Solubilization of histocompatibility and tumour-associated antigens of the P-815 murine mastocytoma cell

Europ. J. Cancer Vol. 12, pp. 263-270. Pergamon Press 1976. Printed in Great Britain

Solubilization of Histocompatibility and Tumour-associated Antigens of the P-815 Murine Mastocytoma Cell* K. J. CLEMETSON, A. GERBER, M. BERTSCHMANN and E. F. LI~SCHER Theodor Kocher Institute, University of Berne, Berne, Switzerland Abstract--The distribution of tumour-associated and histocompatibility antigens from P-815 murine mastocytoma cells was determined during fractionation of homogenized cells. Using inhibition of the cytotoxicity of anti P-815 allo- and xenoantisera rendered specificfor P-815 by absorption in vivo it was possible to foUow the tumourassociated antigen and to compare its behaviour with that of the H-2 antigens. Both antigens could be solubilized in high yield from a crude membrane preparation using 1.2 % DOG. Unlike the anti P-815 xenoantiserum, the anti P-815 alloantiserum appeared to be directed to a large extent against a specific conformation of the tumour antigen which could only be detected on intact cells and not after homogenization. Both the H-2 and the tumour-associated antigens on gel filtration in DOG had an approximate molecular weight of 90,000-100,000 Daltons.

INTRODUCTION

often a major problem is the removal of the detergent while maintaining the antigen in a water-soluble state. Where detergents have been successfully used the main advantage is that the antigen is solubilized in an intact, undegraded form and in high yield. T u m o u r antigens have been solubilized using papain [16,17], trypsin or 3M KC1 [18], by autolysis followed by sonication and papain digestion [19], or using fl-glucosidase [20]; detergents have not yet been extensively used [21]. P-815 is a transplantable, mast cell tumour of the DBA/2 mouse originally induced by methylcholanthrene [22] which can be cultured in vitro. It is characterized by a high malignancy and a weak immunogenicity (for a more detailed description see [23]). Recently, by immunization in allogeneic and xenogeneic systems, followed by absorption of these antisera in vivo in DBA/2 mice, we have been able to produce antisera which are directed specifically against P-815 and are neither cytotoxic to nor are absorbed by normal DBA/2 cells [23]. In this report we describe the use of these antisera in following the solubilization and characterization of

ONE OF the major problems in the effort to establish the antigenic and structural properties of membrane antigens as molecular entities is to obtain their release from the membrane in a high yield and in an active and watersoluble form which is amenable to further biochemical separation. Histocompatibility antigens have been solubilized by treating crude membrane fractions of cells with papain [1-5] and using hypertonic salt solutions, especially 3M KCI [6]. The papain method gives soluble antigen though in low yield. There has been some controversy over how the 3M KC1 acts and the nature of the solubilized antigen [7-9]. A wide range of detergents has been used in attempts to solubilize membrane antigens [10-15]. Some destroy antigenic activity, presumably by causing irreversible conformational changes. Where this does not happen,

Accepted 21 November 1975. *This work was supported by the Swiss National Foundation for Scientific Research. 263

264

K. J. Clemetson et al.

P-815 associated antigen(s) and compare these with the H-2 antigens of this cell. MATERIAL AND M E T H O D S

Culture of P-815 cells P-815 cells were grown either in P-815 medium [24] or in Dulbecco's modification of minimum essential medium (DMEM) (Flow Laboratories) containing 10% horse serum. For membrane isolation, cells were grown in batches of 10 1 to a density of ~ 106 cells/ml in the latter medium.

