Isolation of a papain-solubilised major transplantation antigen from rat leukaemic cells

Journal of lmmunological Methods, 44 (1981) 23--35

23

Elsevier/North-Holland Biomedical Press

ISOLATION OF A PAPAIN-SOLUBILISED MAJOR TRANSPLANTATION ANTIGEN FROM RAT LEUKAEMIC CELLS

ADRIENNE R. THOMPSON l

Department c,f Pathology, University of Sydney, Sydney, Australia (Received 20 August 1980, accepted 15 March 1981)

The major transplantation antigen of the PVG rat, Rt-1 c, has been isolated from PVG leukaemic cells in milligram quantities for use in biological experiments related to transplantation. A 3-stage isolation procedure was developed involving the preparation of plasma membranes, digestion of the membranes with papain to release an active fragment of the Rt-1 c antigen and fractionation of the digest on Sephacryl columns to give an active product. Activity was followed by a newly developed inhibition assay in which extracts of cells were incubated with a reference anti-PVG antiserum, excess unreacted antibody being quantitated by radioactive binding to PVG leukaemic cells glutaraldehydefixed to flexible microtitre plates. In addition to Rt-1 c the preparation contained a second glycoprotein component. The preparation shows allelic specificity in that it inhibits the binding of anti-PVG antiserum to PVG cells, but not the binding of anti-DA serum to DA cells. The preparation represents a 16% yield of the activity of the cells from which it was derived.

INTRODUCTION

T h e m a j o r t r a n s p l a n t a t i o n a n t i g e n s are a h i g h l y p o l y m o r p h i c s y s t e m o f p r o t e i n a n t i g e n s f o u n d on t h e s u r f a c e o f l y m p h o i d cells a n d c o d e d b y genes in t h e m a j o r h i s t o c o m p a t i b i l i t y c o m p l e x . It has b e e n s h o w n t h a t f o r successful t r a n s p l a n t s t h e d o m i n a n t r e q u i r e m e n t is c o m p a t i b i l i t y b e t w e e n d o n o r a n d r e c i p i e n t o f t r a n s p l a n t a t i o n a n t i g e n s c o d e d f o r b y t w o loci, t h e K a n d D loci in t h e m o u s e a n d t h e H L A - A a n d H L A - B loci in m a n . T h e s e species have b e e n t h e m o s t w i d e l y s t u d i e d . Less is k n o w n a b o u t t h e a n a l o g o u s t r a n s p l a n t a t i o n a n t i g e n s o f t h e rat, o r i g i n a l l y k n o w n as Ag-B, H-1 a n d n o w Rt-1. R e c e n t l y it has been r e p o r t e d t h a t t h e r a t m a j o r t r a n s p l a n t a t i o n I Present address: Department of Biochemistry, University of New South Wales, P.O. Box 1, Kensington, N.S.W. 2033, Australia. Abbreviations: BBSS, balanced buffered salt solution; BSA, bovine serum albumin; DTT, dithiothreitol; EDTA, ethylene diaminetetraacetic acid, disodium salt; IgG, immunoglobulin; i.p., intraperitoneal; ME, 2-mercaptoethanol; MW, molecular weight; PBS, phosphate-buffered saline; PVC, polyvinyl chloride; SARG, F(ab)2 of sheep Ig anti-rat IgG, after removal of anti-L chain activity; SDS-PAGE, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. 0 022-1759/81/0000--0000/$02.50 © Elsevier/North-Holland Biomedical Press

