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Haemolytic activity of sheep complement for two assay systems
Veterinary Immunology and Immunopathology, 2 (1981) 393--400 Elsevier Scientific Publishing Company, Amsterdam -- Printed in Belgium
393
HAEMOLYTIC ACTIVITY OF SHEEP COMPLEMENTFOR TWO ASSAY SYSTEMS W. JONAS and M. STANKIEWICZ Wallaceville Animal Research Centre, Research D i v i s i o n , M i n i s t r y of A g r i c u l t u r e and Fisheries, Private Bag, Upper Hutt, New Zealand (Accepted 16 A p r i l 1981 ABSTRACT Jonas, W. and Stankiewicz, M. 1981. Haemolytic a c t i v i t y of sheep complement for two assay systems. Vet. Immunol. Immunopathol., 2: 393-400. Sheep complement (C) is haemolytic for sheep erythrocytes sensitized with r a b b i t antibody (sheep E-rabbit A) provided serum is used as soon as possible a f t e r c o l l e c t i o n . I f l e f t at 4 °C to separate from the c l o t , serum C a c t i v i t y for sheep E-rabbit A is markedly reduced. Heparinized plasma retains i t s haemolytic t i t r e for at least 24 h at 4 °C. Plasma from Mg2+-ethyleneglycol t e t r a a c e t i c acid (EGTA) blood is non-haemolytic, but addition of Ca2+ p a r t i a l l y restores the t i t r e . A high concentration of rabbit A is necessary to sensitize sheep E. Sheep C is haemolytic for human erythrocytes sensitized with sheep antibody (human E-sheep A) in the presence of Mg2+-EGTA. This C a c t i v i t y is stable at 4 °C For 24 h in serum, Mg2 -EGTA plasma and heparinized plasma. Haemolytic a c t i v i t y of serum heated at 50 °C for 30 min was restored by a f a c t o r B containing CM-cellulose f r a c t i o n of foetal lamb serum in the presence of Mg2+-EGTA for human E-sheep A but not sheep E-rabbit A. These findings show that sheep C haemolysis of sheep E-rabbit A requires a Ca2.- Mg2+-dependent pathway that is l a b i l e in v i t r o for 24 h at 4 °C. INTRODUCTION Although sheep erythrocytes sensitized by rabbit antibody (sheep E-rabbit A) are r e a d i l y haemolysed by human and guinea-pig complement (g-p C), sheep C is reported as being poorly or non-haemolytic f o r sheep E-rabbit A (Rice and Crowson, 1950; Barta et a l . , 1975).
During the course of experiments with
sheep C i t was noted that i f the serum C source was used as soon as possible a f t e r c o l l e c t i o n from the sheep, haemolysis of sheep E-rabbit A took place. The purpose of t h i s paper is to report some c h a r a c t e r i s t i c s of sheep C haemolysis of sheep E-rabbit A.
In a d d i t i o n , t h i s sheep C haemolytic a c t i v i t y
is compared with that for human erythrocytes sensitized with sheep antibody, an i n d i c a t o r system that is r e a d i l y haemolysed by sheep C (Feinstein and Hobart, 1969; Rhee et a l . ,
1970).
0165-2427/81/0000--0000/$02.50 © 1981 Elsevier Scientific Publishing Company
394
MATERIALS AND METHODS
Diluent and chelating agents Phosphate buffered saline (PBS, 0.15 M, pH 7.2) was used for some procedures. Veronal buffered saline containing Ca2+ and Mg2+ (Mayer, 1961) was used as the diluent in the haemolytic assays. Stock solutions (0.1 M, pH 7.6) of ethylenediaminetetraacetic acid (EDTA) and ethyleneglycoltetraacetic acid (EGTA) were prepared as described by Fine et al. (1972).
A stock solution of Mg2+-EGTA
(stock 0.1M EGTAcontaining 20 mM Mg2+) was prepared with MgCl2 and the pH adjusted to pH 7.6.
Stock CaC122H20 (30 mM Ca2+) was prepared in saline.
Haemolytic assays Group 0 Rh-positive human erythrocytes (human E) or sheep erythrocytes (sheep E), collected into Alsever's solution were obtained weekly.
The erythrocytes
were washed three times in PBS and made up to 5% in diluent. Six ewes were once injected with human E in Freund's complete adjuvant.
Ten-
day serum was pooled, heated at 56 °C for 30 min and treated with saturated ammonium sulphate (final concentration, 40%). The globulin was washed, reconstituted to the original volume, dialysed against PBS and stored as aliquots at -17 °C.
I t is referred to as sheep A.
'Haemolysin' (rabbit antibody to sheep E) was purchased from the Commonwealth Serum Laboratories, Melbourne, Australia.
Globulin, prepared as above, is
referred to as rabbit A. I n i t i a l l y , dilutions of sheep A and rabbit A were used to sensitize the erythrocytes, but routinely, a 1:25 d i l u t i o n was used. Equal volumes of amboceptor and 5% erythrocytes were mixed immediately before use. Human E sensitized with sheep A are referred to as human E-sheep A: with rabbit A are referred to as sheep E-rabbit A.
sheep E sensitized
The sensitized cells were
not washed before use. Although the 1:25 d i l u t i o n of sheep A caused agglutination of human E, the cells were readily haemolysed. The complement source was s e r i a l l y diluted in 0.4 ml of diluent and 0.05 ml of chelating agent or diluent was added.
