[ 44° ]
A STUDY OF THE BLOOD GROUPS OF THE RABBIT,
WITH REFERENCE TO THE INHERITANCE OF THREE
ANTIGENS, AND THE AGGLUTINABILITY OF THE RED
CELLS CARRYING THEM
BY VALERIE C. JOYSEY
Department of Pathology, University of Cambridge
{Received 27 August 1954)
INTRODUCTION
Work on rabbit blood groups carried out by several workers prior to 1950, has been
summarized by Heard (1953). The sera, however, are not available and the rabbits
possessing the antigens have died, so that the direct correlation between previous
and present work has been impossible.
Kellner & Hedal (1953), working in America, have described an allelomorphic
pair of blood group genes, G and g; while Heard (1953) in this laboratory has
identified and described four blood group antigens, namely Z, Y, X and W. Heard
found that red blood cells of rabbits possessing a particular antigen may be
agglutinated to varying titres by an antiserum containing a single antibody corresponding to that antigen. Cells which were able to demonstrate antibody to a
high titre she called 'strong', while those that demonstrated the same antibody
only to a low titre she called 'poor'. She brought forward evidence to demonstrate
that 'strong' cells possess more functional antigen sites than 'poor' cells.
In the present work Heard's anti-Z and anti- Y have been compared with Kellner
& Hedal's anti-G and anti-£. Antigens Z and G were found to be identical, as
were Y and g.
A study of the inheritance of the antigens Z, Y and W, has been undertaken, and
evidence is brought forward proving that these antigens are controlled by an
allelomorphic trio. The degree of agglutinability of the cells by two of the antibodies
concerned has been shown to be related to the genotype of the rabbit bearing the
cells.
Finally a notation is suggested which will correlate the antigenic systems of Heard
and Kellner & Hedal.
TECHNIQUE
Reagents
(1) Anticoagulant. A solution of o-6% tri-sodium citrate in 3 - 3% aqueous
magnesium sulphate solution was used as an anticoagulant for bleedings of small
volume. For larger volumes (20-40 ml.) 3-4 % tri-sodium citrate solution was used.
(2) Medium in which tests were performed. Lawrence (unpublished) showed that
a non-specific agglutination sometimes occurs when saline is used as a suspending
A study of the blood groups of the rabbit
441
medium for rabbit red cells. Heard, Hinde & Mynors (1949) found that 3-3%
aqueous solution of magnesium sulphate was a satisfactory medium which prevented such anomalies, and this has been used throughout the present work.
(3) Red cell suspension. The standard suspension of erythrocytes used was 2 % by
volume of packed cells. This was made up by matching the density of suspension
with an accurately measured 2 % suspension of sheep cells.
(4) Anti-rabbit globulin serum. The goat anti-rabbit globulin serum used in this
work was obtained from a goat which had received five courses of injections of
rabbit serum. The antiserum after heat inactivation was absorbed with well-washed
rabbit red cells in order to remove agglutinins to unsensitized rabbit red cells. The
antiserum had a titre of 64 when tested against suitably sensitized rabbit cells. The
serum was normally used at a dilution of 1/25.
(5) Test tubes. Tests were performed in glass tubes of two types: (a) 8x55 mm.
called C.F. tubes in this laboratory; (6) 5 x 50 mm. called R.H. tubes in this
laboratory.
Direct agglutination tests
Dilutions were performed with a graduated 1 ml. syringe in C.F. tubes. Serum
dilutions were distributed in aliquots of o-i ml., to which o-i ml. of red cell
suspension was added. The tubes were shaken, and two drops of the serum-cell
suspension transferred to an R.H. tube, and incubated at 370 C. for 1 hr.
Readings were made macroscopically, and microscopically by spreading the cells
on a slide and examining under the low power of a microscope. In the event of there
being discrepancy between the two readings, the microscopic reading was taken as
being the more reliable.
