Reactions of chicken antibodies with immunoglobulins of mouse serum and T cells

,m,,,uno~h‘,n,rir?. Vol. IS. pp 615-622. Prmed ,n Great ,, Pcrpamon Pre,, Ltd 197X

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REACTIONS OF CHICKEN ANTIBODIES WITH IMMUNOGLOBULINS OF MOUSE SERUM AND T CELLS GREGORY

W. WARR, GABRIELLE MARTON, and JOHN J. MARCHALONIS

Cancer

ALEX

SZENBERG*

Biolof) Program. NC1 Frederick Cancer Research Center. P.0 BOY B, Frederick. MD 21701, U.S.A.

C‘hlcken antibodiesraiwd to the (Fab’), fragment ormouse I&i recogniredeterminant\csprc\scd Abstract Ott both normal moue serum and T-cell-associated immunoplohulin. as assessed by immune dit’fuslon and competition r;ldioimmunonssaS. Analysts of reactions aith both normal and myeloma Immunoflohulins Indwatc that chicken anti-(Fah’)2 recognizes determinants expressed on light and hea\) (Fd l’ragmcnt) chains ol’moux 1gG and IeM. The relativeI) low efficiency ofreaction ofpurified ;‘ ha\> and light chains (compared to Intact l$i) with the anti-(Fab’)z suggests that these antibodies recognirre detcrminanls cypressed only v.hen heavy and light chains arc combined in the nattve molecule. Scrolo$cal studies \rith T-cell-associated immunoglohulin shou that thi\ molecule exprcwa determinnnt~ cross-rcacti\c with both light and heavy immunoglohulin polqpeptldc chains. The nntigenlc drtermlnanta wen on the T-cell immunoplobulin lleht and hen\ y chain\ are common to IgM and I&i and arc not 1\otqpe \pcc1fic.

and T cells in mammals do not usually bear membrane immunoglobulin (Ig) determinants demonstrable by the use of antiglobulin reagents raised in other mammalian species. However, such antibodies have been used successfully to isolate or demonstrate surface Ig in solubilized extracts from thymocytes or T cells of rat, man. mouse and pig (Chavin, 1974: Cone & Brown, 1976; Gabison et al., 1977: Haustein & Goding. 197.5; Misra et al.. 1976: Moroz & Hahn. 1973: Marchalonis. 1975; Rieber & Reithmiiller, 1974) and there have been occasional reports of the identification of membrane Ig on T cells ii1 .SiiU using antiglobulin reagents raised in mammalian species (HCmmerling & Rajewsky. 1971; Whiteside & Rabin, 1976: Santana et al.. 1974). The observation that mammalian antisera can be used to demonstrate Ig on the membrane of thymocytes and putative T cells in lower vertebrates (DuPasquier 41 al., 1972; Ellis & Parkhouse, 1975: Emmrich ef al.. 1975; Clemerrrl.. 1977: WarrPtal., 1976: Warr&Marchalonis, 197X) suggests that the antiglobulin reagents raised in phylogenetically distant species can recognize exposed determinants of T-cell-membrane Ig. In support of this hypothesis are the observations that chicken antibodies to rodent and human Igscan readily be used in direct binding assays to demonstrate T-cellmembrane Igs in these species (Jones et u/., 1976; Hiimmerling (‘I u/.. 1976~; Szenberg er al.. 1977). We, therefore, felt it important to determine the nature of the antigenic determinants, on both serum and T-cell-surface Igs, recognized by chicken antisera Thymocytes

to murine Igs, particularly in the light of the demonstration of V,(idiotype-bearing) regions of the Ig heavy chain on T cells (Rajewsky & Eichmann. 1977) and the reported absence of light chain markers (Binz & Wigzell, 1976). We report here that chicken antisera to the (Fab’), fragment of mouse IgG recognize determinants on T-cell Ig tha! are crossreactive with both light and heavy Ig chains.

