection of mouse antiidiotype antibodies specific for xenogeneic idiotypic determinants (e.g., rat or human) is more straightforward. As above, the purified proband antibody is coated directly onto a microtitration plate. Supernatants are then added and any mouse antibodies binding the proband antibody are detected by enzyme-labeled anti-mouse Ig which may have to be previously absorbed with rat or human immunoglobulins in order to prevent possible cross-reaction with the proband rat or human immunoglobulin used to coat the plate. Detection of Antiidiotypic Antibodies to Antibody Producing MouseHuman A L L Heterohybrids. When it is not feasible to obtain purified immunoglobulin as antigen, other procedures for idiotypic detection are required. These approaches can be illustrated by the procedures used to screen for monoclonal antiidiotypic antibodies prepared against idiotypic determinant of immunoglobulins on human acute lymphocytic leukemia (ALL) cells. 34,35 ALL-derived immunoglobulin in the supernatants of mouse-human A L L heterohybrids can be successfully coated to wells that are first coated with anti-human Ig and then adding the heterohybrid supernatant followed by anti-Id supernatant. The assay is then completed by the addition of supernatants thought to contain antiidiotype and finally enzyme-labeled anti-mouse Ig 34 as in the above procedures. Detection of Monoclonal Antibodies Bearing Common ldiotopes. Once a monoclonal antiidiotypic antibody is obtained, it can then be used to screen for the production of other antibodies carrying the same idiotope as that detected on the proband antibody. This technique can be carried out in a variety of ways. In one modification, antibodies to be tested are added to a microtitration plate coated with the monoclonal antiidiotype. As a final step the plate is incubated with AP-conjugated antibody reactive with the isotype of the test antibody. It is important to 36 J. F. Kearney, in "Hybridomas and Monoclonal Antibodies" (W. E. Paul, eds.), p. 751. Raven Press, New York, 1984.
[10]
ANTIIDIOTYPICANTIBODIES
187
note that this procedure cannot be used to assay test mouse antibodies of the same isotype as that of the mouse antiidiotypic antibody. Another approach that can be used is to coat the plates with an antigen that is bound by the test antibodies. The plates are blocked with 1% BSA prior to being incubated with the test antibodies. Next the plates are reacted with AP-conjugated antiidiotype. In an alternative version of this procedure, unconjugated antiidiotype is used and a final layer of APantiisotype that binds to the isotype of the antiidiotype, but not that of the test antigen, is added. All of these ELISA assays are extremely useful in the production and initial screening of hybridoma antibodies, since they allow a large number of different tissue culture supernatants to be examined quickly without waiting for time consuming gamma counting or autoradiography.
lmmunofluorescent Studies The details of the immunofluorescence method have been presented in another chapter [9] in this volume and will not be duplicated here. In this paragraph only the general approach to the study of anti-idiotypic antibodies by immunofluorescence will be discussed. When a population of antibodies shows reactivity in an idiotypic assay such as any of those described above, it is impossible to distinguish between the possibility that a low concentration of antibody is strongly reactive with the antiidiotypic antibodies, or that a high concentration of antibodies are weakly cross-reactive with the antiidiotypic antibodies. A more satisfactory approach to studying antibody populations is to use antiidiotypic antibodies to examine the idiotype of the antibody produced by individual B cells or plasma cells since each cell produces antibody at a single site. This can be done by using the antiidiotypic antibodies to either inhibit antigen specific plaque-forming cells in the Jerne plaque a s s a y 37 o r for immunofluorescence staining of B cells and plasma cells.15 In the case of immunofluorescence, lymphocytes are cytocentrifuged onto glass slides and processed for either surface staining (to detect B cells) or cytoplasmic staining (to detect plasma cells). Next, monoclonal antiidiotypic antibodies conjugated with a fluorochrome are added. 32 In a modification of the procedure the antiidiotypic antibodies themselves are unconjugated and the slides are subsequently stained with fluorescein or rhodamine-conjugated antibodies directed against the constant region of the anti-idiotypic antibodies. It is important in this case that the conjugated antibodies not react with any of the antibodies of the species that 37 j. F. Kearney, R. Barletta, Z. S. Quan, and J. Quintans, Eur. J. Immunol. 11, 877 (1981).
188
SERUM IMMUNOGLOBULINS
[10]
provided the cells being tested. Since almost all monoclonal antiidiotypic antibodies are of mouse origin, this modification of the procedure is largely limited to an examination of the idiotypy of species other than the mouse. When lymphocyte cell surfaces are stained with a heterologous antiserum, it is likely that antibodies bearing several different idiotypes will be detected. When this happens there is no way to tell which of the stained cells are being detected by which of the antiidiotope antibodies in the immune serum. When hybridoma antibodies are used, all cell bound antibodies detected are reacting with the same monoclonal antibody and are likely to be expressing the same epitopes. However, there are some obvious exceptions to this statement, When the panel of hybridomas reactive with al-3 dextran was screened with panels of monoclonal or heterologous antibody by RIA then it could be shown that specificities of the heterologous anti-Id antibodies reacted more truly with the structurally defined idiotypes than did monoclonals. 38 Immunofluorescence is also a convenient way to check for antiidiotypic antibodies in which the idiotope is associated with certain isotypes. In this method, smears are made from a source of B cells, e.g., spleen or polyclonally activated plasma cells and stained first with a fluorescent antibody to a particular isotype followed by staining with the antiidiotype. In this way it is possible to determine rapidly on a large number of clonally derived plasma cells whether the particular idiotype always appears to be restricted to expression with a particular isotype. 39
Idiotypic Specificity of Monoclonal Antiidiotype Antibodies Once it has been established that a monoclonal antibody is specific for the variable regions of an immunoglobulin molecule, it is often of functional and structural importance to determine in which part of the binding site the idiotope determining structures are situated. This determination cannot be done with any assurance because of (1) the long range effects of certain amino acid groups on an epitope, (2) the structural complexity of an idiotope, and (3) the undetermined role played by heavy and light chains on construction of a given idiotope. However, there are certain useful assays that can be carried out. Binding versus Nonbinding Site Idiotopes. Because the V region of an antibody is a complex structure with the binding site often in the shape of 38 B. Clevinger, J. Thomas, J. Davie, J. Schilling, M. Bond, L. Hood, and J. Kearney, in "Immunological Idiotypes" (C. Janeway, E. Sercarz, and H. Wigzell, eds.), p. 159. Academic Press, New York, 1981. 39 G. Morahan, C. Berek, and J. F. A. P. Miller, Nature (London) 301, 720 (1983).