Preparation of crude membranefraction This was carried out essentially by the method of Wallach and Kamat [25]. The cell suspension (10 1) was centrifuged for 20 min at 400 x g and 4°C and washed twice in 0.15MNaCI, 0-005M tris-(hydroxymethyl)amino-methane(tris)/HC1 and once in 0.25M sucrose, 0.005M tris/HC1, pH 7.4. The wet cell weight of the final pellet was determined and the cells resuspended in ten times this amount of 0"25M sucrose, 0.005M tris/HC1, pH 7.4. The cells were disrupted twice by decompression from 60 atmospheres of nitrogen. The efficiency of this process was checked by examination of the homogenate under the microscope. The homogenate was made 0.001M in ethylenediaminetetraacetic acid (EDTA) by adding 0.1M solution and was centrifuged at 2500 x g for 30 min. The supernatant from this was centrifuged for 1 hr at 100,000x g. The sediment from this final centrifugation was resuspended in an equal volume of 0.15M NaC1, 0.005M tris/HC1, pH 7.4 and represents the crude membrane fraction. It was stored frozen at -70°C.

Solubilization of crude membrane fraction using sodium deoxycholate The crude membrane fraction was thawed quickly and solid sodium deoxycholate (DOC) added to give the required concentration. The mixture was stirred rapidly until the DOC dissolved, then more slowly for a further 30 min and was then centrifuged at 100,000 x g for 1 hr. The pellet was resuspended to the original volume in 0.05M phosphate buffer, pH 7.6 and both the pellet and the supernatant were dialysed separately against this buffer for 72 hr with at least four changes of buffer.

Column chromatography in DOC Sephadex G-150 was equilibrated with 0.05M phosphate buffer, pH 7.6 containing 0.6% DOC and was packed in a column

(2.5 x 80 cm) while gradually increasing the DOC concentration to 1"2%. This is necessary to avoid aggregation of the dextran beads in the detergent. After application of the 100,000 x g supernatant, from the P-815 membranes solubilized in 1.2% DOC, to the column, it was eluted with 0.05M phosphate buffer, pH 7.6 containing 1.2% DOC, at a flow-rate of 14 ml/hr, collecting 3 ml fractions. Fractions were dialysed against 0.05M phosphate buffer, pH 7.6 for at least 72 hr with four buffer changes and were stored at -20°C. All buffers used contained 0.05% NaN3 except the final dialysis buffer. Estimation of molecular weight was made by comparison with ovalbumin, bovine serum albumin and bovine gamma globulin (Sigma) run on the same column under the same conditions. The excluded volume was determined using Dextran Blue 2000 (Pharmacia) and the total volume using Na 2 51Cr04, as markers. Protein was determined using the Folin reagent [26]. Lipid was determined by extraction [27] followed by thin layer chromatography on silica gel [28]. This method also extracts and detects DOC.

Antisera Allo- and xenoantisera were prepared and titrated as described in [23]. The C3H anti DBA/2 alloantiserum recognizes principally H-2 and probably also non-H-2 differences between these mouse lines. The C3H anti P-815 recognizes these differences and also antigen specific for P-815 and referred to here as P-815 associated antigen. The presence of more than one type of P-815 associated antigen cannot be excluded. The sheep anti P-815 recognizes these differences and also other, xenoantigens. After absorption of anti P-815 antisera in vivo in DBA]2 mice they recognize only the P-815 associated antigen.

Inhibition of cytolysis assay The cytotoxicity of 20 ~1 of antibody containing 10 LDs0 units (10 times the quantity necessary to lyse 50% of 50,000 ceils) of antibody was assayed after incubation with a serial, twofold dilution of 20/A of antigen. P-815 cells labelled with SlCr were added (20 ~1 of a suspension of 2"5 x 106 cells/ml) followed by 20/A of complement (see 23 for details). The cells were incubated with this mixture for 1 hr at 37°C in an atmosphere of 5% CO2, 95% air. The cytolysis was stopped by addition of 100/~1 of an ice-cold solution of phosphate buffered saline, 0.001M in EDTA.

Solubilization of Histocompatibility and Tumour-assodated Antigens

265

Table 1. Fractionation of homogenized P-815 membranes: Distribution of H-2 and tumour-assodated antigenst H-2 A n t i g e n 2 % Batch no.

