Immunoglobulins of the Marsupial Setonix brachyurus

Immunology, 1974, 27, 1103.

Immunoglobulins of the Marsupial Setonix brachyurus(quokka)

CHARACTERIZATION OF THREE MAJOR SERUM CLASSES,IgG2, IgG1 AND IgM

R. G. BELL, N. R. LYNCH* AND K. J. TURNER

Clinical Immunology Unit, University of Western Australia,Princess Margaret Hospital, Perth, Western Australia, Australia

(Received 21st January 1974; acceptedfor publication 14th May 1974)

Summary. Three immunoglobulin classes, IgG2, IgGI and 1gM, have beenidentified in the serum of the marsupial Setonix brachyurus (quokka). Each of theseclasses has been isolated in pure form and partially characterized physicochemi-cally. These immunoglobulins differed in their electrophoretic mobility, molecularsize, carbohydrate content and in the antigenic determinants of their heavy chains.IgG2 exists in two major subclasses, one of which (IgG2a) was isolated as a homo-geneous preparation. The results of immunoelectrophoretic analyses also suggestthe likely presence of subclasses within the IgG 1 class. This molecular complexityof the quokka humoral immune system is much greater than that of birds and ecto-therms, but is comparable to that shown by eutherian mammals.

INTRODUCTION

Immunoglobulins show an increasing heterogeneity in isotypic class and subclassstructure as one moves from the primitive vertebrates to the more recently evolved mam-mals. All vertebrates thus far examined possess a molecule resembling the mammalianIgM class and this appears to be the only immunoglobulin class in the more primitivegroups such as the Agnathans and most fishes (Marchalonis and Edelman, 1968; Marcha-lonis, 1969, 1971). The Dipnoid fishes represent a further step in this sequence and heretwo immunoglobulin classes are present (Marchalonis, 1969) a feature which this groupshares with amphibians and reptiles (Litman, Frommel, Chartrand, Finstad and Good,1971). It is not until the evolution of homeothermy that the next step to three recognizedheavy chain classes occurs in the birds (Grey, 1967; Leslie and Clem, 1969; Leslie andMartin, 1973). Immunoglobulins of placental (eutherian) mammals have been moreextensively studied and in their case up to five distinct immunoglobulin classes havebeen identified in different species (Grey, 1971; Tabel and Ingram, 1972; Fahey, Wunder-lich and Mishell, 1964a; Johnson and Vaughan, 1967). This group is further charac-terized by the extensive development of subclass specificities within many of the heavy-

* Present address: Children's Asthma Research Institute and Hospital, Denver, Colorado, U.S.A.Correspondence: Dr R. G. Bell, Clinical Immunology Unit, University of Western Australia, Princess Margaret

Hospital, Perth, Western Australia, Australia.

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R. G. Bell, N. R. Lynch and K. J. Turner

chain classes. Thus, in humans, there are four recognized y chain subclasses, two a andtwo ju (Grey, 1971) and subclasses have been defined for many other eutherian species(Fahey, Wunderlich and Mishell, 1964b; Grey, 1971).Although a more extensive immunoglobulin pattern may eventually be defined for

non-mammalian vertebrates, it has been suggested that an extensive radiation ofimmuno-globulin subclass types within eutherians occurred in the early Cenozoic period (Grey,1971). If this is true, there is no reason to expect that birds, who branched off from thereptilian ancestral stock much earlier than mammals, would necessarily be comparablewith eutherians in either class or subclass heterogeneity. Marsupials (metatheria) arebelieved to have diverged from eutherians in the early Cretaceous (Clemens, 1968) at astage when mammalian immunoglobulin classes and subclasses may not have been muchmore varied than the pattern now found in birds. Studies carried out in the Americanopossum (Rowlands and Dudley, 1968; Morse, Burrell and Fisher, 1971) indicated thatthe immune system and the immune response of this species was significantly more primi-tive than that of eutherians. Only two immunoglobulins, a 7S IgG type, and a 19S IgMtype could be found in this species. However, the American opossum is thought to be aprimitive metatherian (Young, 1962) and, as such, may not be representative of the morerecent metatherians who underwent an extensive radiation during the Cenozoic con-temporaneously with the eutherians (Clemens, 1970).The experiments presented here were designed to investigate whether or not a repre-

sentative (Setonix brachyurus, the quokka) of a more modern metatherian line, the Macro-podidae, showed the same type of primitive immunoglobulin pattern as the opossum,or resembled eutherians in immunoglobulin heterogeneity.