24 antigen comprises two molecules coded by two loci corresponding to the K and D loci of the mouse and the A and B loci of man (Goetze et al., 1978). A partial amino acid sequence for the first 27 amino acids of a rat transplantation antigen was published by Blankenhorn et al. (1978) and was shown to haw~ sequence homology with those of mouse, guinea pig and man. For the isolation of the antigen these workers used 10 ~ spleen cells, biosynthetically labelled with tritium, extracted them with detergent and, after immunoprecipitation of the antigen with appropriate antiserum, the proteins were separated on SDS-PAGE and the Rt-1 protein micro-sequenced. At each stage of the isolation and sequencing, detection was by radioactivity and only trace amounts of Rt-I protein were isolated from the 10 ~ cells used, probably about 0.1 ng. A number of biological experiments in rats of importance in the field of transplantation would be possible if larger amounts of Rt-1 antigen were available. This paper describes the preparation of the transplantation antigen Rt-1 c from the cells of the transplantable leukaemia of the PVG rat. Experiments described in this paper show that the leukaemic cell carries as much Rt-1 c antigen as normal PVG lymphoid cells. Moreover, large numbers of leukaemic cells (on average 3 X 109 per rat) can be obtained from the peritoneal cavity after i.p. passage. The PVG leukaemic cells therefore provide a good source of starting material for preparing the Rt-1 e antigen. From studies with other species, two options were available for solubilising transplantation antigens from lymphoid cells, extraction with detergents to release the whole glycoprotein molecule from the membrane, or preparation of a plasma membrar,e fraction from the cells from which a soluble antigenically active fragment of the Rt-1 molecule can be cleaved by papain digestion (Springer et al., 1977). These procedures have been reviewed by Strominger et al. (1976) and Crumpton and Snary (1977}. In preliminary experiments with detergent solubilisation, precipitation of extracted material occurred on storage at --20 °, with a consequent risk of loss of active material. Ultimately, it would be necessary to remove detergent and probably to digest the product with papain in order to prepare a homogeneous, soluble material for subsequent biological experiments. With these considerations in mind, the method involving papain digestion of the cell membranes was preferred rather than detergent solubilisation. MATERIALS AND METHODS Animals, cell-handling media, cell suspensions and indirect radioactive binding assays carried out in tubes, were as described in an earlier paper (Thompson and Roser, 1980). Chemicals

Papain (Sigma) was a suspension containing 27 mg/ml. Sephacryl 200 was supplied by Pharmacia. Haemacell (Behringwerke AG), a degraded gelatin.

25

A n tise ra BN anti-PVG and (BN X PVG) anti-DA antisera were prepared by skin grafting, giving a second skih graft after 14 days and 10 days later injecting 5 X 106 cells i.v., the animals being bled o u t after 14 days. PVG leukaemia was a transplantable leukaemia (Roser and Ford, 1972) serially passaged. It was possible to harvest 2--10 X 109 leukaemic cells from the peritoneal cavity of each rat approximately 14 days after i.p. injection of 107 cells. Extraction of leukaemia cells with detergents The procedures used to extract the Rt-1 (Ag-B) and Thy-1 antigens from t h y m o c y t e s (Letarte-Muirhead et al., 1974) were used. The cells were first extracted with Tween-40, a non-ionic detergent, since ionic detergents break down nuclear membranes and the liberated DNA causes intractable gels. A 'Tween membrane' fraction, spun down from the extract at 160,000 × g was brought into solution by extraction with deoxycholate (DOC). Preparation of plasma membranes from leukaemic cells Leukaemic cells were stored frozen at 109/ml in BBSS. The methods described by Turner et al. (1975) were used to prepare a plasma membrane fraction from leukaemic cells as shown in the flow sheet below: Cells (A) ,

Iextracted with buffered saline . . . . . . . . . . . . . . . . . spun 1500 X g I

i

Extract (B)

Cell residues

u_n 60'000 x g............ l I

Supernate (C)

Plasma membranes papain digestion spun 160,000 X g

i m

Papain digest (D)

Precipitate

Digestion of plasma membranes with papain Papain was pre-actwated as follows {Thompson et al., 1971): papain suspension {300 pl containing 27 mg/ml), 2-mercaptoethanol (ME) (6 pl) to give a final concentration of 0.005 M, and 15 ml Tris buffer (0.01 M containing 0.005 M EDTA, pH 7.4) were incubated at 37°C for 1 h before adding to the membrane suspension.