[10]
ANTIIDIOTYPIC ANTIBODIES
189
a groove or cleft, some antiidiotype antibodies will bind outside of a functional binding site. This can be tested by using an inhibition assay similar to those described above. The proband antibody is bound to a microtitration plate and the monoclonal antiidiotype antibody labeled with a radioisotope or enzyme is then added in the presence of dilutions of inhibitors (haptens) of known reactivity with the proband antibodies. These haptens can be used either by themselves or modified by addition of amino acids or the addition of larger protein carriers. In this way, it is possible to show that a low-molecular-weight hapten such as phosphorylcholine ~7 or 4-hydroxy-3-nitrophenyl-acetyl 3 will inhibit binding by the monoclonal antiidiotype antibody. In this way it can be determined that the idiotope recognized by a given monoclonal antibody is intimately associated with the antigen binding site. On the other hand, if binding can only be inhibited by large molecular weight forms of that antigen then the antibody may be non-binding site related. 17 Furthermore, some antiidiotype antibodies will not be inhibited by antigen in any form: the idiotopes recognized by such antibodies are not associated with the binding site. 4° Light and Heavy Chain Involvement in Formation of an Idiotope. Since idiotypic structures can be determined by light and heavy chain interactions or by light or heavy chain alone, it is possible to determine by direct binding to purified light or heavy chain from the immunizing immunoglobulin which chain expresses an idiotope. However, a more precise method is provided by the use of light and heavy chain recombinant molecules in inhibition assays to determine whether a particular idiotope always associates with one heavy or light chain V region. 41 Most monoclonal antibodies appear to be directed against idiotypes dependent on light plus heavy chains. 32 Although such studies give information on idiotope-Ig chain and/or binding site associations, it is difficult to establish the exact residues that are involved in the formation of an idiotope which can only be determined by X-ray crystallographic analysis of idiotype antiidiotype antibody complexes.
40 M. K. Wittner, M. A. Bach, and H. Kohler, J. Exp. Med. 128, 595 (1982). 41 E. Pillemer and I. L. Weissman, J. Exp. Med. 153, 1068 (1981).
SERUMIMMUNOGLOBULINS
[10]
[10] A n t i i d i o t y p i c A n t i b o d i e s B y DAVID E. BRILES a n d JOHN F. KEARNEY
Introduction In the broad sense, idiotypy is the variation among the variable regions of different immunoglobulins.l Such differences can be detected by amino acid sequence analysis, peptide mapping, isoelectric focusing, differences in antibody specificity, or reactivity with antisera directed against different antibody variable region determinants. Although all of these procedures provide information about variations in variable region structure, the term idiotypy is generally used to refer to variable region characterizations carried out with antibodies to variable region determinants. Antigenic determinants associated with the variable regions of different antibodies are referred to as idiotypic determinants 2 and antibodies that recognize them are referred to as antiidiotypic antibodies. An idiotypic determinant detected by a single hybridoma antiidiotypic antibody (or one of the many antibodies in a heterologous antiidiotypic sera) is said to be an idiotope. 3 The constellation of idiotopes detected by the various antiidiotypic antibodies of a single idiotypic serum, by a group of different antiidiotypic sera, or by a group of hybridoma antiidiotypic antibodies is the idiotype of the antibody under study. This chapter is divided into three parts. The first describes the preparation of antiidiotypic sera. The second describes the immunoassays developed for using antiidiotypic sera to characterize the idiotypes of myeloma proteins, hybridoma antibodies, and antibodies in immune sera. The third section describes the methods that have been developed for use with hybridoma antiidiotypic antibodies.
P r e p a r a t i o n of A n t i i d i o t y p i c Sera
The early idiotypic studies of human and mouse myeloma antibodies were conducted using antiidiotypic sera raised in rabbits, goats, or guinea pigs. The immunization procedures used generally involved the injection
10. M/ikel/i and K. Karjalainen, Cold Spring Harbor Symp. Quant. Biol. 41, 735 (1976). z j. Oudin, Proc. R. Soc. London, Ser. B 166, 207 (1966b). 3 M. Reth, T. Imanishi-Kari, and K. Rajewsky, Eur. J. Immunol. 9, 1004 (1979).
METHODS IN ENZYMOLOGY, VOL. 116
Copyright © 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.