In experiments with the CM-cellulose
fraction, 0.2 ml of the fraction (diluted 1:4) was added to 0.2 ml of s e r i a l l y diluted sheep serum heated at 50 °C for 30 min. Sensitized erythrocytes were added and incubated for 30 min (sheep C, 39 °C; g-p C, 37 °C). CM-cellulose chromatography fraction The method used to prepare factor B from human serum (Lachmann et a l . , 1973)
395
was used with pooled foetal lamb serum (collected from near-term foetal lambs). The CM-cellulose fraction used (11-13 mmhos, 0.49 mg/ml, by gradient polyacrylamide gel electrophoresis contained 5 protein bands (515, 330, 140, 107 and 84 000 daltons).
The fraction had a haemolytic t i t r e with human E-sheep A of
1:32 with 50 °C sheep serum (1:4) in the presence of Mg2+-EGTA. After separation on Sephadex G-200, the haemolytic a c t i v i t y was associated with the 107 000 dalton protein. Complement Blood was obtained from about 65 sheep and used as individual or pooled samples.
For serum, 20 ml of blood was collected into dry Universal bottles and
allowed to clot at room temperature. centrifuged and used immediately. 24 h.
At 1.5 h, some serum was separated,
Some 1.5 h serum was frozen at -20 °C for
At 1.5 h some clotted blood was placed at 4 °C and at 7 and 24 h, serum
was poured o f f , centrifuged and used. For heparinized plasma~ 25 ml of blood was added to I ml of phosphate buffered saline plus heparin (Pularin, Evans Medical Ltd.) giving a final concentration of 5 units/ml.
For EDTAand Mgz+-
EGTA plasma, 18 ml of blood was added to 2 ml of 0.1M EDTAor 20 mM Mg2+-O.1M EGTA respectively.
For the Mg2+-EGTA-Ca2+ samples, one part of 30 mM Ca2+ was
added to 9 parts of Mg2+-EGTAblood within 20 min of collection.
Some samples
were heated at 50 °C f o r 30 min or 56 °C f o r 30 min. G-p blood was allowed to c l o t at room temperature (2 h) and then l e f t overnight at 4 °C; serum was separated, centrifuged and frozen in aliquots at -20 °C. RESULTS
Haemolytic a c t i v i t ~ of serum Sheep serum haemolysis for sheep E-rabbit A was markedly reduced after standing at 4 °C for 24 h whereas the a c t i v i t y for human E-sheep A was unaffected (Table I ) .
(The natural l y t i c t i t r e of sheep C with human E was
1:16-32.) The t i t r e of 1.5 h sheep C with sheep E-rabbit A was not increased above 1:16-32 even i f undiluted rabbit A was used. There was no lysis when rabbit A was used at >1:400.
896
TABLE I Effect of the time sheep serum is l e f t on the clot on haemolytic a c t i v i t y of that serum
Haemolytic assay system Sheep E-rabbit A Human E-sheep A
T i t r e (reciprocal) a f t e r storage '1.5 h at room 4 °C Frozen ~ temperature 7 h 24 h 24 h
D i l u t i o n of amboceptor FI:25 |I:i00 LI:400 [1:25 |I:i00 LI:400
16 8 4 256 64 32
8 2 2 256 64 32
2 0 0 256 64 32
16 8 4 256 64 32
Haemolytic a c t i v i t y of plasma The t i t r e of heparinized plasma with sheep E-rabbit A was 1:16-32 at 1.5 h (Table I I ) .
A f t e r 24 h at 4 °C, the plasma t i t r e was s t i l l
1:16-32 whereas the
serum t i t r e dropped to 1:2 or less. Mg2+-EGTA plasma had the same t i t r e heparinized plasma (Table I I ) .
f o r human E-sheep A as did serum and
The plasma t i t r e s did not drop a f t e r 24 h at
4 °C. TABLE I I Haemolytic a c t i v i t y of plasma used 1.5 h a f t e r c o l l e c t i o n from sheep Haemolytic assay system Sheep E-rabbit A* Human E-sheep A*
Complement t i t r e Dry b o t t l e 16 256
(reciprocal) of blood collected into Heparin EDTAt Mg2+-EGTA T 16 256
0 0
0 256
tFinal concentration, 0.01M EDTA and 2 mM Mg2+-O.01M EGTA *Rabbit and sheep antibody used at 1:25 Requirement for haemolysis Sheep C Haemolytic a c t i v i t y with sheep E-rabbit A and human E-sheep A for a l l samples except Mg2+-EGTA-Ca2+ was abolished by heating and EDTA (Table I I I ) .
Mge+-EGTA
i n h i b i t e d haemolytic a c t i v i t y for sheep E-rabbit A but not human E-sheep A. Haemolytic a c t i v i t y f o r human E-sheep A was restored to 50 °C 1.5 h sheep serum with the CM-cellulose f r a c t i o n ( i n the presence of Mg2+-EGTA) but not for sheep E-rabbit A.