26 The leukaemic (:ell plasma membranes from 30 × 10 '~ cells were suspended in PBS, pH 7.4 (30 ml) containing 1.5 ml of 0.2 M EDTA and 10.5 #l ME, warmed to 37°C, and 15 ml activated papain solution at 37°C added and incubated at 37°C for 15 rain. The digestion was terminated by addition of iodoacetic acid (56.6 mg}. Phenyl methyl sulphonyl fluoride (PMSF} was added to a co n ce nt r a t i on of 10 -3 M in this and subsequent steps throughout the preparation to inhibit further proteolysis by serine esterases. The w~lume of the digest (45 ml) was reduced to 4 ml in an Amicon Diaflo cell with PM 10 filter. This technique was used t h r o u g h o u t for concentrating fractions.

Gel filtration on Sephac~l columns Gel filtration of the papain digest was carried out as described in the legend to Fig. 4. Fractions A to tl were pooled corresponding to the peaks in the elution profile and c o n c e n t r a t e d to appr o xi m at el y 2 ml.

Polyacrylamide gel electrophoresis in sodium dodecyl sulphate (SDS-PAGE) SDS-PAGE was carried out on a 10% polyacrylamide gel slab (9 cm × 10.5 cm) with a 3c7c stacking gel with circulating discontinuous buffer in a Gradipore apparatus (Gradient Laboratories, Sydney} in other respects according to Laemmli (1970). Samples were prepared for electrophoresis by reduction with dithiothreitol {DTT) in urea and SDS followed by alkylation with iodoacetamide (Weber and Osborn, 1969). Samples of control proteins, similarly reduced, were run at the same time. Gels were stained for protein with Coomassie Blue or for glycoprotein with periodate-Schiff's reagent IGlossmann and Neville, 1971 ).

Glutaraldehyde fixation of leukaemic cells to flexible microtitre plates The following procedure for preparing cells on plates was adapted from that of Stocker and Heusser (1979). Leukaemic cells were suspended at 5 × 107/ml in BBSS and aliquots of 5 0 p l transferred using a multichannel pipette (Titertek) to the wells of flexible polyvinyl chloride (PVC) microtitre plates with flat-bottom wells (Cooke L a b o r a t o r y Products). The cells were spun down to the b o t t o m of the wells at 100 × g for 5 min. Glutaraldehyde (0.5%, 50/Jl) was added to each well, giving a final c o n c e n t r a t i o n of 0.25% and allowed to stand for 5 min at room temperature. The glutaraldehyde solution was removed by flicking the plate and the plate washed 4 × by immersion under PBS and flicking to remove the liquid. The wells were filled with 0.3% BSA in BBSS containing 1% azide and could be stored for many m o nth s at 4°C. Before use the storage solution was removed by flicking the plate.

Inhibition assay for Rt-l" activity Rt-1 c activity in extracts was measured by inhibition of binding of BN anti-PVG antiserum to PVG leukaemic cells glutaraldehyde fixed to flexible PVC microtitre plates. The ext r a c t (100/xl} was first incubated with BN anti-

27 PVG serum (100 pl) for 1 h on ice and the residual antibody measured by binding to leukaemic cells (2.5 × 106} on plates. The first incubation could be carried out in tubes, as in the method finally adopted, and any immunoprecipitate spun down in a Beckmann Microfuge for 5 min and 3 aliquots of 50/al (for 3 replicate determinations} transferred to the leukaemic cells on plates. Alternatively the first incubation could be carried out on a polystyrene microtitre plate and an aliquot transferred without centrifuging to the cells on a PVC plate. In either procedure, the residual antibody was incubated with fixed cells on a PVC plate for 1 h on ice and, after washing the plate 3 times by irrigating with Haemaccel in BBSS and flicking off the liquid, the antibody bound to the cells was quantitated by a third incubation for 1 h on ice with 50 pl of an anti-rat IgG reagent made specific for gamma chain as previously described {Thompson and Roser, 1980) and radioiodinated (SARG). After washing as before the plates were cut into rows with scissors and the wells snipped into tubes for gamma counting. RESULTS

Cells of the PVG leukaemia were shown to have essentially the same binding capacity as PVG lymph node cells for BN anti-PVG serum (Fig. 1) and therefore carry similar amounts of Rt-1 c transplantation antigen. PVG 50

o L.O

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Fig. 1. Binding of BN anti-PVG serum to fresh PVG LN cells (× . . . . . . ×), fresh PVG leukaemic cells (A . . . . . A) and to glutaraldehyde-fixed PVG l e u k a e m i c cells (o o), at 2-fold serial dilutions of antiserum. Assay o n polystyrene microtitre plate with 5 X 106 loose cells per well as described in the Methods section. SARG 20/~g/ml, 0.08/.tCi/pg.