[10]
ANTI1DIOTYPIC ANTIBODIES
175
of partially purified antibodies in complete Freund's adjuvant (CFA). 3a In this chapter, we will refer to any immunoglobulin used to elicit an antiidiotypic antibody as a "proband" antibody. Appropriate immunization procedures are no different from those that would be used to prepare antibodies to any macromolecular protein antigen. 4,5 Although a number of different immunization procedures have been used to elicit antiidiotypic antibodies, we have found that a relatively simple method provides adequate results. Rabbits are immunized intraperitoneally with 100-1000 tzg of the proband antibody emulsified in 1 ml of C.FA. 4-6 The more highly purified the antibodies used for immunization are, the higher will be the percentage of the elicited antibodies that recognize idiotypic determinants on the proband molecule. In practice, however, complete purity is not required. In fact, when making antiidiotypic sera to myeloma proteins, purification by three sequential ammonium sulfate precipitations generally works quite well. For mouse antibodies, precipitation from a 47% saturated ammonium sulfate solution is usually satisfactory. Whenever the antigenic specificity of a proband antibody is known, it is desirable to purify it by affinity chromatography from an antigen column. 7-~° A typical preparation of adjuvant for the injection of 3 rabbits would require 1200/zg of purified antibody suspended in 1.6 ml of phosphatebuffered saline (PBS) (0.056 M Na2HPO4, 0.016 M KHzPO4, 0.077 M NaC1, pH 7.2). The PBS-antibody mixture is then added to a 15-ml round-bottom glass centrifuge tube containing 2.4 ml of commercially prepared (Difco) mycobacteria-oil suspension. If a stable emulsion is not obtained, it may be due to the effects of salts or proteins present in the antigen preparation. To obtain a stable emulsion if this occurs, the ratio of oil:aqueous phase can be varied slightly, usually by adding slightly more oil. 3a Abbreviations: ALL, acute lymphocytic leukemia; AP, alkaline phosphatase; BDB, bisdiazotized benzidine; BSA, bovine serum albumin; CFA, complete Freund's adjuvant; ELISA, enzyme-linked immunoadsorbant assay; IdI, individual idiotypic determinants; IdX, cross-reacting idiotypic determinant; PBS, phosphate-buffered saline; SPRIA, solid phase radioimmunoassay; VH, heavy chain variable region; VL, light chain variable region. 4 M. W. Chase, Methods lmmunol. Immunochem. 1, 209 (1967). 5 B. A. L. Hurn and S. M. Chantler, this series, Vol. 70, p. 104. 6 D. E. Briles and R. M. Krause, J. lmmunol. 109, 1311 (1972). 7 j. C. Jaton, D. C. Brandt, and P. Vassalli, in "Immunological Methods" (I. Lefkovits and B. Pernis, eds.), p. 43. Academic Press, New York, 1979. 8 j. S. Garvey, N. E. Cremer, and D. H. Sussdorf, in "Methods in Immunology," 3rd ed., p. 245. Benjamin, Reading, Massachusetts, 1977. 9 D. C. Parker, R. M. Krause, and L. Wofsy, Immunochemistry 10, 727 (1973). i0 B. Chesebro and H. Metzger, Biochemistry 11, 766 (1972).
176
SERUMIMMUNOGLOBULINS
[10]
A pilot bleeding should be made at one month after the primary injection. To determine if antibody has been produced, a small amount of antiserum is mixed with an equal amount of antigen solution (about 1 mg/ ml) in a capillary tube.l~ If no precipitate, or only a weak precipitate, forms (after several hours), the animal should be boosted with a second injection identical to the first. Rabbits are usually bled 7 to 10 days after the second injection. Animals that fail to show detectable responses after two injections are discarded. Animals showing only weak responses after the second injection are given a third injection about 3-4 weeks after the second. High titer antisera will give a positive precipitin reaction within 5-10 min of being mixed with the proband protein in a capillary tube. Since the titer following CFA immunizations generally stays high for several weeks, we generally pool several weekly bleedings from each rabbit before the animal is bled out. When antiidiotype antibodies are made in heterologous species, the recipient animal responds not only to the idiotypic determinants, but also to other determinants on the immunizing immunoglobulin molecule, as well as any contaminating molecules from the species that produced the immunizing immunoglobulin. Thus, the presence of a precipitin reaction between the antiidiotypic sera and the immunizing antigen preparation does not guarantee that the rabbit has begun to make antiidiotypic antibody. In practice, however, whenever a good precipitating serum is obtained, it is almost certain to contain a significant amount of antiidiotypic antibody. Antibodies to constant region determinants and those to contaminating serum molecules can be effectively removed by absorption with pooled normal serum from the species that provided the immunizing antigen. If the immunizing antibody was of a high enough purity so that no antibodies binding other serum components were elicited, then pooled 3'globulin would be the ideal absorbing material. If the proband antibody was of mouse origin, then pooled normal mouse serum from the same inbred strain that provided the proband antibody is used so that any antiimmunoglobulin allotype antibodies will be removed. Originally, antiidiotypic sera were absorbed by simply mixing the antiserum with an excess of normal serum or pooled immunoglobulin and then centrifuging out any precipitate that might occur. T2,~3This procedure proved adequate for studies using precipitation, passive hemagglutination, passive hemagglutination inhibition, direct binding radioimmunoas11 p. H. Maurer, Methods Immunol. Immunochem. 3, 36 (1971). i2 H. G. Kunkel, Fed. Proc., Fed. Am. Soc. Exp. Biol. 29, 55 (1970). 13 H. G. Kunkel, M. Mannik, and R. C. Williams, Science 140, 1218 (1963).
[10]
ANTIIDIOTYPICANTIBODIES
177
says and inhibition radioimmunoassays, and should be satisfactory for direct ELISA and inhibition ELISA assays. However, the presence of complexes in the sera completely prevents the application of such sera to immunofluorescence studies or to studies using antiidiotypic antibodies for inhibition in the Jerne plaque assay. By pasing the immune sera over Sepharose columns bearing the proteins to be used for absorption, the sera can be absorbed without the formation of complexes. 7,8 The absorption is carried out at room temperature and the volume of absorbant required is dependent on the amount of the absorbing immunoglobulin that is attached to the Sepharose, the amount of unwanted antibody in the antiserum, and the affinity of the interaction between the unwanted antibody and the immunoglobulin on the column. The antiserum to be absorbed is generally passed slowly, at room temperature, over a column with a volume similar to the volume of the serum being absorbed. If unwanted antibody remains the absorption is repeated. Care must be taken not to over absorb the antiserum since even "idiotypically specific" antibody may weakly react with some of the non-idiotype-bearing antibodies in normal serum. When anti-idiotypic sera are properly absorbed with normal immunoglobulin, all of the antibodies are removed from the sera except those to the most unique idiotypic determinants of the immunizing molecule. If it is desirable to obtain an antiserum that would detect the more common idiotopes, such as those associated with particular variable gene families, ~4,15 then it is best to absorb with only one, or at most a few, different antibody variable regions. This can be done by absorbing the antiserum with a single myeloma protein of the same heavy and light chain isotypes as the proband antibody. If the absorbing protein was prepared by ammonium sulfate precipitation, it will probably have enough contaminating serum proteins to remove any antibodies to nonimmunoglobulin molecules. When antiidiotypic antibodies are made to rabbit antibodies, it is customary to inject the proband antibodies in adjuvant into allotypically matched rabbits 2 or into guinea pigs tolerized by prior injection with 5 mg of deaggregated (25,000 g for 30 min) normal rabbit immunoglobulin in saline. 16In either technique the only antibodies produced are antiidiotypic antibodies and no absorption is required. Claflin and Davie 17developed a procedure that allowed them to purify 14 M. J. Bosma, C. DeWitt, S. J. Hausman, R. Marks, and M. Potter, J. Exp. Med. 146, 1041 (1977). l~ p. Basta, H. Kubagawa, J. F. Kearney, and D. E. Briles, J. lmmunol. 130, 2423 (1983). i6 K. Eichmann and T. J. Kindt, J. Exp. Med. 134, 532 (1971). t7 j. L. Claflin and J. M. Davie, J. Immunol. 114, 70 (1975).
