Vox Sang. 21: 372-383 (1971)

Allotypes of Serum a,-Macroglobulin P-Lipoprotein and Immunoglobulins in the Domesticated Sheep (Ovis Aries)

C. C. CURTAIN

CSIRO, Division of Animal Health, Melbourne

Abstract. Antisera specific for allotypic markers carried by the serum az-macro- globulin, &lipoprotein and IgGl and IgGZ were produced during cross-immunization experiments with sheep (Ovis aries). Two markers controlled by autosomal co- dominant genes, Ap' and ApZ, were found on the a,-macroglobulin; one marker controlled by an autosomal dominant Lp' was found on the b-lipoprotein, two markers controlled by the autosomal codominants, I g l l and I g P , were found on the IgGl and one marker, controlled by Ig2l and linked to Iggll, was found on the IgG2. The immunoglobulin markers all occurred on the Fc fragments of the immuno- globulin molecules. There was no linkage between the Ap and Lp loci or between these and the immunoglobulin loci. A preliminary survey of the incidences of the markers in various flocks of sheep showed that the incidence of Lp' and Igl' was very much higher in Merinos than in the British breeds of sheep.

Immunoglobulin and lipoprotein allotypes have been found in man and a number of domesticated animal species. They include the Gm and Inv markers of the human immunoglobulins (reviewed in [18]), the Lp and Ag markers of the human serum lipoproteins [l], the As markers of the rabbit immunoglobulins (reviewed in [El ) and the Lp marker in rabbits [lo]. Immunoglobulin allotypes have also been found in mice [8], guinea pigs [Z, 111, cattle [3], pigs [15] and domesti- cated fowls [6, 1'71.

This paper describes the occurence of allotypic forms of the serum a,-macroglobulin, the serum ,!I-lipoprotein and the immunoglobulins in the domesticated sheep (Ovis aries).

Materials and Methods

Animals. The sheep used in cross-immunization experiments were obtained from two outbred flocks, one predominantly of Merino origin (M flock) and the other of cross-bred Border-Leicester Romney-Marsh origin (BR flock).

Serum Protein Allotypes in Sheep 373

Antigens. The antigens used were either whole serum (3 ml emulsified with 1 ml of complete Freund’s adjuvant, Difco) or purified ovine IgGl or IgG2 (3 ml of a I-percent [w/v] solution emulsified with 1 ml of complete Freund’s adjuvant). Five injections of the antigens, spaced a t weekly intervals, were given into the gluteal muscles of each animal.

Protein isolation procedures. IgGl and IgG2 were isolated from sheep serum by gradient elution chromatography [5]. Sheep serum lipoproteins were separated in the preparative ultracentrifuge by flotation on a density gradient [19] and the macroglobulin fraction was separated by gel filtration on ‘Sephadex 6-200’ (5 ml of serum applied to a 2 x 100 cm upward flow column, equilibrated and eluted with 0.14 N NaCI). Fc fragments from IgGl and IgGZ were prepared as described by EDELMAN et al. [7].

Immunological techniques. Ouchterlony immunodiffusion was carried out using Hyland ‘Immuno-plates’ pattern D.

Immunoelectrophoresis was carried out in agar (Difco-Noble) using the method of SCHEIDEGGER [16]. Radioimmunoassay for the detection of anti-immunoglobulin antibodies was carried out using the solid phase radioimmunoassay method of CATT and TRECEAR [4]. The IgGl and IgG2 were labelled with radioactive iodine as described by HUNTER and GREENWOOD [9] to a level of 1.4 atoms of iodine per molecule of protein using carrier-freelzSI (IMS3 Amersham). Polystyrene tubes (Camelec, Adelaide), were rinsed with 1/20 dilution of the antisera as described by CATT and TREGEAR [4] and then rinsed with the labelled immunoglobulins which were dissolved in 0.14 N NaCl (1 mg immunoglobulin/ml). Radioimmunoassay tests were carried out by adding 1 ml of serum to a 1 mg/ml solution of radio-labelled immunoglobulin before reaction with the antibody-coated tubes. After rinsing the tubes once with distilled water the bound radioactivity was determined in a Packard ‘Autogamma’ spectrometer.