Anti-globulin sensitization test
The anti-globulin sensitization test was always performed in parallel with a direct
agglutination titration. The serum cell suspension left in the C.F. tubes (after
removal of the two drops for the direct agglutination test) were incubated for 1 hr.
at 370 C , washed 3 times in magnesium sulphate solution, and resuspended to give
a concentration of approximately 2 %. One drop of the sensitized cells was added
to one drop of goat anti-rabbit globulin serum in an R.H. tube, and incubated for
30 min. at 370 C. The test was read macroscopically and microscopically, but in
this case the macroscopic reading was taken as being the more reliable, since the
agglutinates are very fragile.
Blood grouping tests
'Grouping' tests were always performed in R.H. tubes using serum at a dilution
well below its titre. One drop of cell suspension was mixed with one drop of serum,
and incubated for 1 hr. at 370 C.
The antiglobulin sensitization test was performed and readings were taken as
previously described.
29
Ezp. Biol. 32, 2
VALERIE C. JOYSEY
442
Absorption
Blood to be used for absorptions was bled into 3-4% tri-sodium citrate, centrifuged, the supernatant fluid removed, and the cells washed twice in the suspending
medium. At the last wash the cells were centrifuged for 15 min. and the supernatant fluid removed.
The required volume of packed cells was then transferred to a tube containing
the suspending medium. The cells were resuspended, and repacked for 15 min. in
the centrifuge. The supernatant fluid was removed immediately prior to the
addition of serum to be absorbed. All standard serum absorptions were made with
equal volumes of packed cells, and were performed 6 times, or until absorption was
complete.
ANALYSIS OF RABBIT ISO-ANTISERA OBTAINED FROM
DRS KELLNER AND HEDAL
Dr A. Kellner and Dr E. F. Hedal kindly sent to this laboratory samples of their rabbit'
iso-antisera anti-G and anti-£. These antisera were known to have a direct agglutination titre of 256 (anti-G) and 500 (anti-g). The anti-G serum was known to contain
another unnamed antibody, in the incomplete form, of different specificity from
either anti-G or anti-g. In order to avoid complications due to this incomplete
antibody only direct agglutination reactions were used at this stage of the work.
The cells of forty-four members of a panel of rabbits of mixed breed were tested
by direct agglutination against these sera at a dilution of 1/10 (Table 1).
Table 1. The numbers of animals which give positive reactions with antisera
to Z and G, and Y and g
z-
Z+
Y+
4G+
G-
\
->-
27
3
—>•
0
14
y-
l
g+
-*•
21
0
g-
—•
0
23
It may be seen that the distribution of animals positive to antisera G and g is
almost identical with those positive to Heard's (1953) two antisera Z and Y
respectively. There were, however, three animals (B59; A 17 and 1535) whose
cells were very weakly positive to serum anti-G and yet negative to serum anti-Z.
Nevertheless, the evidence detailed below points strongly to Z being identical
with G, and similarly Y with g.
Kellner & Hedal's conclusion (1953) that all rabbits possess either G or g was
found not to be true of the animals tested in this laboratory.
Of the seven animals in the panel which were negative to antisera Z and Y, four
were also negative to both anti-G and anti-£, while the other three were responsible
for the exceptionally weak reactions with anti-G, discussed above. In order to confirm that G andg may both be absent from a rabbit, Z-negative, Y-negative animals
A study of the blood groups of the rabbit
443
were mated. It was confirmed that the litter was Z—Y— and then the eight young
were tested against anti-G and anti-£.
It may be seen that three members of the litter gave unexpected positive direct
agglutination reactions with serum anti-G (Table 2).
Table 2. The reactions of a Z-negative, Y-negative (i.e. tvw) litter
with unabsorbed antisera to G and g
Anti-G
+
A
Jl
_+
Anti-g
1 1 1 1 1 1 1 1
Member of
ZYlitter
It seemed possible that the difference of reaction between anti-G and anti-Z
might be due to the unnamed incomplete antibody known to be in the anti-G serum,
perhaps acting as a complete antibody to some cells.
To test this hypothesis the following cells were used to absorb different portions
of serum anti-G:
(a) A 45 which reacted to the unnamed incomplete antibody in anti-G but did
not possess the G antigen itself.
(b) Jz of the litter previously described. This was negative to sera both anti-Z
and anti-y, but gave positive direct agglutination with serum anti-G.