178
SERUM IMMUNOGLOBULINS
[10]
idiotypic antibodies reactive with determinants in the antigen binding site of the proband antibody. The procedure is carried out as follows: Antiidiotypic antibodies are made against an immunoglobulin specific for a hapten, such as phosphocholine. The absorbed antiidiotypic serum is then passed over a Sepharose column bearing the proband antibody. The column is washed witha 0.2 M pH 8.3 borate buffer containing 0.15 M NaCI to elute unbound serum proteins. The antibinding site antibodies are eluted from the column by passing the hapten, in this case 10 3 M phosphorylcholine in the same borate buffer over the column. These antibodies are then dialyzed against the borate buffer to remove the hapten. ~7 Antiidiotypic antibodies to mouse myeloma and hybridoma antibodies have also been made in mice. In this case, it has been found that if mice are used that express the same allotype as the injected antibody, the antiidiotypic response is very weak. Better responses are usually obtained if the mouse antibodies are injected into allotypically mismatched mice. The small amount of antiallotype antibody produced is generally absorbed out with a Sepharose column bearing the normal serum obtained from the same mouse strain that provided the proband immunoglobulin.~8
Assay Procedures Double Diffusion in Agar The earliest idiotypic assay in routine use was double diffusion in agar. 2,~2,19This assay has the advantage of not requiring prior absorption of the antiidiotypic sera, since precipitin patterns with idiotype-bearing sera will form a spur behind patterns with sera lacking the aprpopriate idiotype. 2,19,2° In fact, it was this property of the assay that led to the original discovery ofidiotypy. 2,j9 The assay is seldom used now because it is difficult to quantitate, requires large amounts of the antiidiotypic serum and test antibody, and is only applicable to antiidiotypic sera that contain antibodies to a number of distinct idiotopes on the same proband molecule. The assay has an additional disadvantage of giving false negatives, since a precipitin band is often not obtained unless the concentrations of antisera and antigen loaded into a double diffusion pattern are close to equivalence. The assay is usually inappropriate for studies using hybridoma antiidiotypic antibodies because of the frequent failure of monois R. Lieberman, M. Potter, E. B. Mushinski, W. Humphrey, Jr., and S. Rudikoff, J. Exp. Med. 139, 983 (1974). z9 R. J. Slater, S. M. Ward, and H. G. Kunkel, J. Exp. Med. 101, 85 (1955). 20 j. Munoz, Methods IrnmunoL Immunochem. 3, 146 (1971).
[10]
ANTIID1OTYPIC ANTIBODIES
179
clonal antibodies to give precipitation reactions with nonpolymeric antigens.
Passive Hemagglutination and Passive Hemagglutination Inhibition Passive hemagglutination and hemagglutination inhibition provided a major advance in the detection of idiotypy. ~2 Proband or other test immunoglobulins are coupled to red cells, which can then be agglutinated by antiidiotypic antibodies. This procedure is easier to quantitate than double diffusion reactions, antibody-antigen equivalence is not a major concern and the antiserum need not contain antibodies to multiple idiotopes on the same test molecule. Thus, an immune serum, or hybridoma antibody, that recognized only a single idiotope on an immunoglobulin molecule could cause the agglutination of red cells coated with that antibody. One of three reagents is generally used to coat sheep red cells with immunoglobulin: carbodiimide, zl bis-diazotized benzidine (BDB) 22 or the chromic chloride. 23 All three procedures have the potential of making antibody-antibody and red cell-red cell bonds as well as making antibody-red cell bonds. Carbodiimide and BDB make covalent bonds. Since both of these reactions are relatively slow they allow for the production of very reproducible batches of sensitized red cells. The carbodiimide and BDB procedures have the disadvantage that mg amounts of the test antigens are required. The chromic chloride procedure requires much smaller amounts of protein but the reaction is very fast. This can make it difficult to produce similar degrees of aggregation of the antibody and the red cells. This can result in coated cell preparations that vary in their ability to be agglutinated. Details of the passive hemagglutination technique have been given in another chapter [9] in this volume and will not be duplicated here. In the hemagglutination inhibition assay, red cells are coated with the proband immunoglobulin, as for passive hemagglutination, and agglutinated with the smallest amount of the absorbed antiidiotypic serum that yields a strongly positive pattern. Test antibodies suspected of expressing idiotopes shared by the proband antibody are then used to attempt inhibition of agglutination. In general the inhibitors are titrated out with 2- or 3fold dilutions in the microtitration wells prior to the addition of a constant amount of antiidiotypic antibody to each well. Finally the proband-coated red cells are added to each well as for the passive hemagglutination as2~ H. M. Johnson, K. Brenner, and H. E. Hall, J. lmmunol. 97, 791 (1966). 22 C. A. Williams and M. W. Chase, eds., "Methods in Immunology and Immunochemistry," Vol. 1, p. 164. Academic Press, New York, 1967. z3 E. R. Gold and H. H. Fudenberg, J. lrnmunol. 99, 859 (1967).