Results

I . Results of Cross- Immunization with Whole Serum Seven of the twenty-five sheep from the BR-flock injected with

M-flock sera gave precipitin lines with 15 out of 20 sera taken a t random from the M-flock. Two of the antisera gave single lines and five gave double lines. Immunoelectrophoresis of the sera (fig. 1) showed precipitin lines in the a- and b- (or slow a-) regions The anti- serum giving the @-line reacted strongly with the isolated serum lipoprotein fraction and the a-precipitating antiserum reacted with the isolated a-macroglobulin. It was concluded, therefore, that the antisera contained antibodies directed against antigens on the lipo- protein and a,-macroglobulin of sheep serum.

I n further cross-immunization experiments using sera from the M-flock and recipients from the BR-flock another a,-macroglobulin- reacting antiserum was obtained which gave a line of nonidentity on

374 CURTAIN

Fig. 1. Composite immunoelectroplioresis pattern showing the reaction of sheep serum and isolated fractions with anti-allotype antisera. SS, sheep serum; anti SS, anti-sheep serum (rabbit); LP, ,9 lipoprotein; Mg, macroglobulin fraction.

Fig. 2. Pattern of inheritance of Ap' and Ap2 in three generations of sheep. Rams represented by squares, ewes by circles. Ap' white, Ap2 black.

double diffusion in agar with macroglobulin and the antiserum pro- duced in the earlier experiments.

The a,-macroglobulin markers. From experimental matings of sheep from the M-flock involving 4 families of sheep and study of sera from a commercial Romney Marsh stud involving 55 families, it appeared

Serum Protein Allotypes in Sheep 375

Table I. Matings of Ap(l+), Ap(l+, 2- t ) and Ap(2+) sheep

Phenotypes of offspring

17 17

6 7

6 6

6 6

8 7

6 8

10 12

9 9

10 8

8 6

The numbers in italics refer to the number of matings of each type.

Table I I . Matings of Lp(l)+ and Lp(1)- sheep

Matings Lp(l)+ xLp(l)+ Lp(1)- xLp(1)- Lp(l)+xLp(l)-

Number 23 13 19 Phenotypes of

Actual 33 2 - 20 30 2 offspring LP(l)+ Lp(1)- LP(l)+ Lp(1)- LP(1)f Lp(1)-

Expected 35 - - 20 30 -

376 CURTAIN

that the two a,-macroglobulin antigens were controlled by two auto- somal codominant alleles which i t was decided to call Ap1 and Ap2. The results of these matings are set out in table I. The inheritance of Ap1 and Ap2 markers through three generations of sheep is shown in figure 2. All the sheep tested were Ap(l)-t, Ap(2)+ or Ap(1, 2)+, indicating the presence of three genotypes ApllApl, ApllAp2 and Ap2/Ap2.

The /I-lipoprotein markers. A genetic study of the same flocks showed that the /?-lipoprotein antigen was the product of an auto- somal dominant gene which i t was decided to call Lp'. The results of the matings are given in table 11. No evidence was found for linkage between the Ap and Lp loci. It was considered that the Lp(1-) pro- geny arose in the Lp(1 +) x Lp(1 +) and Lp(1 +) x Lp(1-) matings because some Lp(1 -1) sheep are heterozygous, i.e., Lp(1 +,l-).

Distribution of Lp1 and Ap1 alleles in different breeds. Sera from a number of commercial flocks were tested foI the Lp and Ap antigens using double diffusion in agar against anti-Lp(l), anti-Ap(1) and anti- -Ap(2). The results are given in table 111. It can be seen that Lp1 occurs in very high frequency in Merino sheep and to a much lesser extent in the British breeds. The frequencies of Ap1 and Ap2 show very considerable variability from flock t o flock and there appears to be no consistent breed difference.