397
TABLE I I I Effect of heat and chelating agents on haemolytic a c t i v i t y of 1.5 h sheep plasma and serum and on guinea-pig complement Haemolytic system
Dilution of amboceptor
1:25 Sheep Erabbit A
1:400 1:1600 1:6400
Complement source Sheep Plasma (heparin) Mg2+-EGTA-Ca2+ Serum Guinea pig Sheep Plasma (heparin) Mg2+-EGTA-Ca2+ Serum
Human Esheep A
Treatment of complement ~ °C °C Mg2+ ' None 50 56 EDTA 30 min 30 min EGTA
*Haemolytic complement t i t r e
16" 8 16
0 2 0
0 0 0
0 0 0
0 0 0
640 640 320
160 80 0
0 0 0
0 0 0
0 0 0
256 256 256
0 64 0
0 0 0
0 0 0
256 256 256
(reciprocal)
G-p C With the 1:6400 r a b b i t A d i l u t i o n , 50 °C g-p C was n o n - l y t i c with sheep Er a b b i t A (Table I I I ) .
G-p C was non-haemolytic a f t e r heating at 56 °C for
30 min or when used in the presence of EDTA or Mg2+-EGTA. DISCUSSION The data in t h i s paper (summarised in Table IV) indicates that two complement haemolytic pathways are present in sheep.
The previously described Mg2+-dependent
pathway that is mainly responsible for haemolysis of human E-sheep A (Jonas and Stankiewicz, 1980a) is stable in v i t r o at 4 °C for at least 24 h.
A pathway
that is l a b i l e in v i t r o at 4 °C and requires Ca2+ and Mg2+ appears to be responsible for haemolysis of sheep E-rabbit A. Some of the results can also be used to explain the reported poor - or nonhaemolytic a c t i v i t y of sheep C for sheep E-rabbit A.
F i r s t l y , four 50% units
of r a b b i t A amboceptor are conventionally used to s e n s i t i z e sheep E (Bier et a l . , 1945).
This concentration (1:800 with the rabbit A used) did not sensitize
sheep E for l y s i s by sheep C. necessary (Table I ) .
For 'optimal' a c t i v i t y ,
a 1:25 d i l u t i o n was
Secondly, unless sheep serum is used w i t h i n a few hours
of c o l l e c t i o n , the haemolytic a c t i v i t y f o r sheep E-rabbit A w i l l decay, even at 4 °C.
Heparinized plasma w~s, however, a better source of C f o r l y s i s of sheep
E-rabbit A.
I t could be used w i t h i n minutes of c o l l e c t i o n or l e f t f o r 24 h at
4 °C without any reduction in t i t r e .
398
TABLE IV Summary of haemolytic activity of sheep complement for two assay systems Sheep E-rabbit A
Human E-sheep A
High 1:16 - 1:32 Labile Stable No haemolysis No haemolysis No haemolysis
Moderate-high 1:128 -1:256 Stable Stable No haemolysis Haemolysis No haemolysis
No haemolysis No haemolysis Haemolysis
Haemolysis Haemolysis Haemolysis
Antibody (amboceptor) requirement Complement t i t r e Serum, 24 h at 4 °C Plasma (heparinized) 24 h at 4 °C Serum/plasma + EDTA Serum/plasma + Mg2+-EGTA Serum/plasma 50 °C, 30 min Serum/plasma 50 °C, 30 min + CM cellulose fraction + Mg2+-EGTA
Sheep C w i l l haemolyse human E-sheep A in the presence
of a concentration of
Mg2+-EGTA that i n h i b i t s sheep and g-p C haemolysis of sheep E-rabbit A (Table III).
The haemolytic t i t r e
for sheep E-rabbit A could be p a r t i a l l y restored to
sheep Mg2+-EGTA blood by the addition of Ca2+,
As the classical and a l t e r n a t i v e
pathways have been distinguished by use of EGTA or Mg2+-EGTA with serum from the bovine (Pang and Aston, 1977), human and g-p (Sandberg and Osler, 1971; Forsgren and Quie, 1974; P l a t t s - M i l l s and Ishizaka, 1974; Fine, 1977), these results suggest that sheep C haemolysis of sheep E-rabbit A is classical pathway dependent whereas haemolysis of human E-sheep A is a l t e r n a t i v e pathway dependent A l t e r n a t i v e pathway a c t i v i t y is l a b i l e at 50 °C f o r 30 min (Goodkofsky and Lepow, 1971).
The haemolytic a c t i v i t y of sheep C f o r both sheep E-rabbit A and
human E-sheep A (except the Mge+-EGTA-Ca2+ samples, Table I I I ) was l a b i l e to heating at 50 °C.
As the Ca2+-Mg2+ requirement for haemolysis of sheep E-rabbit
A indicated a classical pathway a c t i v i t y , should not have been l a b i l e to 50 °C.
the haemolytic a c t i v i t y possibly
However, g-p C l y s i s of sheep E-rabbit A,
a classical pathway dependent a c t i v i t y (Fine et a l . , 1972) was ' l a b i l e '
to 50 °C
when the rabbit A concentration s e n s i t i z i n g sheep E became l i m i t i n g (Table I I I ) . The CM-cellulose f r a c t i o n of FLS contained a protein that had a molecular weight of 107 000.
The molecular weight of t h i s protein was s i m i l a r to that of
bovine factor B (Pang and Aston, 1978).
The f r a c t i o n was able to restore
haemolytic a c t i v i t y to 50 °C serum for human E-sheep A in the presence of Mg2+EGTA but not to sheep E-rabbit A.