(c) A16 which possessed no antigen reacting with anti-G and was used as a
control.
Testing back produced the results shown in Table 3.
It may be seen that both J2 and ^ 4 5 cells removed the 'impurities' reacting with
^ 4 5 , Jz, J\ and Jy, but left the characteristic anti-G agglutinin, which then had
reactions identical with anti-Z. The three exceptional cells (A 17, 1535 and B59)
which gave rise to the discrepancy between Z and G in Table 1 all gave negative
reactions with direct agglutination and antiglobulin sensitization test with the
absorbed anti-G sera. The very weak reaction with Jz cells shown by anti-G
absorbed by ^45 could not be removed by further absorption with ^ 4 5 . This weak
reaction could not be demonstrated by the antiglobulin sensitization test, as presumably the antibody was washed off the cells. In this it is quite different from the
main antibody reacting with Jz and ^ 4 5 , and must represent a very weak antibody
in the anti-G serum of a third specificity.
It seems likely that Jz and A 45 bear the same antigen, but this could not be fully
established since further absorptions could not be done owing to lack of serum.
Serum anti-G, therefore, contains the typical G agglutinin and at least one other
agglutinin which probably acts as an incomplete antibody with some cells and a
29-2
I
I
I
A I 6 control
by
Absorbed by
All
+
Serum
anti-G
testing
Method
of
DA =direct agglutination;
+=Only two animals tested ;
A G = antiglobulin;
t = Only four nnirnals tested.
-+
3+
-
-
-
+ ++
2-,
--
2- cells
Five
other
Table 3. The reactions of serum anti-G, after absorption with 2-negative cells wlrich had s h m a reaction
with the unabsorbed serum
Twelve
++ ++
++
++
+ +t
++t
++
++
Z + cells
A study of the blood groups of the rabbit
445
complete antibody with others. It also contains a trace of antibody of a third
specificity.
In further experiments, whose object was to verify that G and Z, and g and Y,
were identical, titrations were performed in parallel, to see whether the cells which
are less agglutinable for Z were also less agglutinable for G, and similar comparisons
were made between Y and g.
Table 4. The parallelism in titres obtained with antisera to Z and G;
and Y andg
Test
cells
Anti-y
Anti-£
titre
titre
1963
2472
32
32
Test
cells
Anti-Z
Anti-G
titre
titre
2403
2472
16
32
32
64
64
A2S
64
64
64
64
128
B66
256
256
A21
A2S
32
16
The close parallelism between these results provides additional evidence that Z
is identical with G, and Y with g.
Kellner & Hedal's experiments on haemolysis of rabbit cells by anti-G and
anti-£ in the presence of guinea-pig complement have been repeated and confirmed.
Kellner & Hedal (1953) described G and g as an allelic pair, at least one of which
must be present in every rabbit. While their evidence seems to show this to have
been the case with their own rabbits, it is not true of the stock of this laboratory in
which a number of rabbits have been found which possess neither G (Z) nor g (Y).
These animals have been found to possess Heard's antigen W.
GENETIC RELATIONSHIP BETWEEN ANTIGENS Z, Y AND W
If it is assumed that Z, Y and W are inherited independently, the eight genetic
combinations possible are shown in Table 5.
Table 5. The distribution of rabbits possessing the antigens Z, Y and W
z
ZY
ZW
Y
YW
W
ZYW
0
22
19
19
4
11
18
0
0
It may be seen that among ninety-three members of the panel, all had at least one,
but never more than two of the three antigens. This distribution suggested that
Z, Y and W formed an allelomorphic trio, and therefore breeding experiments
were performed in order to test this hypothesis. Twenty-one types of mating are
possible on this hypothesis, and all these have been tested.
Among the sixty-two families investigated, with 258 young, not a single example
has been found where antigens present in the young were incompatible with those
expected from their known parentage (Table 6).
446
VALERIE C. JOYSEY
The antigens Z, Y and W behave as Mendelian characters controlled by three
allelomorphic genes; the characters cannot strictly be called dominant, for the
heterozygote has somewhat less antigen than the homozygote.