180
SERUM IMMUNOGLOBULINS
[10]
says. One advantage of this asay over direct agglutination is that only a single preparation of red cells is needed to examine the idiotype of a number of test molecules. This eliminates complications in the direct assay that arise from differences in the efficiency of coating the red cells with the different antigen preparations. Another major advantage of the inhibition assay is that it is well suited to studies attempting to detect complete idiotypic identity, or a lack of complete identity, between a proband immunoglobulin and any test immunoglobulin. ~2 In order for a test antibody to completely inhibit the agglutination of proband-conjugated red blood cells by the antiidiotypic sera, it will have to have all of the idiotopes recognized by the antisera on the proband molecule. If the same minimal amount of the test protein and the proband antibody were required for complete inhibition of agglutination, then the molecules would be said to be idiotypically identical as determined by this assay. Kunkel originally used this assay to demonstrate that idiotypically identical myeloma antibodies were seldom, if ever, observed, and that even in normal human sera, individual idiotypic determinants (IdI) of any particular proband antibody were extremely r a r e . 12
Kunkel et al. 24 also demonstrated that if an idiotypically cross-reactive antibody (which we will refer to as the reference antibody), rather than the proband antibody, was used to coat the red cells, the agglutination could be inhibited by any antibody that carried the idiotope(s) that were shared between the proband and the reference antibody (and detected by the idiotypic antiserum). Using this type of assay, it is possible to readily detect idiotypic similarities among different antibodies with the same specificity or among antibodies using the same VH or VL region genes. Idiotypic determinants that are shared by idiotypically nonidentical antibodies are referred to as IdX determinants.
Radioimmuno-Idiotypic Assays Two major types of inhibition radioimmunoassays have been used. In both, the ability of various test antibodies to inhibit the reactivity of the antiidiotypic antisera with a radioactively labeled proband antibody or cross-reactive reference antibody is measured. In an indirect precipitation system, z5 a rabbit antiidiotypic serum and a radioactively labeled mouse proband antibody are mixed together and allowed to incubate for a short time. Next a goat anti-rabbit immunoglobulin serum, that has been ab24 H, G. Kunkel, V. Agnello, F. G. Joslin, R. J. Winchester, and J. D. Capra, J. Exp. Med. 137, 331 (1973). z~ M. G. Kuettner, A. Wang, and A. Nisonoff, J. Exp. Med. 135, 579 (1972),
ll0]
ANTIIDIOTYPICANTIBODIES
181
sorbed so it does not react with mouse immunoglobulin, is added at a concentration that has been previously determined to precipitate all of the rabbit immunoglobulin and any ~25I-labeled antigen bound to it. By counting the amount of radioactivity in either the supernatant or the pellet, it is possible to determine the percentage of the radioactivity bound to the antiidiotypic antibody. When used as an inhibition assay, test antigens are added to the mixture to see if they can block the binding of the labeled proband antibody to the antiidiotypic antibody. The major advantages of this system is that test antibodies that share some, but not all, of the idiotopes of the proband antibody give a plateau of partial inhibition regardless of the amount of inhibitor used. This is usually not a property of the solid phase radioimmunoassay (SPRIA) described below. However this assay has not been widely adopted by other laboratories, in part, because large amounts of anti-rabbit immunoglobulin antibody are required and the fact that it is not as easy to perform as the SPRIA. S PRI A
The major radioimmuno-idiotypic assay currently in use is the SPRIA, 2 in which a 7-globulin fraction of the antiidiotypic serum is allowed to absorb, generally overnight at 4 °, to the surface of vinyl microtitration 26,27plates (disposable polyvinyl Flex " V " Plates, Dynatec Laboratories). The plates are then rinsed with PBS and blocked for at least 1 hr with 1% BSA-PBS to prevent any further nonspecific adsorption, washed with PBS, and the wells filled with 100 txl of 1% BSA-PBS containing 30,000 cpm of either the 125I-labeled28 proband or an idiotypically crossreactive immunoglobulin. Inhibitors can be included in the label mixture to determine their ability to inhibit the binding of the labeled molecule to the antiidiotypic antibodies. After 4-12 hr of reaction at 37 °, the plates are washed with buffer and dried. The dry plates are placed in a special jig (Fig. 1) and the wells are cut from the plates with a hack saw. The radioactivity associated with the wells is then determined in a gamma counter. In practice, the inhibitors are diluted out in the antibody-coated plate after it has been rinsed with PBS, blocked with BSA, and its wells filled with 100 tzl of 1% BSA. To the first well in each row of 8, 100/xl of an appropriate dilution of a test inhibitor is added. Fifty microliter microtitration wands (Dynatech Laboratories) are then used to make 3-fold dilu26 D. Carson and M. Weigert, Proc. Natl. Acad. Sci. U.S.A. 70, 235 (1973). 27 T. N. Marion, A. R. Lawton, III, J. F. Kearney, and D. E. Briles, J. Immunol. 128, 668 (1982). 28 F. C. Greenwood, W. M. Hunter, and J. S. Glover, J. Biochem. 89, 114, (1963).
182
SERUM IMMUNOGLOBULINS MICROTITER
JL_ t
PLATE'-~
-~
÷ TEMPLATE A N D M/CROTITER PLATE ~SSEMBLY
T ~
"t7~'~°ol~l
I
cm
I ! ~
/
~ :~IIT-~I I
('/IT #/A#/. . . . "~.~ !¢/"
[
~
M/CROTITER
I (
i
~ l "
II~ II I I .3 ,,2 III_ t ' - ~
,,
I L
/
~
E--~
' 4"
TEMP'ATE'
1~51/8" hi
/ [ I
~:~
PLADT, - .JsrZ I" RLATE
7 iIII---"
k
( Z 7 4 re, Is)
/ +i ~--8~m i
I)
Il mm
t_t_ ~
T~MPLATE ~ 1 1< 11
~
[10]
r Ii 0 J" #-~i ~ ~T ~3
CUTTING PLAN p
TE .P. TE AFTER c u r r l N G O F F
TOP OF WELLS
/
~
~1/8 ~J
,~
BOTTOMS REMAIN
~ SNUG IN TEMPLATE UNTIL PUSHED OUT AND INTO ~,ALS OR TEST
TUBES--
__
!"-.
I I I
\ SUPPORT
~1~11/6" ~ 1/16 I GUIDE SLOT F O R S A W
METAL X CUTTING GUIOE
FINGER NO TCBES TO AlL) IN REMOVING
TEMP, ATE ANO
MI
ASSE
'!