MATERIALS AND METHODS

Experimental animals(a) Quokkas. Quokkas were captured in their native habitat on Rottnest Island and these

animals were subsequently maintained in large open yards. During the progress of experi-ments animals were housed individually in pens 3 x 2-5 metres. Animals were fed ad libitumon oaten chaff containing vitamin and mineral supplements; bread and fresh greens weredistributed twice weekly and water was freely available.Quokkas were bled from the heart when large volumes (10-15 ml) of whole blood were

required or from a superficial leg vein for smaller volumes (1-3 ml).

Separation proceduresGel filtration was performed on tandem Sephadex G-200 (Pharmacia) columns

(2-5 x 95 cm or 50 x 95 cm), which were equilibrated with 0.I1 M Tris/HCl and 0*1 MNaCl, pH 8.0. Samples were dialysed against this buffer prior to application to the columnand separation by the reverse flow procedure. Elution flow rates varied between 10 and15 ml per hour in different experiments, and 5-7-5-ml fractions were collected andanalysed for protein content by measuring their absorption at 280 nm.

Ion exchange column chromatography was performed using either diethylaminoethylcellulose (DEAE) (Whatman DE- 11) or carboxymethyl cellulose (CMC). Whole serum

or a 50 per cent saturated ammonium sulphate precipitate of whole serum, dialysedagainst 0 005 M P04 buffer, pH 7-5 and containing 1 M urea was added to a column ofDEAE equilibrated with the same buffer. Loading ratios varied between 6 and 8 mg of

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Immunoglobulins of the Quokka

protein (as determined by OD reading) per millilitre of packed DEAE. Fractions wereeluted by a batchwise separation procedure using buffers of 0.005, 0-025, 0 05, 0-08, 0.15and 0-4 M P04 containing 1 M urea at pH 7-5. CMC columns were usually used forseparating components of fractions already purified to some extent. The fraction to bechromatographed was dialysed against the starting buffer (0-02 M P04, pH 6.0) andapplied to a column of CMC equilibrated with this buffer. Batchwise elution with P04buffers of molarity 0.05 (pH 6 0), 0-2 (pH 6.4) and 0-8 (pH 7.0) was then carried out.Columns flowed at a rate of 30-50 ml/hour and fractions of 5-7-5 ml were collected andassayed for protein by reading OD 280 nm.Zone electrophoresis was performed with polyvinyl chloride-polyvinyl acetate (Pevikon

C-870), suspended in 0-1 M veronal buffer, pH 8-6, which had been packed in a perspextray (50 x 11 x 1 cm) lined with plastic sheeting. Samples were applied evenly to the sideof a 2-cm segment of Pevikon cut and removed approximately one-third of the way fromthe cathodic end ofthe block. The segment was replaced and electrophoresis carried out for2-3 days at a constant current of40 mA. After electrophoresis was complete 1-cm segmentswere cut from the block and extracted through a sintered glass funnel with veronal buffer.Protein contents were then estimated on each fraction by the Lowry, Rosenbrough,Farr and Randall (1951) modification of the Folin-Ciocalteau technique.

Preparation of antiseraRabbits were immunized in several subcutaneous sites with either 0-1 ml of whole

quokka serum or 100 Mug of purified IgG2 or IgM emulsified in Freund's complete adjuvant(FCA). After 4 weeks, intramuscular booster injections of the same amount of antigen insaline were given. Test bleeds were taken 2 weeks after this and continued until goodtitres were obtained, at which stage the animals were exsanguinated. Rabbit anti-IgMand IgG2 antisera were used only as light chain antisera after absorption with the im-munizing heavy chain class.Guinea pigs were immunized by the injection into the footpads of 0-1-50 pg of purified

quokka immunoglobulins in FCA. Immunized animals were bled weekly from the heart4 weeks after initial challenge. When immunized by this schedule, guinea-pigs were unableto make antibody to quokka light chains. Subsequent experiments indicated a serologicalcross-reaction between goat antisera directed against guinea-pig light chains and quokkaimmunoglobulins, suggesting a significant degree of homology between the light chains ofthese two species.