2500 x g Supernatant Sediment 87 85 93 96 69 100 64 94 125

1

2 3 4 5 6 7 8 9 Mean Standard error ofthemean

Tumour-associated antigen 2 %

I00,000 x g Supernatant Sediment

11 -11 --4 3 8 13

--24 17 26 20 12 9 26

66 85 69 89 61 74 52 84 66

2500 x g Supernatant Sediment 110 100 57 43 52 30 44 55 132

100,000 x g Supernatant Sediment

17 10 9 8 5 4 4 4 9

--

---12 -20 3 --

20 35 17 18 54 37 20 41 89

90"3

5.6

14.9

71.7

69.7

7.8

3.9

36.7

5.9

1.8

3.4

4.1

11.8

1.4

2.4

7.7

2H-2 d e t e r m i n e d w i t h C 3 H anti DBA/2, t u m o u r - a s s o c i a t e d a n t i g e n with C 3 H anti P-815 in vivo a b s o r b e d a n t i s e r u m : a H o m o g e n a t e taken as 100% ( - - ---- L o w activity/No titre).

After centrifugation (300 x g, 10 min) the radioactivity in 100 pl aliquots of the supernatant was determined using a Nuclear Chicago gamma spectrometer. Results were corrected for the complement control according to the formula: Percentage specific S~Cr release = count/min of sample count/min complement control • 100 count/min antibody control count/min complement control The dilution of antigen giving 50% inhibition of cytolysis is taken as the titre of the antigen and the specific activity of the antigen preparation measured in this assay is defined as: number of LD50 of antibody used titre x volume of antigen taken (in ml) × mg protein/ml

units/rag

RESULTS

The distribution and yield of tumourassociated and histocompatibility antigens at the various stages of preparation of the crude membrane fraction were determined and compared for nine batches of cells (Table 1) using C3H anti DBA/2 and C3H anti P-815 in vivo absorbed (abs.) antisera. The yield of tumourassociated antigen was similarly determined for a further five batches using sheep anti P-815 (abs.) antiserum (Table 2). Yields before and after homogenization were also compared using C3H anti DBA/2 for the H-2 and both C3H anti P-815 (abs.) and sheep anti P-815 (abs.) for the tumour-associated antigens (Table 3). The relative area of plasma and inner (rough and smooth endoplasmic reticulum) membranes was estimated, in the intact cells, to be about 1:3.4, using a morphometric method

[29].

One unit of antigen activity is the quantity which absorbs 1 LD5o of antibody.

Preliminary experiments using the detergents sodium dodecyl sulphate, Triton X-100 and

Table 2. Fractionation of homogenized P-815 cells: Distribution of tumour-associated antigen* % Yield 2500 x g Supernatant Sediment M e a n of 5 batches of cells S t a n d a r d error o f the m e a n

88 9.4

100,000 × g Supernatant Sediment

12

--

42

2.0

--

5.1

* H o m o g e n a t e taken as 100%; A n t i g e n d e t e r m i n e d w i t h sheep anti P-815 in vivo a b s o r b e d antiserum.

266

K. J. Clemetson et al.

sodium deoxycholate to solubilize antigens from the crude membrane preparation of P-815 soon showed that only DOC gave a satisfactory yield of active material and efforts were then concentrated on optimizing this yield. Table 3.

Yield of H-2 and tumour-associated antigens after homogenization*

Antiserum

% Yield in homogenate

C3H anti DBA/2 C3H anti P-815 in vivo absorbed Sheep anti P-815 in vivo absorbed

99 16 92

*Whole cells taken as 100%; H-2 antigen determinations: Mean of 7 batches of cells. Tumour-associated antigen determinations: Mean of 3 batches of cells.