OL
FIG. 1. Apparatus for cutting microtitration wells from microtitration plates. The wells of a microtitration plate are first pressed into a template with 96 holes drilled in an identical pattern to those on a vinyl 96-well plate. The diameter of the holes in the template is small enough so that about 1/8 in. of each well protrudes from the top of the template thus allowing it to be cut offin a later step. The vinyl plate-template sandwich is then placed into a 1/2 in. slot between two stationary blocks. The slot between these two blocks has metal guides down each side that will position the saw blade 1/16 in. from one on the blocks. The vinyl plate-template sandwich is inserted so that the vinyl plate is on the same side of the slot as the path for the saw blade. A hacksaw is then used to cut the wells from the vinyl plate. Once all of the wells are cut the saw blade is disconnected from the saw and pulled out horizontally so as not to dislodge any of the cut wells from the template. The template is then lifted out the top of the slot and the wells punched into plastic tubes compatible with the gamma counter being used. t i o n s t h r o u g h 7 w e l l s . N o i n h i b i t o r is a d d e d to t h e e i g h t h well in e a c h r o w , t h u s p r o v i d i n g a set o f m a x i m u m b i n d i n g c o n t r o l s . T h e p l a t e s h o u l d also c o n t a i n at l e a s t t w o w e l l s that h a v e n o t b e e n c o a t e d w i t h a n t i i d i o t y p i c a n t i b o d y . T h e s e w e l l s will s e r v e as m i n i m u m , o r b a c k g r o u n d , b i n d i n g c o n t r o l s . B e f o r e t h e a s s a y is c a r r i e d o u t , a t e s t t i t r at i o n is p e r f o r m e d to d e t e r m i n e t h e o p t i m a l d i l u t i o n o f t h e a n t i i d i o t y p i c a n t i b o d y to be u sed . I n this t y p e o f a s s a y , it is r a r e f o r o v e r 50% o f t he a d d e d p r o b a n d 125I-counts to b i n d t h e a n t i b o d y o n t h e p l a s t i c s u r f a c e , r e g a r d l e s s o f t h e a n t i b o d y
[10]
ANTIIDIOTYPICANTIBODIES
183
concentration used to coat the plates. For an inhibition assay, the antibody concentration used to coat the plates is one that will bind 2/3 of the number of counts bound by an optimal concentration of antiidiotypic antibody. This will ensure that the amount of antibody adsorbed to the wells is limiting. The antibody used to coat the wells need not be highly purified but should be largely free of contaminating serum albumin, which will block the antibody's adsorption to the plastic microtiter wells. Immunoglobulin isolated from the rabbit anti-idiotypic serum by three sequential precipitations from 50% saturated ammonium sulfate is generally sufficiently free of albumin for use in this assay. The assay is quantitated by determining the amount of each test antigen required to provide 50% inhibition of the uninhibited binding of the labeled proband to the antiidiotypic serum-coated microtiter wells. As a basis of comparison, the binding of the labeled proband antibody is also inhibited with unlabeled proband antibody and the values for each test antigen are presented as the quotient of the number of/xg proband required for 50% inhibition divided by the number of/xg of test antigen required for 50% inhibition. Thus, a test antigen with antibody activity equal to that of the proband would have a value of one and could be said to show idiotypic identity (IdI) by this assay. Any antigen that cross reacts more weakly with the antiidiotypic antiserum from the proband would have a lower value. With the assay set up as described, antibodies that share only a few idiotopes with the proband antibody provide minimal inhibition. To examine the idiotypy of shared determinants, a cross-reactive protein can be used as the 125I-labeled reference protein. 29,3° When this is done, the binding of the reference protein will be able to be completely inhibited by any test protein that carries the idiotopes of the reference protein that are recognized by antiproband antibodies in the idiotypic antiserum. As in the hemagglutination inhibition assay, when the SPRIA is set up in this way, it is said to detect IdX determinants. This assay has one major drawback. When inhibitors are used that share some but not all of idiotopes detected on the reference (or proband) antibody by the antiidiotypic antibodies, they will sometimes give complete inhibition at high concentrations even though they do not contain all of the idiotopes detected by the antiserum. Although this is difficult to appreciate conceptually, it is a real property of the assay. 29 D. E. Briles and R. M. Krause, J. lmmunol. 113, 522 (1974). 30 D. Hansburg, D. E. Briles, and J. M. Davie, J. lmmunol. 119, 1406 (1977).
184
SERUM IMMUNOGLOBULINS
[10]
Testing for Idiotypes o f Antibodies in Immune Serum One of the frequent uses of most of these assays is to examine the expression of idiotypes in the immune serum of animals immunized with particular antigens. The examinations of antibodies in immune serum presents certain ambiguities. When the amount of specific antibody in one serum required to give 50% idiotypic inhibition is more than the required amount of the specific antibody of another serum, it is never clear if the difference is due to the fact that the antibodies in the less inhibitory serum cross-react only weakly with the antiidiotypic serum, or if the less inhibitory serum simply has very few molecules possessing the idiotype in question. Another problem in interpreting data from immune serum studies is that the inhibition observed can be the combined effect of several different serum antibodies, each of which shares different idiotopes with the proband. Both of these problems can be overcome to some degree by using monoclonal antiidiotypic antibodies and by examining the expression of idiotypes expressed by individual B cells and plasma cells (see below).