This suggests that the 107 000 dalton protein
was sheep f a c t o r B. The evidence for the presence of dual C a c t i v a t i o n pathways is of i n t e r e s t r e l a t i v e to the published data on the components of C in sheep serum.
Although
CI has been demonstrated (Colton et a l . , 1968, Barta and Hubbert, 1978), the two other components of the classical pathway (C2 and C4) have e i t h e r not been detected (Barta and Hubbert, 1978) or found in very low levels only (Borsos and Rapp, 1965).
As C2 and C4 were measured in sheep E-rabbit A-based tests
399
(Borsos and Rapp, 1965; Barta and Hubbert, 1978), the t i t r e s might have been higher, i f a high concentration of rabbit A was used and sheep C was assayed immediately a f t e r collection.
A l t e r n a t i v e l y , i f C2 and C4 are genuinely low
(hence the low t i t r e with sheep E-rabbit A), this could be the consequence of an e f f i c i e n t Mg2+-dependent pathway that can be activated by antibody-antigen complexes (Tables I - I I I ,
and Jonas and Stankiewicz, 1980b)o Furthermore, only
low or n e g l i g i b l e levels of C3 (Rice and Crowson, 1950; Barta and Hubbert, 1978) and C5 (Barta and Hubbert, 1978) have been detected in sheep serum.
I t is
possible that sheep C3 and C5 could be detected more e f f e c t i v e l y using a Mg2+dependent pathway and human E-sheep A instead of a classical pathway system with sheep E-rabbit A. ACKNOWLEDGEMENTS We would l i k e to thank Mr Hugh Pulford for technical assistance. Dr M. Stankiewicz g r a t e f u l l y acknowledges a New Zealand Government National Research Advisory Council Post-Doctoral Fellowship. REFERENCES Barta, O. and Hubbert, N.L,, 1978. Testing of haemolytic complement components in domestic animals. Am. J. vet. Res., 39:1303. Barta, 0., Barta, V., Shirley, R.A. and McMurry, J.D., 1975. Haemolytic assay of sheep complement. Zentbl. VetMed., 22:254. Bier, O.G., Leyton, G., Mayer, M.M. and Heidelberger, M. 1945. A comparison of human and guinea pig complement and t h e i r component fractions. J. exp. Med. 81:449. Borsos, T. and Rapp, H.J., 1965. Estimation of molecular size of complement components by Sephadex chromatography. J. Immunol., 94:510. Colton, H.R., S i l v e r s t e i n , A.M., Borsos, T. and Rapp, H.J., 1968. Ontogeny of the f i r s t component of sheep complement. Immunology, 15:459. Feinstein, A. and Hobart, M.J., 1969. Structural relationship and complement f i x i n g a c t i v i t y of sheep and other ruminant immunoglobulin G subclasses. Nature Lond., 223:950. Fine, D.P., 1977. Comparison of ethyleneglycoltetraacetic acid and i t s magnesium s a l t as reagents for studying a l t e r n a t i v e pathway function. Inf. Immunol., 16:124. Fine, D.P., Marney, S.R., Colley, D.G., Sergent, J.S. and Des Prez, R.M., 1972. C3 shunt activation in human serum chelated with EGTA. J. Immunol., 109:807. Forsgren, A. and Quie, P.G., 1974. Opsonic a c t i v i t y in human serum chelated with ethylene g l y c o l t e t r a - a c e t i c acid. Immunology, 26:1251. Goodkofsky, I. and Lepow, I . H . , 1971. Functional relationship of factor B in the properdin system to C3 proactivator of human serum. J. Immunol., 107: 1200. Jonas, W. and Stankiewicz, M., 1980a. Complement a c t i v i t y in serum and Mg2+EGTA plasma from various animals for two haemolytic indicator systems. Immunology Letters, 2:65. Jonas, W. and Stankiewicz, M., 1980b. A modified complement f i x a t i o n t e s t that is subject to minimal anti-complementary a c t i v i t y by sheep serum. N.Z. vet. J . , 28:163.
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Lachmann, P.J., Hobart, M.J. and Aston, W.P., 1973. Complement technology. In: Handbook of Experimental Immunology. Ed by D.M. Weir, 2nd edn., p.5.1. Blackwell Scientific Publications, Oxford. Mayer, M.M. 1961. In: Kabat and Mayer's Experimental Immunochemistry, 2nd edn., Charles C. Thomas, publisher, Springfield, I I I . , p.149. Pang, A.S.D. and Aston, W.P., 1977. Alternative complement pathway in bovine serum: lysis of human erythrocytes. Am. J. vet. Res., 38:355. Pang, A.S.D. and Aston, W.P., 1978. The alternative complement pathway in bovine serum: the isolation of a serum protein with factor B a c t i v i t y . Immunochem., 15:529. P l a t t s - M i l l s , T_A.E. and Ishizaka, K., 1974. Activation of the alternative pathway of human complement by rabbit cells. J. Immunol., 113:348. Rhee, Y.O., Broad, S. and Jonas, W.E., 1970. Agglutinating and complementmediated a c t i v i t i e s of sheep antisera, IgM, slow and fast y-globulin antibodies. Res. vet. Sci., 11:123. Rice, C.E. and Crowson, C.N., 1950. The interchangeability of the complement components of different animal species. I I . In the haemolysis of sheep erythrocytes sensitized with rabbit amboceptor. J. Immunol., 65:201. Sandberg, A.L. and Osler, A.G., 1971. Dual pathway of complement interaction with guinea pig immunoglobulins. J. Immunol., 107:1268.