A range of concentrations of DOC was examined (0.5-3.0%) and distribution of protein and antigenic activity between the supernatant and pellet, after centrifugation at 100,000 x g for 1 hr, was determined (Table 4). It was found that the solubilized protein increased from 69% at 0.5% DOC to a plateau at a maximum of about 80% at 1.5% DOC. Solubilization of H-2 antigens increased between 0.5 and 0.9% DOC and then remained constant. At the highest concentrations of DOC (2"0 and 3.0%) the yield dropped somewhat. The differences were less pronounced with the tumour-associated antigen but a slight improvement in solubilization was found up to 1.2% DOC. Based on this, 1.2% DOC was taken as standard for further experiments. The optimum ionic strength for stability of H-2 antigens was determined by dialysing solubilized antigen against different buffer Table 4.

concentrations in the range 0.002-0.15M phosphate buffer, pH 7.6. Below 0.03M the yield dropped, reaching 62% at 0.002M but between 0.03 and 0.15M little difference was seen, the yield averaging 89% of the nonsolubilized material. The optimum pH for H-2 antigen stability was found by dialysing solubilized antigen against 0.05M phosphate buffers of different pH in the range 6-10, and lay in the range 7.5-8, the yield falling off sharply below 6.5 and above 9. Further experiments were carried out working under these optimized conditions. The molecular weight distribution of protein, lipid and antigens was examined by gel filtration on Sephadex G-150 either after removal of DOC by dialysis or in the presence of DOC. In the second case aliquots were dialysed to remove DOC before assay for antigen activity. Where the DOC was removed from the solubilized material before gel filtration the antigen activity was associated with a high molecular weight protein peak excluded from the gel. In the presence of DOC this excluded protein peak was still present, but the antigenic activity was now included in the gel and appeared as a separate peak (Fig. la and lb). Analysis in detail of fractions from columns run in 1.2% DOC, for various membrane components showed that lipid (at a Ka, of 0.6-0.7) was well separated from the excluded protein peak and from the antigens with a Kay of 0.2-0.3. Assay for antigenic activity of fractions was carried out with the following antibodies: C3H anti DBA/2, C3H anti P-815 (abs.), sheep anti P-815 and sheep anti P-815 (abs.). With both the C3H anti DBA/2 and the C3H anti P-815 (abs.), column fractions inhibited

Distribution of protein and antigens between supernatant and pellet after 100,000 x g for 1 hr at various DOC concentrations*

D O C concentration % Protein in supernatant % H-2 antigen in supernatant %

0.5 69

0.7 71

0.9 76

38

61

88

H-2 antigen in pellet ~o T u m o u r antigen in supernatant % T u m o u r antigen in pellet %

15

6

.

75

83

92

26

20

16

1-2 77

1.5 78

89 .

86 .

92 8.5

. 91 --

2.0 78

3"0 78

80

75

. ND

ND

ND

ND

*H-2 antigen determined with C3H anti DBA/2 antiserum, tumour-associated antigen determined with sheep anti P-815 in vivo absorbed antiserum: Crude membrane fraction taken as 100% : - - -----Low activity/No titre N D ---- Not determined.

Solubilization o f Histocompatibility and Tumour- associated Antigens

O.D. 280 nm 1.0

100

LIPID 0.5 2.0 1.O

do

'

+oo

81~

+20

267

wards analysed by gel filtration on Sephadex G-150 in the absence of DOC, the K,v remained 0.2-0.3. By comparison of the elution volume of the antigen with that of proteins of known molecular weight it was possible to estimate the molecular weight of the H-2 antigen as about 90,000-100,000 Daltons and the tumour antigen as detected with C3H anti P-815 (abs.) or sheep anti P-815 (abs.) antisera as within this range but slightly larger.

1,;o FRACTION

DISCUSSION

IgG

BSA Ov

b

O.D. 28Onto

% 100

1.0

50

0.5

tO

ijvo .b

'

sb

'

+bo

'

14o

'

~,;o

'

FRACTION

Fig. 1. Chromatography of DOC solubilized /)-815 membranes on Sephadex G-150 in DOC containing buffer. (a),/-/-2 antigens determined by the capacity of fractions to neutralize C3H anti DBA/2 antiserum © - - - © and tumourassociated antigen using C3H anti P-815 in vivo absorbed antiserum Q - - - - O . Protein ( O.D. 280 nm). Lipid, semi-quantitative (see "Material and Methods") ZX- - - ~ . Vo, excluded volume; V, total volume. (b) Antigens detected by sheep anti P-815 antiserum © - - © and turnout-associated antigen detected by sheep anti P-815 in vivo absorbed antiserum • O. - Protein ( O.D. 280 nm). Marker proteins: bovine gamma globulin (IgG), bovine serum albumin (BSA) and ovalbumin (Or).