Mouse IeG (predominantly I#3 2a and I& 2h) ma\ prepared from mouse serum bq btnding to protein A 01 .src/ph~~locclr.v N,,W,,., Immobilized to Sepharose-4B (Pharmacla. Uppsala. Sucden) at 5 mg ml packed srl and eluted by 0.5X”,, (\’ I ) acetic acid. 0.15 .\I sodium chloride (Coding. 1976). Thih preparation was free ofcontaminatton by IgM as judged hb immune dit’t’uvon afaln\t /~-‘;pcc~tic antiserum.

me ml. Into acetalte buffer. pH 4.5 (0.2 ‘2-I) and difeqcd \\lth IO me pepsin .C IeG (C‘nlhiochem. CA) for IX hr at 37 C‘ (Edelmnn & fiarchalonls. 1967). The reaction \\;Ls stopped bj diolqsis againat Tris-bufl’cred saline (TBS). pH 8.0 [O. I5 21 sodium chloride. 0.05 .&! Tris-(hydro~~meth~l) amino methane]. and undigested IgG and Fc l’rngments aerc adsorbed by further paasage O\CI- protein-A Sepharo\e (Coding. 1976). The purit] ofthc mouse IgG and the (Fab’), fragments thereof uas contirmed b> pol>ncrylamidc sei electrophoresib (Laemmli. 1970) in sodium dodccyl \ult’atccontaininp but’fers (SDS-PAGE).

Pooled normal mouse serum was fractionated b) lone electrophoresis on starch fblloued by eel tiltratIon on Sepharose-6B. The resulting preparation IfM was judged to he > 95”,, pure h) SDS-PAGE and contaminant\

x Present address: The Walter and Elira Hall Institute of MedIcal Research. P.O. Royal Melbourne Hospital. Victoria 3050. Australia.

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615

61 h

GREGORY

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WARR

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mlfrating with the mobility of ;’ heavy chains were not dctcctahlc. In addition. contamination with I@ and IgA was e\cludcd by immune diffusion againat class specitic antiscra (LItton Bionctic\. Inc. Kensington. MD).

This M;~S isolated from spent cdturc llud in which the cont~nuouJ> cultul-cd monoclonal T lymphoma WEHI 22 had been groan c‘ulturc Iluid wa centrifuged (lO.OOOp for 30 mm) and pussed slouIy (less than 5 ml hr) over an immtlnct;tdsorhc~~t prepared from purified chicken nntimouse igci iFah’), (Szenhcrg vr cii.. 1977) coupled to Scph:trosc4R by cq;mogcn hromrde xti\ation (March (‘r ol.. 1974) at a concentration of 5 mg:ml packed gel. Bound matcr1al M~S ciutcd with ~lycme-HC‘I. pH 1.5 dialyzed into TBS containing O.OO”,, mcrthiolatc and stored at 4 C. No Ig could hc obtained 111control cstrxtions ofculturc medium m WhICIl no T Iymphoma cells had been grown. (‘h;tractcl-i/~ltion of the Ig produced and chcd by WEHI 2, anti trccogni/ed by rahhit or chicken antisera to mouse Ig, has been documcntcd clscuhcrc(H;tustcln cl of., 1975: Moselq <‘I 4.. 1Y77: Vlarchalonis t’l l/i.. lY77). The material present m

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Reactions

of Chicken

Antibodies

10 mg of pooled normal mouse IgG (isolated as described nhow) at 5 mg ml in 0 IS M sodtum chloride were Incubated for 2 hr at 37 C with S”,, (v \) L-mcrcaptoethanol. and then made 1 hl in propionIc acid. After centrifugation (10.000 R for 20 mln) the sample uas separated by gel diffusion on Gacid (Edelman & 200 Sephadex in I :Cf propionic hlarchalonis. 1967). using a 100 x I.h-cm column and a (low-rate ol‘approx 6 ml hr. The reduced IgG resolved into 3 peaks (tdclman bi Marchalonis. 1967). Each peak was dialyred against TBS. concentrated by pressure dialysis and then analyzed b) SDS-PAGE under reducing condttlons. The first two peak5 conrtstcd of heavy chains and the third con\istcd of Ilght chains (Edclman & Marchalonis. 1967).