393
HAEMOLYTIC ACTIVITY OF SHEEP COMPLEMENTFOR TWO ASSAY SYSTEMS W. JONAS and M. STANKIEWICZ Wallaceville Animal Research Centre, Research D i v i s i o n , M i n i s t r y of A g r i c u l t u r e and Fisheries, Private Bag, Upper Hutt, New Zealand (Accepted 16 A p r i l 1981 ABSTRACT Jonas, W. and Stankiewicz, M. 1981. Haemolytic a c t i v i t y of sheep complement for two assay systems. Vet. Immunol. Immunopathol., 2: 393-400. Sheep complement (C) is haemolytic for sheep erythrocytes sensitized with r a b b i t antibody (sheep E-rabbit A) provided serum is used as soon as possible a f t e r c o l l e c t i o n . I f l e f t at 4 °C to separate from the c l o t , serum C a c t i v i t y for sheep E-rabbit A is markedly reduced. Heparinized plasma retains i t s haemolytic t i t r e for at least 24 h at 4 °C. Plasma from Mg2+-ethyleneglycol t e t r a a c e t i c acid (EGTA) blood is non-haemolytic, but addition of Ca2+ p a r t i a l l y restores the t i t r e . A high concentration of rabbit A is necessary to sensitize sheep E. Sheep C is haemolytic for human erythrocytes sensitized with sheep antibody (human E-sheep A) in the presence of Mg2+-EGTA. This C a c t i v i t y is stable at 4 °C For 24 h in serum, Mg2 -EGTA plasma and heparinized plasma. Haemolytic a c t i v i t y of serum heated at 50 °C for 30 min was restored by a f a c t o r B containing CM-cellulose f r a c t i o n of foetal lamb serum in the presence of Mg2+-EGTA for human E-sheep A but not sheep E-rabbit A. These findings show that sheep C haemolysis of sheep E-rabbit A requires a Ca2.- Mg2+-dependent pathway that is l a b i l e in v i t r o for 24 h at 4 °C. INTRODUCTION Although sheep erythrocytes sensitized by rabbit antibody (sheep E-rabbit A) are r e a d i l y haemolysed by human and guinea-pig complement (g-p C), sheep C is reported as being poorly or non-haemolytic f o r sheep E-rabbit A (Rice and Crowson, 1950; Barta et a l . , 1975).
During the course of experiments with
sheep C i t was noted that i f the serum C source was used as soon as possible a f t e r c o l l e c t i o n from the sheep, haemolysis of sheep E-rabbit A took place. The purpose of t h i s paper is to report some c h a r a c t e r i s t i c s of sheep C haemolysis of sheep E-rabbit A.
In a d d i t i o n , t h i s sheep C haemolytic a c t i v i t y
is compared with that for human erythrocytes sensitized with sheep antibody, an i n d i c a t o r system that is r e a d i l y haemolysed by sheep C (Feinstein and Hobart, 1969; Rhee et a l . ,
1970).
0165-2427/81/0000--0000/$02.50 © 1981 Elsevier Scientific Publishing Company
394
MATERIALS AND METHODS
Diluent and chelating agents Phosphate buffered saline (PBS, 0.15 M, pH 7.2) was used for some procedures. Veronal buffered saline containing Ca2+ and Mg2+ (Mayer, 1961) was used as the diluent in the haemolytic assays. Stock solutions (0.1 M, pH 7.6) of ethylenediaminetetraacetic acid (EDTA) and ethyleneglycoltetraacetic acid (EGTA) were prepared as described by Fine et al. (1972).
A stock solution of Mg2+-EGTA
(stock 0.1M EGTAcontaining 20 mM Mg2+) was prepared with MgCl2 and the pH adjusted to pH 7.6.
Stock CaC122H20 (30 mM Ca2+) was prepared in saline.
Haemolytic assays Group 0 Rh-positive human erythrocytes (human E) or sheep erythrocytes (sheep E), collected into Alsever's solution were obtained weekly.
The erythrocytes
were washed three times in PBS and made up to 5% in diluent. Six ewes were once injected with human E in Freund's complete adjuvant.
Ten-
day serum was pooled, heated at 56 °C for 30 min and treated with saturated ammonium sulphate (final concentration, 40%). The globulin was washed, reconstituted to the original volume, dialysed against PBS and stored as aliquots at -17 °C.
I t is referred to as sheep A.
'Haemolysin' (rabbit antibody to sheep E) was purchased from the Commonwealth Serum Laboratories, Melbourne, Australia.
Globulin, prepared as above, is
referred to as rabbit A. I n i t i a l l y , dilutions of sheep A and rabbit A were used to sensitize the erythrocytes, but routinely, a 1:25 d i l u t i o n was used. Equal volumes of amboceptor and 5% erythrocytes were mixed immediately before use. Human E sensitized with sheep A are referred to as human E-sheep A: with rabbit A are referred to as sheep E-rabbit A.
sheep E sensitized
The sensitized cells were
not washed before use. Although the 1:25 d i l u t i o n of sheep A caused agglutination of human E, the cells were readily haemolysed. The complement source was s e r i a l l y diluted in 0.4 ml of diluent and 0.05 ml of chelating agent or diluent was added.