Affinity chromatographyCoupling of specific antisera to Sepharose 4B (Pharmacia) particles was performed by

the method outlined by Mannick and Stage (1971). Normally an (NH4) 2SO4 precipitateof selected antiserum to a particular immunoglobulin class was coupled to cyanogenbromide (CnBr) activated Sepharose by gently mixing these components overnight at 40.Sepharose was activated by adding CnBr dissolved in distilled water in a ratio (w/v) of5: 1 to dry Sepharose (calculated by the method of Mannik and Stage, 1971). The slurryformed was stirred for 20 minutes while the pH was maintained at 110. Sepharoseactivated in this manner was washed with large volumes of distilled water followed by0-2 M Na borate buffer, pH 8-0. Columns of coupled Sepharose were maintained in thecold, samples were added in borate buffer and bound components were then eluted with3 M sodium thiocyanate.

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Sucrose density gradientsThese were performed by the method outlined by Rowley and Turner (1964). Briefly,

1 ml of quokka serum diluted 1 in 3 in saline was layered on top of a continuous gradientwhich ranged from 10-40 per cent sucrose. Samples were then spun for 17 hours (over-night) at 35,000 rev/min in an SW/50 Spinco ultracentrifuge. Fractions were collecteddropwise from the base of the centrifuge tubes.

Determination of the extinction coefficient ofIgG2The concentration of a stock solution of purified quokka IgG2, dissolved in 0-025 M P04

buffer, pH 7 5, was determined by dry weight analysis. The optical density of aliquots ofthis stock solution was measured in duplicate at 280 nm in an Hitachi Perkin Elmer Model124 spectrophotometer. The extinction coefficient E' per " was calculated from thesecombined measurements.

Hexose determinationThe anthrone reaction as outlined by Kabat and Mayer (1961) was used to determine

the hexose contents of samples of purified quokka immunoglobulins whose concentrationhad been determined by optical density. Hexose values ofimmunoglobulins were read froma curve drawn from D-glucose (AR) standards (0-200 pg) which were assayed using theanthrone reaction.

Immunoelectrophoresis and double diffusionImmunoelectrophoresis was performed with a Shandon electrophoresis apparatus

using 1 per cent Oxoid Ionagar in 0 05 M Veronal buffer, pH 8f4. Electrophoresis wascarried out at a constant current of 5-6 mA per slide for 2 hours. Immune diffusion wasallowed to proceed for either 2 days at room temperature or for 1 day at 370. Slides werethen washed for 4 days in 1 per cent NaCl + 1/5000 Na Azide and 1 hour in distilled water,dried at 37° in a gentle air current and finally stained with Amido Black 10B.

RESULTS

Sucrose density gradient fractionation of immune quokka serum showed that antibodyactivity could be isolated in two major peaks (Fig. 1). Using this technique the macro-globulin peak, fraction 1, was clearly separated from a 7S (Rowley and Turner, 1964)peak, fraction 3, which contained most of the antibody activity in this particular experi-ment.

Elution from DEAE-cellulose of a 50 per cent (NH4) 2SO4 precipitate (Fig. 2) ofnormal quokka serum with 0 005 M P04 buffer containing 1 M urea produced a large pro-tein peak which corresponded to 2-3 mg/ml of original serum protein. This peak wasconcentrated and subjected to immunoelectrophoresis, which revealed a single proteinband migrating in the slow gamma region (Fig. 3a). An antiserum produced in rabbitsagainst this material detected several components through its light-chain activity whendiffused against normal quokka serum (Fig. 3b). Most noticeable was a large band in thefast gamma region around the well (IgGl) which fused with the slow gamma component(IgG2), and a smaller inner band (1gM) which migrated on the cathodic side ofthe well.The components IgG2 and IgGl were designated as such by analogy with the electro-phoretic properties of guinea-pig immunoglobulins (Benacerraf, Ovary, Bloch and Frank-

1106


0-2

O0

Fr Fr 2

6

1107

Fr 3 Fr 4

12Tube number

18 24

320

80 0-e

.@ ._

20

FIG. 1. Separation of antibody activity to B. abortus in immune quokka serum by sucrose densitygradient fractionation. Primary response, day 7. Protein as measured by OD (0). Antibody activity,hatched columns.

28

24

E 2.0 -0-005 0\025 005 008 015 0-4

1>1.

0 40 50 60 80 100 120 140 160 180 200 220 240

f Tube number

FIG. 2. Elution profile from DEAE-cellulose of a 50 per cent saturated ammonium sulphate precipitateof 30 ml ofnormal quokka serum. Batchwise separation with phosphate buffers ranging in molarity from0 005 to 0-4 (plus 1 M urea) was used. Arrows mark the time of application of each buffer.