Antigen\ uerc radiolodinated Mith “‘I either by the chloraminc T (Hunter & Greenwood. 1962) or lactoperosidax (Marchalonis. 1969) procedures. The ability ot ;innsera to precipitate .500~10.000 counts min of labelled antigen wa\ determined as follows. Antlbody preparations wrc scl-lally diluted (?-fold). tn a working volume of 200 ~tl of dlluent. 11, IO x 75 mm round-bottom glass test tuba Dllucnt conalhted o1 TBS plub O.Ol”,, Triton X-100 ((‘albiochcm) plus 6 j(g ml of foul IgY prepared from H pool of normal chicken serum A known amount of radiolabelled antigen %:I\ added to each tube. mixed. incubated for I hr at 37 C. and I hr at 4 c‘and then goat antl-fowl IgY serum was added. in an amount predetermined to be capable of prcclpitatm~ > 90”,, of the foul IgY present in the rexnon mlxturc. After further Incubations nt 37 C for l-2 hr. and o\crnight at 4 ( the rcaction mixtures were diluted to approx~matelq 2.5 ml with TBS containing ().()I”,, Triton XIOO. and ccntrll’uged for 20 min at SO00 g at 3 C‘. The hupcrnatant W;I\ aspirated. and the radioactlbity in the precipltatc was determtncd using :I Searle well-type automatic filliim;l-\cintillation spectrometer. The amount of untlbody capable of prcclpitating So”,, of the labelled antigen ~a used an the competition aaaa)s. Nhich werecarried out as t’ollow~ Serial ?-fold dilutions of unlabelled antigens wcrc made in a volume of 200 /II of diluent: dilution antibody was added to each. at ;I concentration determined as described aboLe. After incubation for I hr at 37 C and I hr at 4 C. the rxdlolabclled antiscn was added. Subsequent Incubation and addition ofgoat anti-fowl IgY serum. and preparation of the prcclpitatc lx assa) wcrc exactI> as described above. Approprlatc controls were carried out to determine the dcgrce 01‘ nowapccilic adhcrcncc of radiolabelled antigen to the gla\\ tube5 and to ensure that ncithcr the normal fowl IgY pt-e\cnt in the asp> as carrier. nor the goat anti-fowl IgY wrum. wrc capable of Ircacting uith and preapitating the labclled antqenr. :1,,,,.vwr, White 20-week-old rpccific pathogen-free male chlckens acre purchased from SPAPAS (Storrs. CT). After a S-IO ml prebleed had been taken I’rom a bing vein. they were immuni/cd with the following antigens: mouse (FAB’)_ tlcrlbcd from IgG. mou~c kappa chain (MOPC 41) and moux lambda chain (RPC 20). Animals were given a prmarq subcutaneous inoculum of antigen emulsified in complete Freund‘s adjutant ( I.1 ). and were boosted 2 weeks later with an intrapcritonenl injection ofantigen in saline. 11 ncccasary. ;I wcond boost WH\ pILen 2 weeks later. consisting 01‘ antigen cmulsllied in incomplete Freund’s adjuvant, administered intramuxulnrlq. The dosages were: 2 mg per inoculum for (Fab;) and 1 mg per inoculum for kappa and lambda chatnb. Between 5 and 20 ml of blood were taken from a wing vein ofeach chicken every 2-3 weeks. Serum was separated. and as won as possible precipitated once with l8”,, (WV) sodium alfate (Benedict, 1967); the redissolved precipitate was then pt-ccipitated with I4”,, (w v) sodium sulfate. This last step was

617

with Immunoglohul~ns

repeated. and the precipitate rcdissolvcd in TBS. centrlfugcd at 12.000 g for 20 mm. and fractionated by gel tiltratton on Sepharow6B in TBS. The 100 x 2.5.~cm column \\a\ calibrated Nith proteins of known six. The ma,lmum amount of protein loaded was SO mg. and the column \&it\ run at approx IS ml;hr. The peak corresponding in c’IuIIon position to monomeric Ig uas taken. concentt-atcd b! pressure dialysis. portioned and stored at -20 C until uwci. Normal fowl IgY prepared in this ua) from the wrum 01‘ unimmunized birds ~215 judged to be > 90”,, pu~c ;I\ determined by SDS-PAGE analysis. and WI\ uwd both 111 radioimmunoassay diluent and to immunize :I feat. r\ I -Jarold female goat kept in the Animal Production Arca o1’thc Frederxk Cancer Research Center was immunwd at multiple sites (intramuscularly and suhcutancou\l>) \+lth 5 mg of fowt IgY emulsified in complete Frcund‘s adjutant and boated 3 weeks later ulth the same amount ofimmuno~cn in Incomplete Freund’\ ad.ju\ant. Thercaftcr. the goat wa blctl 100-200 ml from the jugular \cin at 2. to-i-\lccl\ intcr\al\ and the prccipltating tltcr 01. the xrum I’or li)\\l 121’ \\;I\ detrrmmed for use 111radlolmmilnou\\a!