2. Results of Cross- Immunization with IgGl and IgC2 Sixteen of the sheep from the M-flock were injected with BR-IgG1

from 10 individuals and 12 with BR-IgG2 from the same animals. Twelve BR-sheep were injected with IgGl from 8 M-flock sheep and 12 with IgG2 from the same animals. None of the sera tested produced visible precipitin lines on Ouchterlony immunodiffusion although 6 sera (4 from the BR- and 2 from the M-flock) were found to contain antibodies capable of binding 1251-labelled IgGl or IgG2 in the radio- immunoassay test. This binding was judged to be specific because i t was inhibited by the addition of excess unlabelled immunoglobulin. Using this inhibition effect as a test i t was found that each of two sera (Merino) contained non-cross-reacting antibodies t o antigens of the IgG1, and four of the sera contained an antibody which reacted with an antigen of the IgG2 molecule but not with the IgG1. A set of inhibition curves for each of the antigen-antibody systems is given in figure 3. For convenience, labelled IgG was used for routine testing rather than labelled IgGl and IgG2.

Tabl

e ZZZ. D

istr

ibut

ion

of L

p',

Ap'

an

d A

p2 in

dif

fere

nt b

reed

s of

she

ep

Phen

otyp

es

Freq

uenc

ies

Tot

al

Lp(

1)"

Lp(

1)-

Ap(

l)+

A

p(l,

2)+

A

p(2)

' O

,bL

p(l)-

O

oLp(

1)-

Ap'

A

P2

Dor

set

Hor

n Fl

ock

1 Fl

ock

2 C

orri

edal

e Fl

ock

1 Fl

ock

2 So

uthd

own

Floc

k 1

Floc

k 2

Floc

k 1

Floc

k 2

Floc

k 1

Floc

k 2

Floc

k 3

Floc

k 1

Floc

k 2

Floc

k 3

Floc

k 4

Bor

der

Leic

este

r

Rom

ney

Mar

sh

Mer

ino

5 6 23

16

14

12

7 8

7 2 17

.86

27.2

7 82

.14

72.7

3 0.

625

0.72

7 0.

375

0.27

3 28

22

9 7

18

R ;. 25

L

19

5

18

0 5'

30

4 Y

w rn

D-

12

2 0 7 18

J 7

2 7 2 4

22.2

2 0

77.7

8 10

0.00

0.

667

0.58

3 0.

333

0.41

7

4 2 21

17

8 3

11

12

6 4 16

.00

10.5

3 84

.00

89.4

7 0.

540

0.47

4 0.

540

0.52

6

0 0 18

12

6 3

6 6 6 3

0 0 10

0.00

10

0.00

0.

500

0.50

0 0.

500

0.50

0

5 0 3

25

40

31

15

18

15

10

19

16

5 3 3

13.3

3 0 8.

82

86.6

7 10

0.00

91

.18

0.66

7 0.

687

0.67

7

0.33

3 0.

313

0.32

3 40

34

46

25

35

62

10

16 7 5

29

17

19

32

20

16

15

26

82.1

5 60

.97

83.3

3 92

.54

17.8

5 39

.03

16.6

7 7.

46

0.69

6 0.

610

0.63

1 0.

672

0.30

4 0.

390

0.36

9 0.