cytotoxicity in the same region (Fig. la). The sheep anti P-815 was inhibited by fractions eluting in a higher molecular weight region but when sheep anti P-815 (abs.) antiserum was used, the fractions inhibiting cytotoxicity were about the same as for the alloantisera, though the maximum was shifted to a slightly higher molecular weight. Fractions containing antigenic activity were pooled, dialysed and concentrated by ultrafiltration on a Diaflo PM 10 membrane. Recovery of antigenic activity was about 20% with a purification of about eight times in terms of antigenicity/protein. Concentration of antigenic fractions by freeze-drying gave improved yields of H-2 antigens ( > 4 0 % ) but relatively poor yields ( < 10%) of tumour-associated antigens. When antigen-containing fractions were pooled, dialysed to remove DOC, and concentrated by ultrafiltration, and were after-

These studies indicate that both histocompatibility and tumour-associated antigens of P-815 murine mastocytoma cells are present on the plasma membrane and can be solubilized in high yield using DOC. When the cells are homogenized, the yields of H-2 antigens and of tumour-associated antigen (as determined using the in vivo absorbed xenoantiserum) in the homogenate are virtually quantitative. This would exclude the possibility that these antigens are expressed on internal membranes since, as these have a surface area some 3-4 times greater than the plasma membrane, then the yield in the homogenate should be greater than that originally present on whole cells. Using the in vivo absorbed alloantiserum to determine tumour-associated antigen in the homogenate a very much lower yield was found (16% of that on whole cells). There are two possible explanations for this: the tumourassociated antigen may be solubilized during the homogenization process and the in vivo absorbed xenoantiserum may recognize this soluble species to a much greater extent than does the similarly treated aUoantiserum. However, in following the cell fractionation there does not appear to be much difference in the distribution of antigen between supernatant and pellet as detected by either antiserum. A difference would be expected if higher solubility were the explanation. The alternative explanation is that the in vivo absorbed alloantiserum detects a particular conformation or arrangement of the tumour-associated antigen on the intact cell and cannot detect this after homogenization. The in vivo absorbed xenoantiserum does not differentiate between these situations, perhaps due to its being directed against more antigenic determinants. If the activity in the homogenate is taken as 100% then the yields of tumour-associated antigen in the crude membrane fraction (sediment 100,000 x g) are about the same (40%) when assayed by both antisera.

268

K. J. Clemetson et al.

Yields of H-2 antigens did not change between whole cells and homogenate, and during cell fractionation over 90% of the original antigenic activity could be accounted for. In the case of the tumour-associated antigen, only about 50% of the activity of the homogenate could be detected in the fractionated material. This lower yield is most likely accounted for by loss of tumour-associated antigen due to factors such as conformational change during fractionation or enzymatic degradation. The difference in ability of the absorbed alloantiserum to recognize the tumourassociated antigen after cell homogenization is probably also responsible for the higher variation in the determination of antigen during cell fractionation (Table i). Both types of antigen are readily soluble in DOC and at the optimum concentration of DOC virtually no antigen remains associated with membrane or higher molecular weight material; all the activity is included on a Sephadex G-150 filtration, run in the presence of DOC. When the DOC is removed from the solubilized material by dialysis, the antigens are reincorporated into larger molecular entities which are excluded from Sephadex G-150 but which retain their antigenicity. This reincorporation seems to be due to the presence in detergent-solubilized membrane antigens of a lipophilic region which has been removed in antigens solubilized with papain [10]. When the antigens are separated from the bulk of the excluded protein and from lipid, then this reintegration does not occur on removal of DOC. After some time (days) at 4°C, or after freezing and thawing, aggregation does occur and is accompanied by a loss of antigenic activity. The molecular weight of both antigens appeared to lie in the range 90,000-100,000 Daltons, as determined by gel filtration in the presence of DOC, with the tumour-associated antigen slightly higher than the H-2. These molecular weights may be exaggerated due to binding of DOC to lipophilic regions of the