28 leukaemic cells are thus a relatively plentiful source for e x t r a c t i o n o f this g l y c o p r o t e i n . Fig. 1 also shows similar binding o f BN anti-PVG serum to fresh and g l u t a r a l d e h y d e - f i x e d PVG leukaemic cells, so t h a t fixed leukaemic cells m a y be used as targets for radioactive binding assays. A p a r t f r o m convenience, fixed cells have the advantage that assays (:an be carried o u t in d e t e r g e n t s and tests s h o w e d t h a t binding was n o t inhibited b y d e o x y c h o l a t e up to a c o n c e n t r a t i o n o f at least 0.5%. It was also f o u n d that the binding assay could be carried out on leukaemie cells g l u t a r a l d e h y d e - f i x e d to flexible PVC plates with f i a t - b o t t o m e d wells, provided tile cell n u m b e r s did not exceed 2 × 10 (' per well. T h e Rt-1 antigen in cell e x t r a c t s could be q u a n t i t a t i v e l y e s t i m a t e d by an inhibition assay in which BN anti-PVG serum was first i n c u b a t e d with the e x t r a c t and the excess a n t i b o d y q u a n t i t a t e d by binding to fixed l e u k a e m i c cells on PVC plates. Fig. 2 shows t h a t the e x t e n t of inhibition is linear with increasing a m o u n t s o f e x t r a c t w h e n the first i n c u b a t i o n is carried o u t in d e o x y c h o l a t e (an ionic d e t e r g e n t ) . Similar inhibition was observed in T w e e n 40 (a non-ionic d e t e r g e n t ) or in BBSS.

Ic) x 201T a_ /3

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Fig. 2. I n h i b i t i o n o f b i n d i n g of BN a n t i - P V G s e r u m to P V G l e u k a e m i c cells by e x t r a c t s of PVG l e u k a e m i c cells in s o d i u m d e o x y c h o l a t e solutions. T h e curve s h o w s i n h i b i t i o n b y t h e cell e x t r a c t in 2% d e o x y c h o l a t e s o l u t i o n of b i n d i n g of BN a n t i - P V G s e r u m ( d i l u t e d to 1/64), the first i n c u b a t i o n of serial d i l u t i o n s o f e x t r a c t being carried o u t in t u b e s (,". . . . . . z'x) as described in t h e M e t h o d s section. Residual a n t i b o d y in an a l i q u o t was q u a n t i t a t e d b y m e a s u r i n g t h e b i n d i n g to P V G l e u k a e m i c cells g l u t a r a l d e h y d e - f i x e d to flexible PVC m i c r o t i t r e plates (2 × 106 cells p e r well). In this a n d s u b s e q u e n t g r a p h s residual b i n d i n g is p l o t t e d , so t h a t t h e m i n i m u m b i n d i n g r e p r e s e n t s m a x i m u m i n h i b i t o r y activity ( S A R G 13.1 p g / m l , 0.58 laCi/pg).

29

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~ 3

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~ 2 .~o" o ....