These were carried out in agarose tn barbital buffer. pH X.6 (0.05 M). in the presence of 1.0 :1-/ wdium chloride l’ot chicken antIbodies and in the absence of added xxilum chloride for analyses using rabbit antiwra.

KESl LT‘S

The reactions of chicken antibodies to mouse (Fab’),, kappa and lambda chains were investigated in terms of their ability to precipitate known antigens. We have used both gel diffusion analyses and radioimmune precipitation titration assays. Figure 2 shows a typical result of the immune diffusion reactions of anti-(Fab’), against a variety of mouse Igs: normal IgG and IgM, and TEPC 15 (KZ) and MOPC 104E (@) myeloma proteins. All four antigens showed clear precipitation lines; both TEPC I5 and MOPC l04E showed incomplete cross-reactivity (deficient) with respect to IgG. Similarly, MOPC l04E is deficient in its cross-reaction with normal IgM. Normal serum IgM and IgG showed a reaction of complete identity with the anti-(Fab’)z (Table I ). In addition, all the detectable reactivity of the chicken anti-(Fab’), antibodies to IgG could be removed by adsorption with normal pooled mouse IgM bound to extensive non-isolype Sepharose 4B, suggesting specific cross-reactions in the Fd region of the heavy chain. This antiserum did not show a precipitin line with the i. myeloma RPC 20, but did react with MOPC 315 (ri). suggesting again a heavy chain Fd crossreaction. The results of a more comprehensive analysis of the reactions of anti-(Fab’), serum undertaken by immune diffusion are shown in Table 1. These results (Table I) demonstrate that the antirecognized both heavy and light chain (Fab’), determinants on mouse Igs. For example, kappa chains (MOPC 41) showed reactions of partial identity with both IgM and IgG. and a reaction of non-identity with MOPC l04E (@). It was not possible to answer the question of whether or not these heavy-chain determinants w’ere in the V, or CH regions. Radioimmune precipitation reactions of the anti-(Fab’)Z.

titration of the anti-x and anti-i

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antibodies (Fig. 3) showed essentially the same patterns of activity as observed in immune diffusion analysis. However. these results (Fig. 3) again demonstrate that these chicken antibodies show varying degrees of cross-reactivity. which are particularly clear in the case of anti-light chain antibodies. For example. while the anti-ii precipitated k--chain more efficiently than i-chain. and vice versa for the anti-i antibody (Fig. 3). the reactions of the anti-r; witli MOPC‘ lO4E (/.;I) and of tlic anti-i with normal IgM and (Fab’), (Fig. 3) are most reasonably explained on the basis of some cross-reaction between K and i chains. To assess in a quantitative manner the degree of cross-reaction between Igs as detected by the anti-(Fab’), antibodies, we performed competition radioimmunoassays. Figure 4 shows the inhibition of precipitation of iZ5 I-labelled (Fab’)? with chicken anti-(Fab’), antibody assayed using homologous antigen [(Fab’)2], normal mouse IgM. and a variety of myeloma proteins. In addition. normal rabbit IgG. which reacts with the anti-(Fab’), was tested in this experiment. It can be seen that none of the myelomas inhibited precipitation to a greater extent than 3@-40”,,. On the other hand. all the myelomas tested

Serum IgM

Serum I@(;

WARR <‘Ir/i. showed some inhibition, at between 100 and 1000 times lower efficiency than that shown by the (Fab’),. It is impossible to exclude minor contamination with other Igs as a cause of this inhibition. The normal mouse IgM showed significant inhibition. tending towards completion. but with approximately %-fold lower efficiency than the (Fab’)2. It should be noted that the slope of inhibition obtained for the IgM was significantly different from that for the (Fab’),. suggesting that contamination with IgG was not responsible for the observed reaction, and that the chicken anti (Fab’)2 does not react in an identical fashion with 11and ;’ chain Fd regions. The inhibition curve obtained with normal mouse IgG was indistinguishable from that seen with the (Fab’)? fragments derived from the same preparation (data not shown). The rabbit IgG showed an interesting pattern of inhibition. which rose to 50”,, with the same efficiency as mouse (Fab’):, but plateaued at this (50”,,) level (Fig. 4). The most reasonable explanation of this result is that lagomorph (rabbit) IgG shares many (about half) of the antigenic determinants expressed on mouse IgG, as seen by the chicken antimouse (Fab’):. The question of whether or not the anti-(Fab’): antibodies recognized heavy or light chain determinants on mouse Igs was approached by determining the degree to vvhich isolated ;’ and light chains could inhibit the precipitation 01 L2’I-labelled (Fab’), by the anti-(Fab’), antibody. As shown in Fig. 5. it-can be seen that both ;’ and light chains derived from normal mouse IgG inhibited precipitation of (Fab’): to a signilicant degree. Tl~e efticiency ol‘inliibition by ;’ and light chains was approximately equal on a molar basis. However, intact IgG was a much more potent Inhibitor of precipitation than either ;’ or light chains (40- to l00-fold difference at 30”,, inhibition). suggesting that the anti-(Fab’)2 recognized either (a) heavy and light chain determinants that are expressed only in the native molecule. or (b) determinant(s) resulting from an interaction between heavy and light chains. Rc~rrc~tior~vof c,hic,kcrr tm/ihoclir.\ n.irh mm~.\~ T-wll