In experiments with the CM-cellulose
fraction, 0.2 ml of the fraction (diluted 1:4) was added to 0.2 ml of s e r i a l l y diluted sheep serum heated at 50 °C for 30 min. Sensitized erythrocytes were added and incubated for 30 min (sheep C, 39 °C; g-p C, 37 °C). CM-cellulose chromatography fraction The method used to prepare factor B from human serum (Lachmann et a l . , 1973)
395
was used with pooled foetal lamb serum (collected from near-term foetal lambs). The CM-cellulose fraction used (11-13 mmhos, 0.49 mg/ml, by gradient polyacrylamide gel electrophoresis contained 5 protein bands (515, 330, 140, 107 and 84 000 daltons).
The fraction had a haemolytic t i t r e with human E-sheep A of
1:32 with 50 °C sheep serum (1:4) in the presence of Mg2+-EGTA. After separation on Sephadex G-200, the haemolytic a c t i v i t y was associated with the 107 000 dalton protein. Complement Blood was obtained from about 65 sheep and used as individual or pooled samples.
For serum, 20 ml of blood was collected into dry Universal bottles and
allowed to clot at room temperature. centrifuged and used immediately. 24 h.
At 1.5 h, some serum was separated,
Some 1.5 h serum was frozen at -20 °C for
At 1.5 h some clotted blood was placed at 4 °C and at 7 and 24 h, serum
was poured o f f , centrifuged and used. For heparinized plasma~ 25 ml of blood was added to I ml of phosphate buffered saline plus heparin (Pularin, Evans Medical Ltd.) giving a final concentration of 5 units/ml.
For EDTAand Mgz+-
EGTA plasma, 18 ml of blood was added to 2 ml of 0.1M EDTAor 20 mM Mg2+-O.1M EGTA respectively.
For the Mg2+-EGTA-Ca2+ samples, one part of 30 mM Ca2+ was
added to 9 parts of Mg2+-EGTAblood within 20 min of collection.
Some samples
were heated at 50 °C f o r 30 min or 56 °C f o r 30 min. G-p blood was allowed to c l o t at room temperature (2 h) and then l e f t overnight at 4 °C; serum was separated, centrifuged and frozen in aliquots at -20 °C. RESULTS
Haemolytic a c t i v i t ~ of serum Sheep serum haemolysis for sheep E-rabbit A was markedly reduced after standing at 4 °C for 24 h whereas the a c t i v i t y for human E-sheep A was unaffected (Table I ) .
(The natural l y t i c t i t r e of sheep C with human E was
1:16-32.) The t i t r e of 1.5 h sheep C with sheep E-rabbit A was not increased above 1:16-32 even i f undiluted rabbit A was used. There was no lysis when rabbit A was used at >1:400.
896
TABLE I Effect of the time sheep serum is l e f t on the clot on haemolytic a c t i v i t y of that serum
Haemolytic assay system Sheep E-rabbit A Human E-sheep A
T i t r e (reciprocal) a f t e r storage '1.5 h at room 4 °C Frozen ~ temperature 7 h 24 h 24 h
D i l u t i o n of amboceptor FI:25 |I:i00 LI:400 [1:25 |I:i00 LI:400
16 8 4 256 64 32
8 2 2 256 64 32
2 0 0 256 64 32
16 8 4 256 64 32
Haemolytic a c t i v i t y of plasma The t i t r e of heparinized plasma with sheep E-rabbit A was 1:16-32 at 1.5 h (Table I I ) .
A f t e r 24 h at 4 °C, the plasma t i t r e was s t i l l
1:16-32 whereas the
serum t i t r e dropped to 1:2 or less. Mg2+-EGTA plasma had the same t i t r e heparinized plasma (Table I I ) .
f o r human E-sheep A as did serum and
The plasma t i t r e s did not drop a f t e r 24 h at
4 °C. TABLE I I Haemolytic a c t i v i t y of plasma used 1.5 h a f t e r c o l l e c t i o n from sheep Haemolytic assay system Sheep E-rabbit A* Human E-sheep A*
Complement t i t r e Dry b o t t l e 16 256
(reciprocal) of blood collected into Heparin EDTAt Mg2+-EGTA T 16 256
0 0
0 256
tFinal concentration, 0.01M EDTA and 2 mM Mg2+-O.01M EGTA *Rabbit and sheep antibody used at 1:25 Requirement for haemolysis Sheep C Haemolytic a c t i v i t y with sheep E-rabbit A and human E-sheep A for a l l samples except Mg2+-EGTA-Ca2+ was abolished by heating and EDTA (Table I I I ) .
Mge+-EGTA
i n h i b i t e d haemolytic a c t i v i t y for sheep E-rabbit A but not human E-sheep A. Haemolytic a c t i v i t y f o r human E-sheep A was restored to 50 °C 1.5 h sheep serum with the CM-cellulose f r a c t i o n ( i n the presence of Mg2+-EGTA) but not for sheep E-rabbit A.