-o ~-"

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1"8 ~ 1/32 1/6L, L_ 1/'128 1/256 1/ 12 NIL / 1/16

3bLUTION OF EXTRAET OR [ELLS Fig. 3. I n h i b i t i o n o f b i n d i n g of BN a n t i - P V G s e r u m to l e u k a e m i c cells g l u t a r a l d e h y d e fixed to flexible PVC m i c r o t i t r e plates b y various f r a c t i o n s f r o m t h e p r e p a r a t i o n of a p a p a i n digest o f l e u k a e m i c cells. T h e f r a c t i o n s tested ( A - - D in t h e flow diagram in t h e M e t h o d s s e c t i o n ) were t h e l e u k a e m i c cells t h e m s e l v e s (A) (x x), t h e e x t r a c t o f cells in b u f f e r e d saline (B) {o . . . . . e), t h e s u p e r n a t a n t (C) a f t e r r e m o v a l o f t h e cell residues (A . . . . . . ~), a n d t h e p a p a i n digest (D) a f t e r c e n t r i f u g a t i o n t o r e m o v e u n d i g e s t e d m e m b r a n e s ( o - - - - - - o ) . T h e u p p e r NIL m a r k r e p r e s e n t s n o a d d e d e x t r a c t a n d t h e l o w e r NIL m a r k r e p r e s e n t s n o a d d e d e x t r a c t or a n t i s e r u m ( S A R G 6.4 p g / m l , 0.23 ~Ci/pg).

There was little difference in results with the two procedures for the first incubation (on plates or in tubes, see Methods section). However, the latter was adopted in assays for soluble antigen. The conditions for papain digestion differed considerably from those used by Turner et al. {1975) for HLA antigens. In particular, the large amount of papain (2 mg to membrane protein (10 mg)} was reduced by a factor of 10 and the papain pre-activated before digestion of the leukaemic cell membrane preparation. The time course of the digestion was followed by inhibition assays and was complete in 15 min and this time was adopted for proteolysis by activated papain in subsequent experiments. The activity of various fractions during isolation of a membrane preparation and its digestion with papain are compared using inhibition assays (Fig. 3), allowing for volume differences of the fractions. Comparable inhibitory activity in the cells and the extract after removal of the cell residues indicated complete release of active material from the cells. After spinning down the 'membranes' at 160,000 X g, the supernatant contained very little of the activity (Fig. 3), while activity resides in the 'membranes', as was demonstrated in a separate experiment. The activity after digestion with papain was approximately one-third of that of the cells themselves. This was assessed by comparison of the dilution of cells or digest resulting in 50% inhibition of binding {Fig. 8). The 'NIL' level in Fig. 4 reveals a degree of non-specific binding of second antibody to the plates in the absence of either extract or

30

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-~ ~._3--~ 7-,-~r---j 1~0 20 30 "3 SO 60 TUBE NU~BF~ Fig. 4. Molecular sieve chromatography on a Sephacryl 200 column of a papain digest of membranes from PVG leukaemic cells. The sample was prepared as described in the Methods section (and shown in the f l o w diagram). The column o f Sephacry] 200 (40 c m x 2.6 era) was packed as recommended by the manufacturer. The elution buffer was 0.01 M Tris, 0 . ] g M sodium chloride, 0.02% sodium azide, pH 7.4. 5 m l fractions were collected at a f l o w rate of a p p r o x i m a t e l y 25 m l / h . The e x t i n c t i o n o f the eluate at 280 nm is shown by the solid line. A molecular weight calibration curve for material eluted from the column ( . . . . . . ) was derived from the elution positions, shown by X's, of control proteins human serum albumin (MW 67,000), ovalbumin (MW 43,000) and c h y m o t r y p sinogen (MW 25,700). The curve for the Rt-1 e activity o f the fractions, measured by the i n h i b i t i o n assay and shown in detail in Fig. 6, is superimposed in the above figure ( - - - - - - ) , the position of minimum binding, representing maximum Rt-1 c activity, appearing in fraction D (tubes 31--36).

serum. This background binding did not impair the use of the assay in following the purification of the Rt-1 c antigen. The E2s0 elution profile on molecular sieve chromatography on a Sephacryl column of protein components of the papain digest of leukaemic cell membranes (Fig. 4) is quite consistent for different experiments. The fractions pooled, as shown in this figure, when analysed for Rt-1 activity by the inhibition assay showed a minimum binding, representing a maximum of Rt-1 activity, in fraction D, with close agreement of 3 replicate analyses as shown in Fig. 5. This graph is also superimposed on the elution profile in Fig. 4, together with a molecular weight calibration line derived from the chromatographic behaviour of a limited number of control proteins on the same column. Together these curves show that material with Rt-1 antigenic activity appears in fraction D and has a molecular weight in the region expected by analogy with HLA antigens, viz. 46-49K (Turner et al., 1975).