Ig

We were delighted that sufficient T-cell Ig was to allow the detection of a visible precipitin

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Reactions of Chicken

I@

6

Antibodies

619

with Immunoglobulins

RPC 20 (A) 1

207

25 OI IgG (Fab’)2

MOPC 104E (Au)

MOPC 41 (XL

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Fg Globulin Fig. 3. Radioimmune precipitation analysis of the reactions ofchicken rtnti-(Fah’)l. anti-r; and nnti-i sera with rL’I-labelled mouse tmmunoglobulins. The globulin fractions ofeach serum werediluted ;I\ shoun. snd the preciptuttion rewtron was carried out as described tn Materials and Methods

albeit a weak one. The ability of the anti(Fab’):, to precipitate T-cell Ig in immune diffusion analysis is shown in Fig. 6. This figure also clearly shows that the anti-(Fab’), did not recognize components present in fetal calf serum, and that normal chicken globulin did not react with T-cell lg. The ability of chicken antibodies to mouse Igs to react with ’ z5I-labelled T-cell Ig was also routinely tested by radioimmune precipitation titrations. Preparations of antibody from five chickens immunized with (Fab’), and two immunized with kappa chain all showed the ability to precipitate T-cell Ig to a maximum of between 20 and SO”,, of total acid-precipitable radioactivity. Three of these titrations are shown in Fig. 7. reaction,

The antigenic cross-reactions of T-cell Ig wsere investigated by radioimmune competition assay, in which the precipitation of lZ 51-labelled T-cell Ig was inhibited with various normal and monoclonal Igs of mouse origin, The results of one such experiment are shown in Fig. 8. The homologous antigen (IgG) and normal serum IgM inhibited precipitation virtually to IfJOY,,and although the slopes of the inhibition curves were different. their efficiency of inhibition was similar (cf. results with (Fab’),, Fig. 4). Of the myelomas tested, RPCZO (j.)_ MOPC 41 (K) and MOPC 104E (Lb<) showed poor inhibition, rising no higher than 25”,,. while MOPC 315 (;.a) showed inhibition rising to W,,. but of 30- to 60-fold lower efficiency than that of IgG or IgM at the same level of inhibition (Fig. 8).

gg of Globulin

Fig. 4. Competition radinlntmunoassa~, using purified inlmuno~iobulins to inhibit the precipitation of “‘I-lebelled mouse I@ (Fah’)z frapmcnts by chicken anti-(Fah’),. These titrations were cart-ted out as described in Matertals and Methods. The concentratton of inhibiting immunoglobulin t)(e) is shoun on the abscissa.

pg inhibitor

Fig. 5. Competition radioimmunoas\~t). using uhole mouse I@ or purified heavy t;,) and light (L) polypeptide chwns to tnhrhit the precipitatton 01 “‘I-labelled mouse IgG (Fah’), fragments by chicken :mti-(Fab’),.