antigens compared to the hydrophilic proteins used as standards, since this phenomenon is known to occur. However, on gel filtration of partially purified antigen in the absence of DOC the same Kav was observed. This molecular size is similar to that reported for DOC-solubilized HL-A antigens (88,000 Daltons) [11] or for DOC-solubilized Ag-B antigens (100,000 Daltons) [14] or for part of the H-2 antigen solubilized by NP-40 and determined by polyacrylamide gel electrophoresis (88,000 Daltons) [30], although histocompatibility antigens solubilized by other methods [4] give molecular weights in the 35,000-45,000 Daltons range. Data on the molecular weights ofundegraded tumour specific/associated antigens are largely lacking; Davies et al., [19] have shown that the tumour-specific antigen of EL4 cells is somewhat larger than the H-2 antigens but did not estimate the molecular weights. A molecular weight of 55,000 Daltons has been reported [16] for papain solubilized tumour antigen from an aminoazo dye-induced rat hepatoma, although probably the intact molecule is larger. These studies show that DOC is a good solubilizing agent for this tumour-associated antigen and can give both H-2 and tumour antigens in an "intact" form which can be separated from the bulk of other membrane components. Although the antigenic activity detected by in vivo absorbed allo- and xenoantisera specific for P-815 cells has been ascribed to a "tumourassociated" antigen, it has not yet been established whether this (or these) antigen(s) are tumour-specific transplantation antigens or if they are other immunologically specific determinants expressed by the P-815 cell. It has been shown in the preceding paper that these antigens are typical of the P-815 cell and are not due to antigenic drift or modification of our particular cell line. Acknowledgements--We would like to thank Miss S. Widmer for excellent technical assistance.

REFERENCES 1. 2. 3.

F. LILLYand S. G. NATHENSON,Solubilization of the H-6.A isoantigen of the mouse. In Advance in Transplantation. (Edited by J. DAUSSET,J. HAMBUROER and G. MATHI~)p. 279. Munksgaard, Copenhagen (1968). A. SANDERSON,HL-A substances from human spleens. Nature (Lond.) 220, 192 (1968). D . L . MANN, G. N. ROGENTINE,J. L. FAHEY and S. NATHENSON,Human lymphocyte membrane (HL-A) alloantigens: Isolation, purification and properties. J. Immunol. 103, 282 (1969).

Solubilization of Histocompatibility and Tumour-associated Antigens

4. 5.

6. 7.

8. 9.

I0. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

22. 23.

24. 25.