31

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g× o

o

3-

IK

11 ~

A_B C. o E F q , .1~0 __L_ 20

310

.~*0 J - - 50I

6'0 NIL

TUBE NUMBER

Fig. 5. Rt-1 c activity in the e]uate from gel filtration on Sephacryl 200 of a papain digest of the membranes from PVG ]eukaemic cells (see Fig. 4). The tubes under the major peaks were bulked into fractions A--H as indicated, concentrated and used to measure in

the inhibition assay the capacity of the fractions to inhibit the binding of BN anti-PVO serum to PVG leukaemic cells glutaraldehyde-fixed to PVC flexible microtitre plates. Triplicate analyses are shown (SARG 4.5 t~g/ml, 0.48 pCi/pg). An aliquot of this material was reduced, alkylated and examined by SDSpolyacrylamide gel electrophoresis on a 10% gel. When stained with Coomassie blue it was shown to contain two major bands which also stained for glycoprotein (Fig. 6). One of these bands corresponded, by comparison with the mobility of reference proteins, to a molecular weight of 36 K and the other to a d o ubl et of 42 K. The molecular weight of the heavy chain of the papain-cleaved HLA molecule ranges between 34 K and 37 K (Turner et al., 1975) and the 36 K band appears to be the active Rt-1 c fragment. The nature of the second 42 K glycoprotein band is not known. The other major protein band running faster and therefore of lower molecular weight than the c h y m o t r y p s i n control protein (25.7 K) corresponds with the molecular weight of 12 K of ~2-microglobulin, which constitutes the smaller chain of Rt-1 c which would be separated from the heavy chain during preparation of the sample for SDS-PAGE. Traces of other protein bands are also present. The Rt-1 product, fraction D off a Sephacryl column, was tested for its immunological specificity. This was established in an e x p e r i m e n t in which its capacity to inhibit the binding of BN anti-PVG serum to PVG lymph node cells was d emo n st r a t e d (Fig. 7), whereas it was unable to inhibit the binding of (BN × PVG) anti-DA serum to DA lymph node cells which carry the Rt-1 a allele. The activity of the preparation was compared in the usual inhibition assay with that of fresh PVG lymph node cells (Fig. 7). The inhibition curves were n o t significantly different for 100 pl of Rt-1 ¢ preparation and 120 X 106 PVG lymph n o d e cells. The total Rt-1 e preparation of 2 ml would have an inhibitory activity equal to 24 X 108 PVG lymph node cells. It will be recalled t h a t PVG leukaemic cells have similar am o unt s of Rt-1 c antigen to PVG lymph node cells (Fig. 1). Since 15 x 109 leukaemic cells were used in the preparation o f the Rt-1 ¢ product, this would imply t hat 16% of the total

32

I

a

b

c

B

C

L)

E

c

a

Fig. 6. SDS-polyacrylamide ~el electrophoresis of fractions from the preparation of the major transplantation antigen of the PVG rat (Rt-le). The photograph shows (a) control proteins (top to bottom) human serum albumin (MW 67,000), ovalbumin (MW 43,000) and chymotrypsinogen (MW 25,700); (b) saline extract of PVG leukaemic cells; (c) papain digest of membranes from leukaemic cells (fraction D of flow diagram in Methods section)~ B, C, D, E, fractions from gel filtration of Sephacryl 200 (see Fig. 4). Movement is from top to bottom of the gel photograph. Fracti(ms were prepared for electrophoresis by reduction and alkylation as described under Methods, together with other conditions for electrophoresis. The arrow indicates the band of MW 36 K corresponding to the heavy chain of the papain-solubilised antigenic fragment of RT-1 c.