Production and Specificity Testing of Monoclonal Antiidiotype Antibodies
Production of Monoclonal Antiidiotypic Antibodies We have successfully used an immunization protocol originally described by Lieberman et al. 18for the production of allogeneic antiidiotype antibodies in mice. This procedure has been satisfactory not only for the production of antiidiotype antibodies against mouse and human idiotypes, but also as a general method for production of monoclonal antibodies to a variety of foreign proteins and cells. The procedure involves local subcutaneous immunizations, followed by fusion of the lymph node cells obtained from nodes draining the sites of immunization. The protocol for immunization consists of 4-5 50 t~l injections at 3 day intervals. Generally, 1-100/~g of purified antibody is injected per mouse, distributed in six subcutaneous sites: rear foot pads, inguinal regions and subaxillary regions. The first immunization consists of the appropriate amount of antibody in PBS emulsified with an equal volume of CFA (Difco). The next 3-4 immunizations consisting of the same amount of antigen in 50 ~1 of physiological saline administered into the same sites. One day after the last immunization the draining lymph nodes are removed. Single cell suspensions are made by teasing the lymph nodes with fine forceps, and the cells obtained are fused with a
[10]
ANTUDIOTYP1C ANTIBODIES
185
non-secreting myeloma such as Ag8.653 (obtainable from American Type Culture Collection) by methods previously described. 31,32 This protocol has worked successfully for constructing hybridomas to both mouse and human immunoglobulin idiotypes. 33 Hybridoma antibodies have also been prepared to idiotypes expressed by surface antibody of human B cell neoplasms. This has been done by injection of the B lymphoma cells themselves or by injecting antibody produced by cell lines obtained by fusing lymphomas with nonsecreting plasmacytoma lines. 34,35
Screening for Monoclonal Antiidiotypic Antibodies Enzyme-Linked Immunoabsorbent Assay (ELISA). The details of the ELISA method have been presented in another chapter [8] in this volume and will not be duplicated here. The advantages of an ELISA assay over a SPRIA are (1) the stability of the conjugated protein (some have been used for 5-6 years without significant loss of stability). (2) The lack of necessity to take special safety precautions because of the non-radioactive nature of the marker used. (3) The possibility of using lower cost equipment for accurate quantitation of the assay. Isologous or Allologous Antiidiotype Antibodies. Isologous or allologous antiidiotypic antibodies are made by immunizing mice with mouse antibodies obtained, respectively, from the same or a different inbred line as the injected mouse. In the particular modification developed to screen for culture wells containing isologous or allologous (mouse anti-mouse) antiidiotypic antibodies, the microtitration plate is first coated with the proband antibody, and then blocked with 1% BSA, prior to incubation with tissue culture supernatants suspected of containing antiidiotypic antibody. The plate is then washed in borate buffered saline and incubated with a mixture of several antiisotype antibodies that detect all mouse isotypes except the light and heavy chain isotype of the proband antibody. Finally, the plate is incubated with an appropriate substrate. The assay system will detect any antibody in the tissue culture supernatant that reacts with the proband antibody whether it reacts with constant 31 J. F. Kearney, A. Radbruch, B. Liesegang, and K. Rajewsky, J. lmmunol. 123, 1548 (1979). 32 R. Stohrer, M. C. Lee, and J. F. Kearney, J. lrnmunol. 131, 1375 (1983). 33 H. Kubagawa, M. Mayumi, J. F. Kearney, and M. D. Cooper, J. Exp. Med. 156, 1010 (1982). 34 M. Mayumi, H. Kubagawa, G. A. Omura, W. E. Gathings, J. F. Kearney, and M. D. Cooper, J. Immunol. 129, 904 (1982). 35 K. Thielemans, D. G. Maloney, T. Meeker, J. Fujimoto, C. Doss, R. A. Warnke, J. Bindl, J. Gralow, R. A. Miller, and R. Levy, J. lmmunol. 133, 495 (1984).
186
SERUMIMMUNOGLOBULINS
[10]
region, or with idiotypic determinants. 36 To determine whether or not the antibody in the supernatant is reacting with constant or variable region determinants, the same supernatant is also added to microtitration wells coated with different members of a panel of mouse monoclonal antibodies that have a different specificity than the proband antibody but show the same light and heavy chain isotypes as the proband antibodies. Those monoclonal antibodies that react only with the proband antibody are idiotypically specific for the V region. Once a putative antiidiotypic antibody is detected by this procedure it is recloned and retested by the same assay. Xenogeneic Antiidiotype Antibodies. The detection of mouse antiidiotype antibodies specific for xenogeneic idiotypic determinants (e.g., rat or human) is more straightforward. As above, the purified proband antibody is coated directly onto a microtitration plate. Supernatants are then added and any mouse antibodies binding the proband antibody are detected by enzyme-labeled anti-mouse Ig which may have to be previously absorbed with rat or human immunoglobulins in order to prevent possible cross-reaction with the proband rat or human immunoglobulin used to coat the plate. Detection of Antiidiotypic Antibodies to Antibody Producing MouseHuman A L L Heterohybrids. When it is not feasible to obtain purified immunoglobulin as antigen, other procedures for idiotypic detection are required. These approaches can be illustrated by the procedures used to screen for monoclonal antiidiotypic antibodies prepared against idiotypic determinant of immunoglobulins on human acute lymphocytic leukemia (ALL) cells. 34,35 ALL-derived immunoglobulin in the supernatants of mouse-human A L L heterohybrids can be successfully coated to wells that are first coated with anti-human Ig and then adding the heterohybrid supernatant followed by anti-Id supernatant. The assay is then completed by the addition of supernatants thought to contain antiidiotype and finally enzyme-labeled anti-mouse Ig 34 as in the above procedures. Detection of Monoclonal Antibodies Bearing Common ldiotopes. Once a monoclonal antiidiotypic antibody is obtained, it can then be used to screen for the production of other antibodies carrying the same idiotope as that detected on the proband antibody. This technique can be carried out in a variety of ways. In one modification, antibodies to be tested are added to a microtitration plate coated with the monoclonal antiidiotype. As a final step the plate is incubated with AP-conjugated antibody reactive with the isotype of the test antibody. It is important to 36 J. F. Kearney, in "Hybridomas and Monoclonal Antibodies" (W. E. Paul, eds.), p. 751. Raven Press, New York, 1984.