Table 6. The inheritance of the antigens Z, Y andW.
Number of
Type of mating
ZZxZZ
ZZxZY
ZZxZW
ZZx YY
ZZx YW
ZZxWW
ZYxZY
ZYxZW
ZYx YY
ZYxYW
ZYxWW
ZWxZW
ZWx YY
ZWx YW
ZWxWW
YYx YY
YYx YW
YYxWW
YWx YW
YWx WW
WWxWW
fa m i l l
No. of babies in litters of genotypes
M
tested
ZZ
8
4
5
29
6
6
2
I
I
3
3
3
1
3
1
1
2
3
ZY
1
1
zw
YY
YW
WW
10
6
12
1
1
5
3
3
4
5
Table of matings
8
4
7
1
5
5
6
5
2
0
11
8
4
3
0
11
2
2
1
.
6
0
11
6
4
6
15
4
6
1
8
3
4
4
1
4
18
DOSAGE EFFECT OF GENES OF THE Z, Y AND W SYSTEM
As a result of direct agglutination titrations on rabbit cells Heard (1953) showed
that:
(a) Cells bearing the same antigen were agglutinated to different titres by a serum
containing a single antibody. Those cells that demonstrated antibody to a high titre
were said to be ' strongly agglutinable', and those that were able to demonstrate the
same antibody to a low titre were said to be 'poorly agglutinable'.
(b) ' Strongly agglutinable' cells appear to have a larger number of functional
antigenic sites on the erythrocytes than ' poorly agglutinable' cells as demonstrated
by absorption experiments.
Examination of the data for Z and Y suggested that the least agglutinable cells
are those with the genotypes ZW and YW, and the most agglutinable cells are
homozygous for Z and Y. In order to investigate this possible relationship between
the genotype of a cell, and its degree of agglutinability, a series of direct agglutination titrations was performed for Z and Y antigens. The antiglobulin test was not
used in these titrations, as it was found that the titre of a serum differed little
between different types of cells when the antiglobulin sensitization test was performed. Titrations have not been performed for W, as no high titred 'complete'
anti-W sera are yet available.
A study of the blood groups of the rabbit
The titrations were performed by the method outlined in the section on technique. At each titration at least one constant control cell was used, namely a very
poorly agglutinable cell, 54 for Y and 2473 for Z.'
Using a modified form of Race & Sanger's (1950) method of scoring, the titration
readings were converted into numerical values, i.e.:
F=io,
=8,
+ =5,
= 3.
+) = 6,
W=2,(W)=i.
The values obtained in each titration were totalled to give a ' score' for each type
of cell. To prevent the scores from becoming too large the values below a fixed
base-line were ignored. This base-line was taken as the (+ +) end-point for the
standard poorly agglutinable type of cell. An example of scoring is shown in
Table 7.
Table 7. The method of scoring used in titrations
Serum dilutions
Cells
1/8
1/16
++
(+)
(to)
+ +V
+ +V
1/2
i/4
54
++
12
+ +V
1/32
Score
1/64
1/128
(++)
to
1/256
12
+ +V
—
48
f base-line
Each titration was performed at least twice, and the values obtained were averaged
for each type of cell. In the case of the control cells the maximum discrepancy
obtained between repeated titrations was 5 points for Z and 3 points for Y, whereas
the greatest discrepancy for the cells of any rabbit was 12 for Z and 11 for Y.
Therefore the greatest error in this work represents a difference in titre of slightly
more than one doubling dilution. The average values were plotted on a graph
(Fig- 1).
It may be seen that in the case of the Y antigen the range of scores shown by the
Y Y cells, which are the most agglutinable, does not overlap that of the Y W cells,
which are the least agglutinable. The YZ cells occupy an intermediate position
between the YY and YW cells, and overlap the range of YW cells.
The relationship is not so clear with regard to the Z antigen. There appears to be
little, if any, difference between the ranges of ZY and Z W cells, although ZZ cells
are clearly more agglutinable than either.