397
TABLE I I I Effect of heat and chelating agents on haemolytic a c t i v i t y of 1.5 h sheep plasma and serum and on guinea-pig complement Haemolytic system
Dilution of amboceptor
1:25 Sheep Erabbit A
1:400 1:1600 1:6400
Complement source Sheep Plasma (heparin) Mg2+-EGTA-Ca2+ Serum Guinea pig Sheep Plasma (heparin) Mg2+-EGTA-Ca2+ Serum
Human Esheep A
Treatment of complement ~ °C °C Mg2+ ' None 50 56 EDTA 30 min 30 min EGTA
*Haemolytic complement t i t r e
16" 8 16
0 2 0
0 0 0
0 0 0
0 0 0
640 640 320
160 80 0
0 0 0
0 0 0
0 0 0
256 256 256
0 64 0
0 0 0
0 0 0
256 256 256
(reciprocal)
G-p C With the 1:6400 r a b b i t A d i l u t i o n , 50 °C g-p C was n o n - l y t i c with sheep Er a b b i t A (Table I I I ) .
G-p C was non-haemolytic a f t e r heating at 56 °C for
30 min or when used in the presence of EDTA or Mg2+-EGTA. DISCUSSION The data in t h i s paper (summarised in Table IV) indicates that two complement haemolytic pathways are present in sheep.
The previously described Mg2+-dependent
pathway that is mainly responsible for haemolysis of human E-sheep A (Jonas and Stankiewicz, 1980a) is stable in v i t r o at 4 °C for at least 24 h.
A pathway
that is l a b i l e in v i t r o at 4 °C and requires Ca2+ and Mg2+ appears to be responsible for haemolysis of sheep E-rabbit A. Some of the results can also be used to explain the reported poor - or nonhaemolytic a c t i v i t y of sheep C for sheep E-rabbit A.
F i r s t l y , four 50% units
of r a b b i t A amboceptor are conventionally used to s e n s i t i z e sheep E (Bier et a l . , 1945).
This concentration (1:800 with the rabbit A used) did not sensitize
sheep E for l y s i s by sheep C. necessary (Table I ) .
For 'optimal' a c t i v i t y ,
a 1:25 d i l u t i o n was
Secondly, unless sheep serum is used w i t h i n a few hours
of c o l l e c t i o n , the haemolytic a c t i v i t y f o r sheep E-rabbit A w i l l decay, even at 4 °C.
Heparinized plasma w~s, however, a better source of C f o r l y s i s of sheep
E-rabbit A.
I t could be used w i t h i n minutes of c o l l e c t i o n or l e f t f o r 24 h at
4 °C without any reduction in t i t r e .
398
TABLE IV Summary of haemolytic activity of sheep complement for two assay systems Sheep E-rabbit A
Human E-sheep A
High 1:16 - 1:32 Labile Stable No haemolysis No haemolysis No haemolysis
Moderate-high 1:128 -1:256 Stable Stable No haemolysis Haemolysis No haemolysis
No haemolysis No haemolysis Haemolysis
Haemolysis Haemolysis Haemolysis
Antibody (amboceptor) requirement Complement t i t r e Serum, 24 h at 4 °C Plasma (heparinized) 24 h at 4 °C Serum/plasma + EDTA Serum/plasma + Mg2+-EGTA Serum/plasma 50 °C, 30 min Serum/plasma 50 °C, 30 min + CM cellulose fraction + Mg2+-EGTA
Sheep C w i l l haemolyse human E-sheep A in the presence
of a concentration of
Mg2+-EGTA that i n h i b i t s sheep and g-p C haemolysis of sheep E-rabbit A (Table III).
The haemolytic t i t r e
for sheep E-rabbit A could be p a r t i a l l y restored to
sheep Mg2+-EGTA blood by the addition of Ca2+,
As the classical and a l t e r n a t i v e
pathways have been distinguished by use of EGTA or Mg2+-EGTA with serum from the bovine (Pang and Aston, 1977), human and g-p (Sandberg and Osler, 1971; Forsgren and Quie, 1974; P l a t t s - M i l l s and Ishizaka, 1974; Fine, 1977), these results suggest that sheep C haemolysis of sheep E-rabbit A is classical pathway dependent whereas haemolysis of human E-sheep A is a l t e r n a t i v e pathway dependent A l t e r n a t i v e pathway a c t i v i t y is l a b i l e at 50 °C f o r 30 min (Goodkofsky and Lepow, 1971).
The haemolytic a c t i v i t y of sheep C f o r both sheep E-rabbit A and
human E-sheep A (except the Mge+-EGTA-Ca2+ samples, Table I I I ) was l a b i l e to heating at 50 °C.
As the Ca2+-Mg2+ requirement for haemolysis of sheep E-rabbit
A indicated a classical pathway a c t i v i t y , should not have been l a b i l e to 50 °C.
the haemolytic a c t i v i t y possibly
However, g-p C l y s i s of sheep E-rabbit A,
a classical pathway dependent a c t i v i t y (Fine et a l . , 1972) was ' l a b i l e '
to 50 °C
when the rabbit A concentration s e n s i t i z i n g sheep E became l i m i t i n g (Table I I I ) . The CM-cellulose f r a c t i o n of FLS contained a protein that had a molecular weight of 107 000.
The molecular weight of t h i s protein was s i m i l a r to that of
bovine factor B (Pang and Aston, 1978).
The f r a c t i o n was able to restore
haemolytic a c t i v i t y to 50 °C serum for human E-sheep A in the presence of Mg2+EGTA but not to sheep E-rabbit A.
This suggests that the 107 000 dalton protein
was sheep f a c t o r B. The evidence for the presence of dual C a c t i v a t i o n pathways is of i n t e r e s t r e l a t i v e to the published data on the components of C in sheep serum.