328

56

41

42

67

378

40 -

30-

- E 20- - 0 OI -

6 P 10-

CURTAIN

40 -

30-

2 20- - 8 - - 6 P 10-

I I

1 2 3 1 2 3 cpm, a 1 0 - 3 cprn, x 10 -

Fig. 3. Radioimmunoassay for allotypes of ovine IgGl and IgG2, the curves showing binding of 1251-labelled IgG on to the walls of polystyrene tubes coated with: A (1) anti-IgGl(1) in the presence of increasing amounts of TgG1(1)+ IgG1; (2) anti-IgGl(2) in the presence of increasing atnountv of IgG1(2)+ IgG1; (3) anti- IgGl(1) in the presence of increasing amounts of IgG1(2)+ IgG1, and (4) anti- IgGl(2) in the presence of increasing amounts of IgG(l)+ IgG1. B (1) anti-IgG2(1) in the presence of increasing amounts of IgG2(1)+ IgG2, and (2) anti-IgGZ(1) in the presence of increasing amounts of IgGZ(1)- IgG2. The 1Z5I-IgG was obtained from a pool positive for IgGl(I), IgGl(2) and JgGZ(1).

Inheritance of antigens. Experimental matings involving 6 families of sheep and a study of sera from a commercial Romney Marsh stud involving 80 matings showed that the IgGl antigens were the pro- ducts of two autosomal codominant alleles which it was decided t o call Igl and Igl 2. The details of the matings are set out in table IV. The inheritance of the IgG2 antigen appeared to be closely linked t o that of Igll and t o be controlled by an autosomal dominant gene which it was decided to call Ig2l. The complex formed by Ig l l and Ig21 will be referred to as Igl1;21.

Distribution of the IgGl alleles and Ig2l in different breeds. The results of a survey using the radioimmunoassay of a number of commercial flocks are given in table V. The Merino flocks generally have a high frequency of the Ig11;21 complex compared with those of British origin.

Serum Protein Allotypes in Sheep 379

Molecular localization of Igl l , Ig12 and Ig2I. Radioimmunoassay experiments were carried ou t using Fc and Fab fragments isolated from IgGl and IgG2. Binding of radio-labelled immunoglobulin was inhibited only by the Fc fragment from the appropriate specific immunoglobulin. The inhibition curves with the Fc fragments were identical with those of the whole immunoglobulins.

Table IV. Matings of IgGl(1 1 ); 2(1-t), IgGl(1 i , 2 I - ) ; 2(1+) and IgGl(2 i ) qlieep

IgGI(1 I ); 2(1+)x IgCl(1+); 2(1-k), 10

Actual Expected

Actual Expected

IgGl(2 t) x IgG1(2+), 21

IgG1(1+); 2(1+ ) A

IgGl(2 -I ), 6 Actual Expected

IgG1(1+,2 t); 2(1 +) x IgGI(2+), 10

Actual Expected x2 = 0.25

IgG1(1+,2+); 2(1 +)x IgG1(1+); 2(1+), 14

Actual Expected x2 = 0.8

IgGl(l-I-$i-); 2(1+)X IgG1(1+,2+); 2(1+), 25

Actual Expected x 2 = 2.0

13 13

-

-

-

-

-

8 10

12 9

-

-

~

~-

8 8

7 8

12 10

14 18

The numbers in italics refer to the number of matings of each type. In no case

did IgG2(1+) occur in the absence of IgG1(1+).

380 CURTAIN

Table V. Distribution of Ig1*;21 and I g P in different breeds of sheep

Breed Phenotypes Gene frequencies Total

zgi(i+); 1~1(1+, 1~1(2+) rgil; 21 zg12

2(1+) 2+); 2(1+)

Dorset Horn Flock 1 Flock 2 Flock 3

Corriedale Flock 1

Southdown Flock 1 Flock 2

Flock 1 Flock 2

Flock 1 Flock 2 Flock 3

Flock 1 Flock 2 Flock 3 Flock 4

Border Leicester

Romney Marsh

Merino

16 1 2 9

15 21 17

0.361 0.250 0.232

0.639 0.750 0.768

36 36 28

4 1 2 15 0.323 0.677 31

7 3

15 12

22 17

0.329 0.281

0.671 0.719

44 32

2 0

7 2

10 14

0.289 0.063

0.711 0.937

19 16

4 2 3

12 15 10

14 22 13

0.333 0.244 0.308

0.667 0.756 0.692

30 39 26

35 21 56 45

23 17 21 35

0.738 0.686 0.842 0.702

0.262 0.314 0.158 0.298

63 43 79 89

Discussion

The study of the distribution of protein variants in a number of species has been of value in two directions. Firstly, it has been used to reveal similarities and differences between populations : secondly, it has led t o greater insight into the precise genetic mechanisms con- trolling the expression of certain physiologically important proteins. The most detailed studies of this kind have been carried out using the allotypic markers of human, rabbit and mouse immunoglobulin [8, 12, 13, 181.