K


(a)

(b)

(i)

(ii)

(iii)

(iv)

IgG2 IgM TgGlFIG. 3. Immunoelectrophoretic analysis of DEAE Fraction 1. (a) Centre well, DEAE-cellulose,0-005 M fraction. (i) Guinea-pig anti-quokka transferrin; (ii) rabbit anti-whole quokka serum. (b)Centre well, normal quokka serum; (iii) and (iv) rabbit anti-DEAE Fraction 1 (0005 M P04) absorbedwith y2 heavy chains. The formation of two distinct bands at the cathode indicating the existence oftwo light chain classes.

Fr I i6

5

Fr

Q

0 20 40 60 80 100 120 140 160 180 200 220 240Tube number

FIG. 4. Sephadex G-200 elution profile of 45 ml of normal quokka serum which had been resolved on a200 x 5 cm column. Flow rate, 12 ml/hour.

lin, 1963). The small inner band was identified as quokka macroglobulin by demonstrat-ing that this component was present in the excluded fraction of a Sephadex G-200 filtrateof normal serum and was therefore designated IgM.

Macroglobulins were purified by concentrating to 5 ml the proteins (Fig. 4, Fraction 1)excluded from a Sephadex G-200 column (5 x 200 cm) which had been loaded with 45 mlof normal quokka serum. This concentrated macroglobulin peak was then dialysed against0-1 M veronal buffer, pH 8f6, and subjected to zone electrophoresis on a Pevikon block.The resulting protein profile (Fig. 5a) was resolved into three major fractions. A proteinreacting with anti-light chain sera was isolated in pure form in Fraction 1 (Fig. 5b) butwas contaminated by a lipoprotein component in Fraction 2. This procedure isolatedfrom 0-2-0-3 mg of IgM/ml of original serum sample.

1108


18 (a)

006Fr I Fr 2\. Fr3

~0~04C I

°002 Point of *1application .p A

0 IM% A0 10 20 30 40 50Negative Tube number Pbsitive

(b) (i)

(ii)

FIG. 5. (a) Elution profile of Sephadex G-200 Fraction I after electrophoresis on a Pevikon block(PVC). Electrophoresis proceeded for 2 days at a constant current of 40 mA and 1-cm fractions werecut, washed and analysed for protein by the Folin-Ciocalteau method. The proteins contained inFraction 1 are shown in (b) after immunoelectrophoresis. (b) Centre well IgM (G-200 Fraction I,PVC Fraction I). Antisera: (i) rabbit and anti-quokka light chain serum; (ii) rabbit and wholequokka serum.

Isolation of the immunoglobulin identified in Fig. 3 as IgG1 proved to be more difficultthan the isolation of either JgG2 or IgM. Preliminary experiments had indicated that thisimmunoglobulin possessed a higher molecular weight than JgG2 and therefore con-centration by Sephadex G-200 filtration was the initial step (Fig. 4). The middle peakwhich corresponded to the 7S component could be divided into two portions, FractionIIA, comprising the ascending portion of the peak, and the major, trailing portion,Fraction IIB. Although separation by this procedure was by no means absolute, moreIgG1 was present in Fraction IIA and more JgG2 was present in Fraction IIB. FractionIIA was concentrated by negative pressure dialysis and further fractionated by ion-exchange chromatography on a CMC column. The first fraction that eluted with a 0-02 mPG4 buffer was found to contain IgG1 at about 80-85 per cent purity. This fraction wasfinally separated from the major contaminant, IgG2, by zone electrophoresis on Pevikon._An alternative procedure involved the repeated recycling on Sephadex G-200 of theascending portion of the G-200-IIA fraction (Fig. 4). Both methods produced a pureIgGl fraction as tested by double diffusion analysis, but yields were of the order of0-1-0-2 mg lgGl/ml of serum.

1109

R. G. Bell, N. R. Lynch and K. J. Turner+

(a)

(b)

(c)

(d)

(c)

(f)

FIG. 6. Identity of quokka IgGI, IgG2 and IgM by immunoelectrophoresis. Wells in each case containnormal quokka serum. Upper pattern, troughs (a) and (b) contain guinea-pig anti-quokka IgG2serum. Middle pattern, troughs (c) and (d), guinea-pig anti-quokka IgGl serum. Lower pattern, troughs(e) and (f), guinea-pig anti-quokka IgM serum.