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Fig. X. Competition radlolmmunoassaq u\,ng put-liid mouse immunoglobulins to Inhibit the precipitation 01‘ 11’1labelled T-ccl1 ~mmunoglobulin by chicken antl-( t.ah I~

Although this experiment clearly demonstrated the sharing of antigenic determinants between mouse serum Igs and the T-cell Ig, it did not allow the localization of these shared determinants to the heavy or light chain. This problem was investigated by testing the ability of purified mouse :’ and light chains to inhibit the precipitation of ’ 251-labelled T-cell Ig by the chicken anti-(Fab’)2 (Fig. 9). These results clearly demonstrate that the chicken anti-(Fab’)? antibody recognized light and heavy chain crossreactive determinants on T-cell lg. As was the case for inhibition of precipitation of (Fab’), by this antibody, purified ;’ and light chams are much less efficient than intact IgG in inhibiting the precipitation of T-cell Ig, again suggesting that the anti-( Fab’), may recognize determinants expressed only when the heavy and light chains are in their native conformation and/or combination. At the 30”,, inhibition level purified light chain is I; 10th as effective as intact IgG. a degree which cannot be explained by the observed trace contamination with ;’ chain. ;’ chain was 1/40th

30 20 lo-

pg Inhibitor 9. Competition r~~dioimrn~lnons\~~~ u51n.g M hole mouse IgG or the lsolatqi heavy (;,) and light (L) polypeptlde chain\ to inhibit the prcapitation ot ‘: il-labelled T-cell imlilunoglobulin b\i chicken antl-( Fab’) ,. Fig.

effective as intact IgG. The fact that n&her inhibition goes to completion also argues against the possibility that the observed inhibition results from contamination of the chain preparations with IgG. Since the chicken anti-(Fab’), antibody can recognize T-cell Ig, the question of whether or not such T-cell Ig was identical with (Fab’), was investigated. A gel immunodiffusion analysis (Fig. 10) demonstrated that T-cell Ig is antigenically deficient (i.e. did not show a reaction of identity) with mouse IgG. as

0

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Fig. 7. Analyst\ b) rndIolmmunc PI-wpltatlon 01 Ihe reactions of t-o chlckcn anti-(Fab’), preparationa (328 and 3 IX) and one chicken antl-~, (MOPC‘ 41) preparation wth “‘I-labellcd T-cell ~mm~lnoflobulin. LIdail\ 01‘ the renction

Reactions of

Chxken

Antibodies

DISCLSSIO\

The present results confirm in a direct imthat manner reports munochemical chicken antiglobulin antibodiescan detect mammalian T-cell Ig (Jones rt (I/., 1976: Hammerling er al., 1976~; Szenberg et ul.. 1977: F. Loor, personal communication) and in addition (I) document some of the reactivities ofchicken antibodies to mouse lg. and (2) demonstrate serological cross-reaction between murinc T-cell-derived Ig and both heavy and light chains of serum lg. The reactions by the chicken antibodies to (Fab’): of mouse serum Igs merit comment in terms of the Clearly. determinants observed cross-reactions. common to ti and i light chains are recognized and crossreactions between heavy chains also occur. While serum IgG and IgM showed apparent reactions of complete identity as assessed by immune diffusion analysis and adsorption experiment, the RIA data (Fig. 4) suggest that these molecules did not react in an identical manner with thechicken anti (Fab’)>. and we cannot completely exclude the possibility that reactivities specific to the ;’ chain are also present at a low level. It has not proved possible, from this study, to conclude whether these antibodies recognize determinants common to heavy and light chains. A number of problems arise in interpreting all serological results and should be considered here. One is the presence of impurities in the immunizing antigen. especially at the < I”,, level. which is very difficult to exclude. The problem is acute when trying to assess the signiticance of inhibition by myeloma proteins in competition radioimmunoassay, particularly when they show I OO- to IOOO-fold less efficient inhibition than that seen with pooled normal Igs. A reactions with second problem in interpreting mycloma proteins is that the chicken antibodies may be recognizing determinants specified by (pooled) V,,. V,>and V,, in the immumzing antigen (e.g. (Fab’)2 from normal IgG). Because monoclonal myeloma proteins might only express one or a few ofthese determinants, the significance of low levels of inhibition is difficult to assess. Results of specificity studies using chicken antibodies raised against whole myeloma proteins suggest that the reagents show considerable reactivity against isotypic determinants of the immunizing protein. Suitable absorptions should. theretore. render them class-specific. On the basis of the presented data, we conclude that chicken antibodies to mouse IgG (Fab’)2 fragments recognize the following determinants on serum Igs. (I) Determinants expressed by ti and i light chains, although anti-i reactivity is weak relative to the reactivity with K chain determinants. It is not known whether these are localized at the V or C regions. (2) Determinants expressed on the Fd fragment of the ;’ chain. and which cross-react with determinants on other heavy chain classes (as demonstrated here most clearly for /1 chain). It is not known whether these determinants are expressed on V, or Cn, and the site of their cross-reaction on the IgM molecule has not been determined. (3) Determinants expressed most efficiently in the native Ig molecule. It is not known whether these determinants are present on individual heavy or light