The Lp and Ap markers could possibly be of value in augmenting the population data already obtained from studies on haemoglobins, blood groups and transferrins [20]. The data for the distribution of these and the immunoglobulin markers given in tables I11 and V

Serum Protein Allotypes in Sheep 381

suggest considerable variation between flocks and breeds although, on the whole, the samples were too small and the mating patterns not sufficiently random t o justify statistical analysis.

In relation t o the immunoglobulin markers, there is a special interest in the 7s immunoglobulin sub-classes of ruminants. Unlike many other species examined, the IgGl as well as IgA appears t o be a major secretory immunoglobulin occurring in exocrine secretions, such as colostrum and saliva. IgGl is also the complement-fixing immuno- globulin whilst IgG2 does not fix complement and does not occur in secretions to any extent. The fact that I g l l and Ig2l occurred together or were both absent from all the sera examined suggests that the genes for IgGl and IgG2 are very close together on the same chromo- some. A detailed study of immunoglobulin allotypes in the mouse showed a similar close linkage between the mouse a-chain and the three y-chain cistrons [13]. An extensive study in man by NATVIG et al. [14] and VAN LOGHEM et al. [21] of the IgA and IgG markers (Am and Gm loci) have also shown close linkage between the cistrons for the human a, y1 y z and ya chains. Studies of rare recombinants bet- ween the closely linked y-chain cistrons in man [14] have given an indication of the order in which these occur viz. y2, y3, y l . A com- parative study of immunoglobulin allotypic markers and a search for rare recombinants could be of very considerable value in determining the probable order in which the classes and subclasses in the different species arose. Furthermore, a knowledge of the order of the heavy chain cistrons might enable direct interspecies comparisons to be made, particularly amongst the 7.9 subclasses thus leading to a more rational nomenclature for these.

.4 cknowledgments

The author is grateful to Mr. H. CAMP and Miss B. WATT for technical assistance and to Mr. H. YELLAND of Glcntrevor Stud, Newlyn, Victoria for permission to take blood from his flock and for valuable pedigree data. The author is also grateful to members of the staff of the Victorian Department of Agriculture, Veterinary Research Institute for the sera used in the large-scale allotypic marker surveys.

References

1. ALLISON, A. C. and BLUMBERG, B. S.: An isoprecipitation reaction distinguish-

2. BENACERRAF, B. and GELL, P. G. H.: Delayed hypersensitivity to homologous ing human serum-protein types. Lancet i : 634-637 (1961).

y-globulin in the guinea pig. Nature, Lond. 189: 586-587 (1961).

382 CURTAIN

3. BLAKESLEE, D. L. and STONE, W. H.: Immunogenetics of IgG allotypes of cattle. Fed. Proc. 28: 436 (1969).

4. CATT, K. and TREGEAR, G. W.: Solid-phase radioimmunoassay in antibody- coated tubes. Science 158: 1570-1571 (1967).

5. CURTAIN, C. C.: A new immunoglobulin subclass in the sheep. Immunology, Lond. 16: 373-380 (1969).

6. DAVID, C. S.; KAEBERLE, M. L. and NORDSKOG, A. W.: Genetic control of immunoglobulin allotypes in the fowl. Fed. Proc. 28: 436 (1969).