Although
CI has been demonstrated (Colton et a l . , 1968, Barta and Hubbert, 1978), the two other components of the classical pathway (C2 and C4) have e i t h e r not been detected (Barta and Hubbert, 1978) or found in very low levels only (Borsos and Rapp, 1965).
As C2 and C4 were measured in sheep E-rabbit A-based tests
399
(Borsos and Rapp, 1965; Barta and Hubbert, 1978), the t i t r e s might have been higher, i f a high concentration of rabbit A was used and sheep C was assayed immediately a f t e r collection.
A l t e r n a t i v e l y , i f C2 and C4 are genuinely low
(hence the low t i t r e with sheep E-rabbit A), this could be the consequence of an e f f i c i e n t Mg2+-dependent pathway that can be activated by antibody-antigen complexes (Tables I - I I I ,
and Jonas and Stankiewicz, 1980b)o Furthermore, only
low or n e g l i g i b l e levels of C3 (Rice and Crowson, 1950; Barta and Hubbert, 1978) and C5 (Barta and Hubbert, 1978) have been detected in sheep serum.
I t is
possible that sheep C3 and C5 could be detected more e f f e c t i v e l y using a Mg2+dependent pathway and human E-sheep A instead of a classical pathway system with sheep E-rabbit A. ACKNOWLEDGEMENTS We would l i k e to thank Mr Hugh Pulford for technical assistance. Dr M. Stankiewicz g r a t e f u l l y acknowledges a New Zealand Government National Research Advisory Council Post-Doctoral Fellowship. REFERENCES Barta, O. and Hubbert, N.L,, 1978. Testing of haemolytic complement components in domestic animals. Am. J. vet. Res., 39:1303. Barta, 0., Barta, V., Shirley, R.A. and McMurry, J.D., 1975. Haemolytic assay of sheep complement. Zentbl. VetMed., 22:254. Bier, O.G., Leyton, G., Mayer, M.M. and Heidelberger, M. 1945. A comparison of human and guinea pig complement and t h e i r component fractions. J. exp. Med. 81:449. Borsos, T. and Rapp, H.J., 1965. Estimation of molecular size of complement components by Sephadex chromatography. J. Immunol., 94:510. Colton, H.R., S i l v e r s t e i n , A.M., Borsos, T. and Rapp, H.J., 1968. Ontogeny of the f i r s t component of sheep complement. Immunology, 15:459. Feinstein, A. and Hobart, M.J., 1969. Structural relationship and complement f i x i n g a c t i v i t y of sheep and other ruminant immunoglobulin G subclasses. Nature Lond., 223:950. Fine, D.P., 1977. Comparison of ethyleneglycoltetraacetic acid and i t s magnesium s a l t as reagents for studying a l t e r n a t i v e pathway function. Inf. Immunol., 16:124. Fine, D.P., Marney, S.R., Colley, D.G., Sergent, J.S. and Des Prez, R.M., 1972. C3 shunt activation in human serum chelated with EGTA. J. Immunol., 109:807. Forsgren, A. and Quie, P.G., 1974. Opsonic a c t i v i t y in human serum chelated with ethylene g l y c o l t e t r a - a c e t i c acid. Immunology, 26:1251. Goodkofsky, I. and Lepow, I . H . , 1971. Functional relationship of factor B in the properdin system to C3 proactivator of human serum. J. Immunol., 107: 1200. Jonas, W. and Stankiewicz, M., 1980a. Complement a c t i v i t y in serum and Mg2+EGTA plasma from various animals for two haemolytic indicator systems. Immunology Letters, 2:65. Jonas, W. and Stankiewicz, M., 1980b. A modified complement f i x a t i o n t e s t that is subject to minimal anti-complementary a c t i v i t y by sheep serum. N.Z. vet. J . , 28:163.
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Lachmann, P.J., Hobart, M.J. and Aston, W.P., 1973. Complement technology. In: Handbook of Experimental Immunology. Ed by D.M. Weir, 2nd edn., p.5.1. Blackwell Scientific Publications, Oxford. Mayer, M.M. 1961. In: Kabat and Mayer's Experimental Immunochemistry, 2nd edn., Charles C. Thomas, publisher, Springfield, I I I . , p.149. Pang, A.S.D. and Aston, W.P., 1977. Alternative complement pathway in bovine serum: lysis of human erythrocytes. Am. J. vet. Res., 38:355. Pang, A.S.D. and Aston, W.P., 1978. The alternative complement pathway in bovine serum: the isolation of a serum protein with factor B a c t i v i t y . Immunochem., 15:529. P l a t t s - M i l l s , T_A.E. and Ishizaka, K., 1974. Activation of the alternative pathway of human complement by rabbit cells. J. Immunol., 113:348. Rhee, Y.O., Broad, S. and Jonas, W.E., 1970. Agglutinating and complementmediated a c t i v i t i e s of sheep antisera, IgM, slow and fast y-globulin antibodies. Res. vet. Sci., 11:123. Rice, C.E. and Crowson, C.N., 1950. The interchangeability of the complement components of different animal species. I I . In the haemolysis of sheep erythrocytes sensitized with rabbit amboceptor. J. Immunol., 65:201. Sandberg, A.L. and Osler, A.G., 1971. Dual pathway of complement interaction with guinea pig immunoglobulins. J. Immunol., 107:1268.