with Immunoglobulins

62

I

chains, but clearly the interaction of these chains in the native molecule leads to their most efficient expression. Chicken antibodies to mouse Ig possess the interesting property of being able to recognize Ig in siru on the T-cell membrane (Jones rt ul.. 1976: Hammerling et al., 1976~; Szenberg r~ ul.. 1977). presumably because they react with determinants not detected by most anti-mouse globulin reagents raised in other mammalian species. However, the precise structure of the antigenic determinants which chicken antibodies recognize on mouse Igs remains unknown. The most reasonable presumption is that they reside in the polypeptide portion of the molecule, rather than in the carbohydrate moiety. The reasons for this are ( I) there is little if any carbohydrate on the (Fab’), portion of mouse IgG (Fougereau 01 trl.. 1976). (2) Antibodies to the purified K chain myeloma protein MOPC 41 are reactive with mouse serum (and T-cell derived) Igs. MOPC 41 has been completely sequenced (Gray ct (11.. lY67) and it is carbohydrate-free. (3) Myeloma proteins such as MOPC l04E. MOPC 3 I5 and TEPC I5 are poor inhibitors of the reactions, as compared with normal Igs. These myelomas are heavily glycosylated. but whereas the protein portion of the molecules is monoclonal, the glycosyl moieties should still show the microheterogeneity characteristic of all glycoproteins (Spiro. 1973). In addition. absorption of the chicken anti-(Fab’)? with a nonlymphoid alloantigen-bearing cell (the in I’;/,‘() grown U.V. irradiation-induced fibrosarcoma I 12) did not diminish the capacity of this antibody to precipitate “‘I-labelled T-cell lg. suggesting that cross-reactions with murine cell membrane alloantigens were not involved. This absorption coupled with the inhibition data using purified light and heavy chains and the lack resembling of precipitation of components alloantigens by the chicken antisera militate against the possibility that the reaction with T-cell Ig represents a cross-reaction with a non-Ig component such as /j2 microglobulin (Gottlieb (lt (I/.. 1977). Further evidence that chicken antibodies to mouse (Fab’), bind to Fab-region polypeptide determinants comes from the observation that chicken anti-Fab. like anti-idiotypic antibody. will block binding 01‘ the arsonate hapten by idiotype-bearing murine antibodies to this hapten (Warr G. W. & Marchalonis J. J., unpublished observation). Definitive answers to the nature of the antigenic determinants on T-cell Ig require detailed chemical analyses of the molecules. Such studies are in progress and the results will be given in detail elsewhere (Moseley c’/ t/l.. in preparation). The results presented here also clearly demonstrate the serological cross-reactivity of T-cell-derived Ig with mouse serum Ig. and furthermore demonstrate the expression of both heavy and light chain crossreactive determinants on T-cell Ig. The T-cell-derived Ig is antigenically deficient with respect to IgG as seen by chicken anti-(Fab’)2. whereas serum IgM and Igcl did show reactions of identity. These serological demonstrations of heavy and light chain determinants on T-cell Ig confirm numerous other reports that Tcell Ig is comprised of both heavy and light chains. as assessed by both biochemical and serological methods (Hammerling er UI.. 1976u.h; Warner. I Y74:

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GREGORY

Marchalonis~ 1975. 1976, 1977; Marchalonis & Warr, 1978: Cone. 1976; Misra et al.. 1976; Gabison rt al., 1977; Moroz & Lahat, 1974; Boylston & Mowbray, 1974). Current information suggests that murine Tcell-associated Ig consists of heavy chains similar to, but lacking, the strict isotypic determinants of bl chains in non-covalent association with x-like light chains (Moseley rt al.. 1977; HBmmerling of al., 1976/1; Cone & Janeway, 1977). Although the covalent nature of Tcell Ig can only be resolved unequivocally by chemical methods the chicken antibodies to mouse Ig clearly provide a useful reagent for investigating and isolating T-cell-associated Ig cross-reactive material.

REFERENC‘ES

W. WARR

<‘i al