a c t i v i t y o f t h e cells was r e c o v e r e d in t h e p r o d u c t , n e g l e c t i n g s a m p l e s r e m o v e d for testing. DISCUSSION A l t h o u g h t h e Rt-1 a l l o a n t i g e n s o f t h e r a t have b e e n i s o l a t e d in less t h a n n a n o g r a m q u a n t i t i e s f o r p a r t i a l a m i n o acid s e q u e n c i n g ( B l a n k e n h o r n et al., 1 9 7 8 ) , t h e m e t h o d s used c a n n o t be d i r e c t l y a p p l i e d t o p r e p a r i n g m i l l i g r a m quantities of these proteins for biological e x p e r i m e n t a t i o n . For example, to scale up t h e m e t h o d s used b y B l a n k e n h o r n et al. ( 1 9 7 8 ) to h a n d l e t h e im-

33

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1/16 1/32 1/6~ 1/12S

DILUTION OF EXTRAI:T OR EELLS

Fig. 7. Specificity o f i n h i b i t o r y activity o f Rt-1 c p r e p a r a t i o n a n d c o m p a r i s o n o f i n h i b i t o r y activity in t h e p r e p a r a t i o n with t h a t of whole P V G cells. I n h i b i t i o n b y Rt-1 c p r e p a r a t i o n {fraction D f r o m gel f i l t r a t i o n o n S e p h a c r y l 200, Figs. 4 and 5) of t h e b i n d i n g of BN a n t i - P V G s e r u m to fresh PVG LN cells (~ . . . . . A) a n d o f t h e b i n d i n g of (BN × P V G ) a n t i - D A s e r u m to fresh DA LN cells ( e - - - - - - - o ) . I n h i b i t i o n by P V G LN cells o f t h e b i n d i n g of BN a n t i - P V G s e r u m to fresh P V G LN cells (o o). T h e assay was carried o u t in tubes, s p i n n i n g d o w n t h e cells o r e x t r a c t a f t e r t h e first i n c u b a t i o n w i t h a n t i s e r u m b e f o r e t r a n s f e r r i n g an a l i q u o t to measure b i n d i n g of residual a n t i b o d y t o fresh P V G or D A cells ( S A R G 13 p g / m l , 0.18 pCi/pg).

munoprecipitation of solubilised antigen from the 30 × 109 cells used in the present experiments would have required 800 ml of alloantiserum. For preparative purposes, conventional biochemical methods are more suitable. A procedure is described based on that of Turner et al. (1975) for isolating HLA antigens. Using PVG leukaemia cells which may be obtained in large numbers, milligram quantities of Rt-1 ¢ antigen may be obtained in a limited number of steps, viz., preparation of plasma membrane from the cells, digestion of the membranes with papain and separation of an active fraction by gel filtration on Sephacryl. The product, analysed by SDS-PAGE, contains the two chains of the Rt-1 antigen, the glycoprotein of molecular weight 36 K corresponds to the heavy chain of the papain-digested fragment of the HLA antigen {Turner et al., 1975) and the other band has the mobility expected for the ~-microglobulin chain. The Rt-1 c preparation reveals one other glycoprotein c o m p o n e n t of u n k n o w n identity. The molecular weight of this 42 K band appears too large to be an Ia product: Other bands represent only minor contaminants. It was considered that this material meets the purity required for biological experiments and that for this purpose further purification with inevitable reduction in yield was not warranted although methods such as affinity chromatography on Iectin columns and electrophoresis could be used.