7. EDELMAN, G. M.; HEREMANS, J. F.; HEREMANS, M.-TH. and KUNKEL, H. G.: Immunological studies of human y-globulins: relation of the preeipitin lines of whole y-globulin to those of the fragments produced by papain. J. exp. Med.

8. HERZENBERG, L. A. : A chromosome region for gnmma,A and beta,* globulin H chain isoantigens in the mouse. Cold Spr. Harb. Symp. quant. Biol. 29: 455-462 (1964).

9. HUNTER, W. M. and GREENWOOD, F. C.: Preparation of lSII-labelled human growth hormone of high specific activity. Nature, Lond. 194: 495-496 (1962).

10. KELUS, A. S.: Lp’ - allotypic determinant of rabbit /Mipoprotein. Nature, Lond. 218: 595-596 (1968).

11. KELUS, A. S.: Allotypic marker GPy,-l of guinea-pig immunoglobulins. Nature, Lond. 223: 398-399 (1969).

12. KELUS, A. S. and GELL, P. G. H.: Immunoglobulin allotypes of experimental animals. Progr. Allergy vol. 11, pp. 141-184 (Karger, Basel 1967).

13. MINNA, J. D.; IVERSON, G. M. and HERZENBERG, G. H.: Identification of a gene locus of yg, immunoglobulin H-chains and i ts linkage to the H-chain ehromo- some region in the mouse. Proc. nat. Acad. Sci., Wash. 58: 188-194 (1967).

14. NATVIG, J. B.; KUNKEL, H. G. and GEDDE-DAHL, T.: Genetic studies of the heavy chain subgroups of yG globulin. Recombination between the closely linked cistrons ; in KILLANDER, Gamma globulins. Proceedings of the Third Nobel Symposium, pp. 313-328 (Almqvist & Wiksell, Stockholm 1967).

15. RASMUSEN, B. A.: Isoantigens of gamma globulin in pigs. Science 148: 1742- 1743 (1965).

16. SCHEIDEGGER, J. J. : Une micro-m6thode de l’immuno-6lectrophor8se. Int. Arch. Allergy 7: 103-110 (1955).

17. SKALBA, D.: Allotypes of hen serum proteins. Nature, Lond.204: 894-895 (1964).

18. STEINBERG, A. G.: Genetic variations of human immunoglobulins. The Gm and Inv types; in GREENWALT, Advances in immunogenetics, p. 75 (Lippincott, Philadelphia 1967).

19. STOKES, R. P.; JACOBSSON, A. and WALTON, K. W.: The isolation of low- density 8-lipoprotein from small volumes of human serum. J. Atheroscler. Res.

20. STORMONT, C.; SUZUKI, Y.; BRADFORD, G. E. and KING, P.: A survey of hemo- globins, transferrins and certain red cell antigens in nine breeds of sheep. Genetics 60: 363-371 (1968).

21. LOGHEM, E. VAN; NATVIG, J. B. and MATSUMOTO, H.: Genetic markers of im- munoglobulins in Japanese families. Inheritance of associated markers belong- ing to one IgA and three IgG subclasses. Ann. hum. Genet. 33: 351-359 (1970).

112: 203-223 (1960).

7: 187-196 (1967).

Serum Protein Allotypes in Sheep 383

Note Added in Proof

Following recent deliberations of the Allotypes Sub-committee of the Nomen- clature Committee of the International Union of Immunological Societies, i t has been decided to use the following notation in future reference to the sheep irnrnu- noglobulin markers:

This paper Future

Markers

Genes

Im - y l (a) Irn - y l ( b ) Im - y 2 ( a )

Irn - y 1" Im - y ~b

Im - y 2n

Gene Complex Ig 1 ' : 2' Im - y l " : - y 2 "

It is hoped that this nomenclature will be applied to new allotypie systems in other species.

Author's address: Dr. C. C. CURTAIN, CSIRO, Division of Animal Health, Animal Health Research Laboratory, Private Bag No. 1, P.O. Parkuille, Victoria, 3052 (Australia)