If we accept Heard's (1953) conclusion that strongly agglutinable cells in the
rabbit have a large number of antigen sites, then it appears that the cells homozygous for Z and Y possess more Z and Y antigen sites than the corresponding
heterozygous cells. Furthermore, in cells heterozygous for Y, most YZ cells have
more Y sites than Y W cells.
Similar 'dosage' effects have been recognized in human blood groups in the
MNSs, 'Rh', Kell, Duffy and P systems (Landsteiner & Levine, 1927; Sanger &
448
VALERIE C. JOYSEY
Race, 1951 ; Race, Taylor, Boorman & Dodd, 1943; Lawler & Race, 1950;
Malone & Dunsford, 1951; Race, Sanger & Lehane, 1952; Plant, Ikin, Mourant,
Sanger & Race, 1953; Fisher, 1953; Mourant, 1947; Race, 1953).
Antigen Z
ZW
ZY
Genotypes of cells
Antigen Y
ZZ
YZ
YY
Genotypes of cells
Fig. 1. Graph to illustrate the differences in titre obtained by titration of anti-Z and anti- Y
aera with cells of different types
The effect here seems analogous to that in the Rh system in man, where cDE/cde
cells are more agglutinable with anti-i? sera than CDe/cDE cells, where possibly
the homozygous D competes for substrate with the E antigen more successfully than
does heterozygous D (Lawler & Race, 1950). Similarly, in rabbits W appears to
compete with Y for substrate more effectively than does Z, and therefore the number
of Y sites is lower in Y W than YZ cells.
NOTATION
Heard's temporary notation of Z, Y and W is not the most convenient way to
express an allelomorphic trio. The notation of Kellner & Hedal (1953) has precedence over that of Heard, but the use of G an&g leaves difficulty as to the naming
of the third allele. It is therefore suggested that the alleles be called: G° (identical
with G and Z); Gb (identical with g and Y); G° (identical with W).
The use of this system leaves room for the addition of any further alleles that may
subsequently be found.
A study of the blood groups of the rabbit
a
449
b
It seems possible that G , G , and G* may be the same as Castle & Keeler's (1933)
allelomorphic trio, Hi, Hz, and O.
G° can most probably be identified with O since complete antibodies to Gc are
rare, and therefore this antigen would not have been easily demonstrable before
the advent of the antiglobulin sensitization test. It has not been possible to compare
gene frequencies with those of other authors, due to deliberate selection and inbreeding in the present stock.
SUMMARY
1. Kellner and Hedal's antigen G is identical with Heard's antigen Z, and
similarly g with Y.
2. Kellner & Hedal's (1953) conclusion that all rabbits must possess at least one
of the antigens G or g was found not to be true of animals in this stock. Animals
lacking both antigens were found to possess Heard's antigen W.
3. Z, Y and Wform an allelomorphic trio.
4. Heard's evidence strongly suggests that the most agglutinable cells have more
antigen sites (for the antibody in question), than less agglutinable cells. Evidence
provided here shows that in the case of the Z and Y antigens homozygous cells are
more agglutinable than heterozygous cells, and therefore probably have more sites
of the Z or Y antigens respectively. Also the number of Y antigen sites is greater
in YZ than in Y W cells. It was not possible to demonstrate a similar difference for
Z between ZY and ZW cells.
5. It is suggested that the notation of this allelomorphic trio in rabbits should be
standardized as follows: Ga = Kellner & Hedal's G and Heard's Z; Gb = Kellner &
Hedal's £ and Heard's Y; G°=Heard's W.
I should like to express my gratitude to the Agricultural Research Council for
provision of a grant, to Dr R. R. A. Coombs for supervision and criticism of this
work, to Dr D. Heard for training in techniques used, and to Dr R. R. Race,
Dr Ruth Sanger and Dr A. E. Mourant for criticism of the manuscript.
I am also grateful to Dr A. Kellner and Dr E. F. Hedal for provision of samples
of their anti-G and anti-g sera.
ADDENDUM
Since the completion of this paper, the fifty-three offspring of thirteen more matings
in ten of the mating categories shown in Table 6 have been tested. All offspring of
these matings possessed the expected Z, Y and W antigens.
450
VALERIE C. JOYSEY
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