34 T h e p r o c e d u r e for assaying solubilised antigen was d e v e l o p e d f r o m inhibition assays carried o u t in t u b e s on g l u t a r a l d e h y d e - f i x e d cells (Letarte-Muirhead et al., 1 9 7 4 ; S t o c k e r and Heusser, 1979). Assays m a y be carried o u t in ionic or n o n - i o n i c d e t e r g e n t s or in the a b s e n c e o f d e t e r g e n t s . Washing the plates by irrigation and flicking o f f the wash liquid, as well as c u t t i n g the wells straight into t u b e s for g a m m a c o u n t i n g o f f e r c o n s i d e r a b l e e c o n o m y o f t i m e a n d e f f o r t . M o r e o v e r , the plates m a y be s t o r e d for use as r e q u i r e d , avoiding the need for p r e p a r i n g fresh cells. R e p r o d u c i b i l i t y is e x c e l l e n t and in fact b e t t e r t h a n with t u b e assays or on plates with loose cells since t h e r e can be no a c c i d e n t a l loss o f cells during washing. A f u r t h e r technical p o i n t c o n c e r n s t h e r a d i o a c t i v e binding assay. It m a y be used as a sensitive ' t r a c e ' d e t e c t i n g s y s t e m as d e s c r i b e d by Morris and Williams ( 1 9 7 5 ) and this is p r e f e r r e d for i n h i b i t i o n assays. In all the p r e s e n t e x p e r i m e n t s a s t a n d a r d n u m b e r o f c o u n t s o f labelled s e c o n d a n t i b o d y ( S A R G ) was used. H o w e v e r , a r e d u c t i o n in the a m o u n t o f cold S A R G was m a d e in p a r t i c u l a r e x p e r i m e n t s to increase the sensitivity o f d e t e c t i o n . F o r this r e a s o n c o m p a r i s o n o f c o u n t s b o u n d by cells in d i f f e r e n t e x p e r i m e n t s is n o t possible. T h e papain-solubilised Rt-1 c antigenic f r a g m e n t s h o w s specific: allelic activity in vitro in inhibiting b i n d i n g to cells bearing the Rt-1 c allele o f the m a j o r t r a n s p l a n t a t i o n antigen o f a l l o a n t i s e r u m p r o d u c e d against t h o s e cells. H o w e v e r , it d o e s n o t inhibit c o r r e s p o n d i n g binding to cells c a r r y i n g t h e Rt-1 a allele o f a n t i s e r u m against R t - l a - b e a r i n g cells. E x p e r i m e n t s are p l a n n e d in this l a b o r a t o r y to test in vivo activity by i m m u n i s i n g with the antigen p r e p a r a t i o n and d e m o n s t r a t i n g its c a p a c i t y to i n f l u e n c e t h e allograft response. ACKNOWLEDGEMENTS T h e w o r k was s u p p o r t e d b y a g r a n t f r o m t h e N a t i o n a l H e a l t h a n d Medical R e s e a r c h Council o f Australia. I wish to t h a n k P r o f e s s o r Bruce R o s e r and Dr. Susan Dorseh for suggesting t h e p r o j e c t and for h e l p f u l discussions. REFERENCES Blankenhorn, E.P., J.M. Cecka, D. Goetze and L. Hood, 1978, Nature 274, 90. Crumpton, M.J. and D. Snary, 1977, Contemp. Top. Mol. Immunol. 6, 53. Glossmann, H. and D.M. Neville, 1971, J. Biol. Chem. 246, 6339. Goetze, D., R. Sporer and L.A. Manson, 1978, Rat Newsletter 4, 14. Laemmli, U.K., 1970, Nature 227,680. Letarte-Muirhead, M., R.T. Acton and A.F. Williams, 1974, Biochem. J. 143, 51. Morris, R.J. and A.F. Williams, 1975, Eur. J. Immunol. 5,274. Roser, B. and W.L. Ford, 1972, Aust. J. Med. Sci. 50, 165. Springer, T.A., D.L. Mann, A.L. DeFranco and J.L. Strominger, 1977, J. Biol. Chem. 252, 4682. Stocker, J.W. and C.H. Heusser, 1979, J. Immunol. Methods 26, 87. Strominger, J.L., D.L. Mann, P. Parham, R. Robb, T. Springer and D. Terhorst, 1976, in Lymphocyte Diversity, Cold Spring Harb. Syrup. Mol. Biol. XLI, 323.

35 Thompson, A.R. and B. Roser, 1980, Pathology 12, 189. Thompson, E.O.P., R.W. Sleigh and M.B. Smith, 1971, Aust. J. Biol. Sci. 24,525. Turner, M.J., P. Cresswell, P. Parham and J. Strominger, 1975, J. Biol. Chem. 250, 4512. Weber, K. and M. Osborn, 1969, J. Biol. Chem. 244, 4406.