FIG. 7. Antigenic identity of quokka immunoglobulin heavy chains as shown by double diffusion inagar. Upper pattern: (a) guinea-pig anti-quokka IgGl; (b) and (c) quokka IgM (PVC Fraction I);(d) guinea-pig anti-IgG2; (e) and (f) quokka IgGl (Sephadex G-200 11A, recycled); (g) quokkaIgG2 (DEAE 0-005 M). Lower pattern: (h) quokka IgM (PVC Fraction 2); (i) quokka IgG2 (as for g);(j) guinea-pig anti-quokka IgM; (k) quokka IgGl (as for e and f). The dark precipitate around (h) is alipoprotein contaminant present in PVC Fraction 2.

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STUDIES ON ANTI ENIC RELATIONSHIPS

Immunoelectrophoretic patterns developed by using antisera raised in guinea-pigs toquokka IgG2, IgGl and IgM are shown in Fig. 6 and agar double diffusion patterns inFig. 7. The specificity and antigenic 'identity' of each of these classes is clearly demon-strated by the lack of cross-reactivity with each antiserum.

PHYSICOCHEMICAL CHARACTERISTICS OF IgGl, IgM AND IgG2

A 1 x 100 cm column of Sepharose 6B was calibrated for molecular weight deter-minations by filtration of the following standards: bovine serum albumin (mol. wt =67,600); human IgG (mol. wt = 155,000); Catalase (mol. wt = 240,000) and ferritin(mol. wt = 540,000). The molecular weights of quokka JgG2 and IgGl obtained by useof this column were 130,000 and 240,000 respectively. These are average values forduplicate preparations of each immunoglobulin.

Hexose contents of quokka immunoglobulins are shown in Table 1. Extinction co-efficients were assessed only for IgG2 as this was the only immunoglobulin which couldbe purified in sufficient quantity. TheEperCent of 12-3 for IgG2 was thereafter used asa standard for estimating all quokka immunoglobulins spectrophotometrically.

TABLE 1PHYSICOCHEMICAL PROPERTIES OF QUOKKA IgG1, IgG2 AND IgM

Hexose contentImmunoglobulin Molecular

class weight pg/mg Percentage El, cm, 280nm

IgG2 130,000 24-8 2-5 12-3IgG1 240,000 14-5 1*5IgM > 200,000 63-0 6-3

SUBCLASS COMPONENTS

Most preparations of IgG2 from DEAE columns were observed to give at least twoprecipitin lines when reacted with guinea-pig anti-quokka y2 (Fig. 8a). One of thesecomponents appeared to be present in higher concentration than the other in normalquokka serum and a series of experiments in which guinea-pigs were immunized with 1and 0-5 jug of IgG2 (DEAE Fraction 1) produced antisera with more activity against themajor component (designated IgG2b) and less against the other (IgG2a). One of theguinea-pig antisera raised by this schedule was precipitated with 50 per cent (NH4) 2SO4and conjugated to Sepharose 4B as described earlier. Varying amounts of a DEAE0-005 M PO4 fraction, containing only IgG2, was added to this column until conditionswere found where only IgG2b was bound and the excess IgG2a was eluted with thestarting buffer. The yield of protein after concentration was small, but it was sufficientto raise an antiserum in guinea-pigs specific for IgG2a and to show that absorption of thiscomponent from serum did not remove IgG2b (Fig. 8b).

Analysis of quokka IgGl did not proceed to the same degree as IgG2, mainly due tothe difficulty experienced in isolating reasonable quantities of this immunoglobulin in

1111

1112 R. G. Bell, N. R. Lynch and K. J. Turner

(a) - +

IgG2b --XI- i

IgG2c

IgG2b

IgG2a (iv)

(b)