[10]
ANTIIDIOTYPICANTIBODIES
187
note that this procedure cannot be used to assay test mouse antibodies of the same isotype as that of the mouse antiidiotypic antibody. Another approach that can be used is to coat the plates with an antigen that is bound by the test antibodies. The plates are blocked with 1% BSA prior to being incubated with the test antibodies. Next the plates are reacted with AP-conjugated antiidiotype. In an alternative version of this procedure, unconjugated antiidiotype is used and a final layer of APantiisotype that binds to the isotype of the antiidiotype, but not that of the test antigen, is added. All of these ELISA assays are extremely useful in the production and initial screening of hybridoma antibodies, since they allow a large number of different tissue culture supernatants to be examined quickly without waiting for time consuming gamma counting or autoradiography.
lmmunofluorescent Studies The details of the immunofluorescence method have been presented in another chapter [9] in this volume and will not be duplicated here. In this paragraph only the general approach to the study of anti-idiotypic antibodies by immunofluorescence will be discussed. When a population of antibodies shows reactivity in an idiotypic assay such as any of those described above, it is impossible to distinguish between the possibility that a low concentration of antibody is strongly reactive with the antiidiotypic antibodies, or that a high concentration of antibodies are weakly cross-reactive with the antiidiotypic antibodies. A more satisfactory approach to studying antibody populations is to use antiidiotypic antibodies to examine the idiotype of the antibody produced by individual B cells or plasma cells since each cell produces antibody at a single site. This can be done by using the antiidiotypic antibodies to either inhibit antigen specific plaque-forming cells in the Jerne plaque a s s a y 37 o r for immunofluorescence staining of B cells and plasma cells.15 In the case of immunofluorescence, lymphocytes are cytocentrifuged onto glass slides and processed for either surface staining (to detect B cells) or cytoplasmic staining (to detect plasma cells). Next, monoclonal antiidiotypic antibodies conjugated with a fluorochrome are added. 32 In a modification of the procedure the antiidiotypic antibodies themselves are unconjugated and the slides are subsequently stained with fluorescein or rhodamine-conjugated antibodies directed against the constant region of the anti-idiotypic antibodies. It is important in this case that the conjugated antibodies not react with any of the antibodies of the species that 37 j. F. Kearney, R. Barletta, Z. S. Quan, and J. Quintans, Eur. J. Immunol. 11, 877 (1981).
188
SERUM IMMUNOGLOBULINS
[10]
provided the cells being tested. Since almost all monoclonal antiidiotypic antibodies are of mouse origin, this modification of the procedure is largely limited to an examination of the idiotypy of species other than the mouse. When lymphocyte cell surfaces are stained with a heterologous antiserum, it is likely that antibodies bearing several different idiotypes will be detected. When this happens there is no way to tell which of the stained cells are being detected by which of the antiidiotope antibodies in the immune serum. When hybridoma antibodies are used, all cell bound antibodies detected are reacting with the same monoclonal antibody and are likely to be expressing the same epitopes. However, there are some obvious exceptions to this statement, When the panel of hybridomas reactive with al-3 dextran was screened with panels of monoclonal or heterologous antibody by RIA then it could be shown that specificities of the heterologous anti-Id antibodies reacted more truly with the structurally defined idiotypes than did monoclonals. 38 Immunofluorescence is also a convenient way to check for antiidiotypic antibodies in which the idiotope is associated with certain isotypes. In this method, smears are made from a source of B cells, e.g., spleen or polyclonally activated plasma cells and stained first with a fluorescent antibody to a particular isotype followed by staining with the antiidiotype. In this way it is possible to determine rapidly on a large number of clonally derived plasma cells whether the particular idiotype always appears to be restricted to expression with a particular isotype. 39
Idiotypic Specificity of Monoclonal Antiidiotype Antibodies Once it has been established that a monoclonal antibody is specific for the variable regions of an immunoglobulin molecule, it is often of functional and structural importance to determine in which part of the binding site the idiotope determining structures are situated. This determination cannot be done with any assurance because of (1) the long range effects of certain amino acid groups on an epitope, (2) the structural complexity of an idiotope, and (3) the undetermined role played by heavy and light chains on construction of a given idiotope. However, there are certain useful assays that can be carried out. Binding versus Nonbinding Site Idiotopes. Because the V region of an antibody is a complex structure with the binding site often in the shape of 38 B. Clevinger, J. Thomas, J. Davie, J. Schilling, M. Bond, L. Hood, and J. Kearney, in "Immunological Idiotypes" (C. Janeway, E. Sercarz, and H. Wigzell, eds.), p. 159. Academic Press, New York, 1981. 39 G. Morahan, C. Berek, and J. F. A. P. Miller, Nature (London) 301, 720 (1983).
[10]
ANTIIDIOTYPIC ANTIBODIES
189
a groove or cleft, some antiidiotype antibodies will bind outside of a functional binding site. This can be tested by using an inhibition assay similar to those described above. The proband antibody is bound to a microtitration plate and the monoclonal antiidiotype antibody labeled with a radioisotope or enzyme is then added in the presence of dilutions of inhibitors (haptens) of known reactivity with the proband antibodies. These haptens can be used either by themselves or modified by addition of amino acids or the addition of larger protein carriers. In this way, it is possible to show that a low-molecular-weight hapten such as phosphorylcholine ~7 or 4-hydroxy-3-nitrophenyl-acetyl 3 will inhibit binding by the monoclonal antiidiotype antibody. In this way it can be determined that the idiotope recognized by a given monoclonal antibody is intimately associated with the antigen binding site. On the other hand, if binding can only be inhibited by large molecular weight forms of that antigen then the antibody may be non-binding site related. 17 Furthermore, some antiidiotype antibodies will not be inhibited by antigen in any form: the idiotopes recognized by such antibodies are not associated with the binding site. 4° Light and Heavy Chain Involvement in Formation of an Idiotope. Since idiotypic structures can be determined by light and heavy chain interactions or by light or heavy chain alone, it is possible to determine by direct binding to purified light or heavy chain from the immunizing immunoglobulin which chain expresses an idiotope. However, a more precise method is provided by the use of light and heavy chain recombinant molecules in inhibition assays to determine whether a particular idiotope always associates with one heavy or light chain V region. 41 Most monoclonal antibodies appear to be directed against idiotypes dependent on light plus heavy chains. 32 Although such studies give information on idiotope-Ig chain and/or binding site associations, it is difficult to establish the exact residues that are involved in the formation of an idiotope which can only be determined by X-ray crystallographic analysis of idiotype antiidiotype antibody complexes.
40 M. K. Wittner, M. A. Bach, and H. Kohler, J. Exp. Med. 128, 595 (1982). 41 E. Pillemer and I. L. Weissman, J. Exp. Med. 153, 1068 (1981).