FIG. 8. (a) Definition of quokka IgG2 subclasses. Upper pattern: centre well, normal quokka serum;(i) polyvalent guinea-pig anti-quokka IgG2 showing three distinct precipitin arcs, IgG2a, IgG2b andIgG2c are identified; (ii) weak anti-IgG~a. Lower pattern: (iii) polyvalent guinea-pig anti-quokkaIgG2; (iv) guinea-pig anti-quokka IgG2a. Note the complete crossover of IgG2b by IgG2a at thecathodic end of the precipitin bands, indicating separate antigenic determinants on these subclasses. (b)Absorption offrom IgG2a whole quokka serum: (e) and (g) Polyvalent guinea-pig anti-IgG2; (f) guinea-pig anti-IgG2a; (h) normal quokka serum diluted 1/20; (i) guinea-pig anti-quokka IgG2a; (j) no serum;(k) normal quokka serum absorbed with guinea-pig anti-quokka IgG2a (final dilution of quokkaserum equals approximately 1/15. No reaction of anti-IgG2a (f) and (i) with quokka serum absorbedwith anti-IgG2a (k) yet a greater dilution (h) (1/20) of normal quokka serum reacts with anti-IgG~aat (i). Polyvalent anti-IgG2, for example, retains activity with both absorbed (k) and unabsorbed (h)quokka serum.

pure form. However, heavy chain specific antisera usually identified two precipitin arcswith common antigenic determinants. It would appear likely, therefore, that there are atleast two subclasses of IgGl as well as IgG2, although neither of these have been isolatedin pure form.

DISCUSSION

Three distinct immunoglobulin classes found in the serum of normal quokkas have beencharacterized in this paper. Each class possessed shared antigenic determinants (lightchains), and each had specific heavy chain determinants which were unique to that class.Unlike the situation in eutherians IgGl and IgG2 did not share Fc heavy chain antigenicdeterminants and were therefore not subclasses of IgG. For this reason IgG1 and IgG2have been accorded separate isotypic class status. In normal animals IgG2 appeared tobe the major immunoglobulin class and this molecule was characterized by a typically


heterogeneous pattern of physicochemical features. This was clearly shown in its electro-phoretic mobility where, although the bulk of the IgG2 migrated in the slow gammaregion, a significant portion migrated well into the beta region where it was the mostrapidly migrating immunoglobulin class. A similar heterogeneity was shown in SephadexG-200 filtration where IgG2 heavy chain-specific components were spread from the ex-cluded protein fraction (mol. wt >200,000) to the albumin peak. * These findings resemblethose of Thomas, Turner, Eadie and Yadav (1972) in that antibody activity absorbableby anti-quokka y-chain antibodies was found in all three major peaks of Sephadex G-200filtrates of quokka serum. This size heterogeneity was also shown by serum IgA (Bell,Stephens and Turner, 1974), although this class did not appear in the third SephadexG-200 peak.Quokka macroglobulin was characterized by a relatively fast y mobility and, due to its

low serum concentration, usually formed an arc close to the well on immunoelectro-phoresis. The other serum immunoglobulin identified in this study, IgGl, possessed a morerapid mobility on electrophoresis which extended from the gamma to the beta region.Like IgM, it had a restricted precipitin arc on immune diffusion when compared withthat ofIgG2. Estimates of the molecular weight of IgGI were hampered by the apparentchange in molecular weight which occurred during the isolation of this class. After filtra-tion ofwhole quokka serum on Sephadex G-200, IgGI eluted after the breakthrough peakbut slightly ahead of IgG2 (Fig. 4, Fraction IIA), suggesting a molecular weight greaterthan 140,000 but less than 200,000. Two separate preparations of IgGl, one prepared byrecycling a Sephadex G-200 IIA fraction of whole serum on Sephadex G-200, and theother by ion-exchange chromatography of a Sephadex G-200 IIA fraction followed byblock electrophoresis, gave identical molecular weights of 240,000 on a calibrated Sepha-rose 6B column. Estimates of the weight of IgGl, based on biological activity in wholeserum (Lynch and Turner, unpublished observations) gave a molecular weight of 178,000,which is in accordance with the elution pattern ofIgGl in whole serum. Dimerization isunlikely to be occurring unless the whole molecule had a molecular weight of less than130,000 and the true molecular weight of IgGl remains conjectural.

Subclasses were unequivocally identified in the IgG2 globulin class where one subclass,IgG2a, was isolated and another major subclass IgG2b, could be defined by antigenicanalysis. The formation of spurs on immunoelectrophoresis indicated the likelihood that atleast one other IgG2 subclass (IgG2c) also existed. The capacity of IgGl heavy chain-specific antisera to form multiple precipitin bands on immunoelectrophoresis indicatedthat subclasses probably existed within this heavy chain class as well.

Previous studies of metatherian immunoglobulins and immune responses (Rowlandsand Dudley, 1968; Thomas et al., 1972) concluded that metatheria were more primitivein their immune apparatus than eutherians. This conclusion was based primarily on theapparent slower rate of conversion of IgM to IgG antibody in primary and secondaryresponses in metatheria compared with eutherians. However, this is a relatively poormeasure ofimmune competence and metatherians can mount both rapid and high-titredprimary and secondary responses which are comparable with those of eutherians. Further-more, not all eutherians show the 'classical' rapid conversion of IgM antibody to IgG andthis is more a function of the species, the antigen, as well as the dose site and schedule,than it is of eutherians as a group. When eutherians are compared to ectotherms such asreptiles, fishes or amphibians, there is an obvious and important difference between the

* Unpublished observation.

1113

1114 R. G. Bell, N. R. Lynch and K. J. Turner

kinetics and conversion rates of antibody formation. However, these represent differentends of a spectrum, and there seems little doubt that on this sort of scale the quokka, atleast, is in a position which is very close to the eutherian type compared to ectotherms. Thecurrent studies indicate that the complexity of the humoral immune system is muchgreater than that of birds as well as that of ectotherms. The complexity of the immuno-globulin spectrum possessed by quokkas where IgM, IgGl, IgG2 and IgA (Bell et al., 1974)exist as separate classes in serum is very much analogous to the eutherian pattern. Thefurther existence of a complex of subclasses within at least two of the main isotypic classes,IgG2a, IgG2b and IgG2c, IgGla, and IgGlb and IgGl homocytotropic antibody (Lynchand Turner, unpublished results) irrevocably aligns metatherians with the eutherians asa group where a considerable development of complexity in the humoral immune (andpresumably in the basic cellular mechanisms) system has taken place.The existence ofIgM, IgA and an IgG (IgY) like system ofimmunoglobulins in chickens

as well as in eutherians and metatherians suggest that this may well have been a commonpattern in all groups prior to their respective separations. This would be compatible withthe suggestions of Grey (1971) that immunoglobulin subclasses (IgG subclasses) haveevolved separately in the various eutherian families. The data presented here which showdifferences in the molecular weights of IgG2 and IgGl both from each other and fromtheir eutherian counterparts also suggest a separate development probably after theeutherian/metatherian division had taken place. If this is so then the metatherian sub-classes have also evolved at a later date than the eutherian/metatherian divergence andsubsequent to the development of IgGl and IgG2. Considering the period over whichboth groups have evolved there is no alternative but to assume that in both groups therehas been a virtually contemporaneous development of immunoglobulin classes and sub-classes.

ACKNOWLEDGMENTS

This work was supported by research grants from the Saw Medical Research Founda-tion, the Commonwealth Scientific Industrial Research Organization, the National Healthand Medical Research Council of Australia and the Asthma Foundation of WesternAustralia. Some facilities were provided from a C.S.I.R.O. grant to Professor H. Waring,Department of Zoology, University of Western Australia, for marsupial research. Theskilled technical assistance of Mrs Lee Hazell and Miss Jillian Butler is gratefullyacknowledged.

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CLEMENS, W. A. (1968). 'Origin and early evolution ofmarsupials.' Evolution, 22, 1.

CLEMENS, W. A. (1970). 'Mesozoic mammalianevolution.' Ann. Rev. Ecol. and Syst., 1, 357.

FAHEY, J. L., WUNDERLICH, J. and MISHELL, R.

(1964a). 'The immunoglobulins of mice. I. Fourmajor classes of immunoglobulins in mice: 7Sy2-,7Syl-, ylA (ft2a)-, and 18SyIM-globulins.' J. exp.Med., 120, 223.

FAHEY, J. L., WUNDERLICH, J. and MISHELL, R.(1964b). 'The immunoglobulins of mice. II. Twosubclasses of mouse 7Sy2-globulins: y2a- and y2b-globulins.'_7. exp. Med., 120, 243.

GaEY, H. M. (1967). 'Duck immunoglobulins. I. Struc-tural studies on a 5 7S and 7-8S y-globulin.' J.Immunol., 98, 811.

GREY, H. M. (1971). Phylogeny of immunoglobulins.'Biochemical Evolution and the Origin of Life (ed. by E.Schoffeniels), p. 96. North-Holland, Amsterdam.

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immunoglobulins. I. Evidence for six immuno-globulin classes.'j. Immunol., 98, 923.

KABAT, E. A. and MAYER, M. M. (1961). ExperimentalImmunochemistry, 2nd edn. Charles C. Thomas,Illinois.

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Immunoglobulins of the Marsupial Setonix brachyurus