P. CRESSWELL,R. J. ROBB, M. J. TURNER and J. L. STROMINGER, Papainsolubilized HL-A antigens: Chromatographic and electrophoredc studies of the two subunits from different specificities. J. biol. Chem. 249, 2828 (1974). M. HEss and D. A. L. DAVIES, Basic structure of mouse histocompatibitity antigens. Europ. J. Biochem. 41, 1 (1974). R . A . REISFELD, M. A. PELLEGRINOand B. D. KAHAN, Salt extraction of soluble HL-A antigens. Science 172~ 1134 (1971). D . L . MANN, The effect of enzyme inhibitors on the solubilization of HL-A antigens with 3M KC1. Transplantation 14, 398 (1972). S.K. OH, M. A. PELLEGRINOand R. A. REISFELD,Hypertonic salt extraction of HL-A antigens: Assessment of protease activity. Proc. Soc. exp. Biol. Med. (N.Y.) 145, 1272 (1974). M. PRAT, G. TARONE and P. M. COMOGLIO, Antigenic and immunogenic properties of membrane proteins solubilized by sodium deoxycholate, papain digestion or high ionic strength. Immunochemistry 12~ 9 (1975). T . A . SPRINGER, J. L. STROMINGERand D. MANN, Partial purification of detergent-soluble HL-A antigen and its cleavage by papain. Proc. nat. Acad. Sci. (Wash.) 71, 1539 (1974). D. SNARY, P. GOODFELLOW, M. J. HAYMAN, W. F. BODMER and M. J. CRUMPTON, Subcellular separation and molecular nature of human histocompatibility antigens (HL-A). Nature (Lond.) 247, 457 (1974). J . R . DAWSON,J. SILVER, L. B. SHEPPARDand D. B. AMOS, The purification of detergent-solubilized HL-A antigens by affinity chromatography with the hemagglutinin from Lens culinaris. J. lmmunol. 112, 1190 (1974). I. BERNIER,A. DAUTmNY,J. COLUMBAmand P. JOLL~S, Detergent-solubilized HL-A antigens from human platelets: A comprehensive study of various purification techniques. Bioehim. biophys. Acta 356, 82 (1974). M. LETARTE-MUIRHEAD,R. T. ACTON and A. F. WILLIAMS, Preliminary characterization of Thy-1.1 and Ag-B antigens from rat tissues solubilized in detergents. Bioehem. J. 143, 51 (1974). D. SAUSER, C. ANCKERS and C. BRON, Isolation of mouse thymus-derived lymphocyte specific surface antigens. J. Immunol. 113, 617 (1974). R . W . BALDWIN,J. R. HARRIS and M. R. PRICE, Fractionation of plasma membrane-associated tumour-specific antigen from an aminoazo dyeinduced rat hepatoma. Int. J. Cancer 11, 385 (1973). R. BILLING and P. I. TERASAKI, Human leukemia antigen. II. Purification. J. nat. Cancer Inst. 53, 1639 (1974). R. S. METZGAR, T. MOHANAKUMAR,R. W. GREEN, D. S. MILLER and D. P. BOLOGNESI, Human leukemia antigens: Partial isolation and characterization. J. nat. Cancer Inst. 52, 1445 (1974). D . A . L . DAVIES, V. S. G. BAUGr~, S. BUCKHAMand A. J. MANSTONE,Separation of the specific antigen of a mouse lymphoma from histocompatibility antigens. Europ. or. Cancer 10, 781 (1974). R . W . BALDWIN,J. G. BOWENand M. R. PRICE, Solubilization of membraneassociated tumour-specific antigens by fl-glucosidase. Biochim. biophys. Acta 367, 47 (1974). E. K~DAR, A. AARONOVand D. SULITZEANU,Analysis of cell membrane antigens using radioiodinated, purified antibodies. I. Solubilization and preliminary fractionation of membrane antigens of cells transformed by SV40 virus. Israel J. med. Sci. 9, 266 (1973). T . B . DUNN and M. POTTER, A transplantable mast-cell neoplasm in the mouse. J. nat. Cancer Inst. 18, 587 (1957). M. BERTSCHMANN,K. J. CLEMETSONand E. F. L/0SCHER, Preparation and properties of antisera directed against antigens of the P-815 mastocytoma cell not shared by its syngeneic host, the DBA/2 mouse. Europ. J. Cancer 12, 255 (1976). R. SCHINDLER,M. DAY and G. A. FISCHER, Culture of neoplastic mast cells and their synthesis of 5-hydroxytryptamine and histamine in vitro. Cancer Res. 19, 47 (1959). D. F. H. WALLACH and V. B. KAMAT, Preparation of plasma-membrane fragments from mouse ascites tumor cells. In Methods in Enzymology. (Edited by E. F. NEUFELD and V. GINSBURG) Vol. VIII, p. 164. Academic Press, New York (1966).

269

270

K. J. Clemetson et al. 26. 27.

28. 29. 30.

O . H . LOWRY, N. J. ROSEBROUOH, A. L. FARR and R. J. RANDALL, Protein measurement with the Folin phenol reagent, or. biol.Chem. 193, 265 (1951). O. RENKONEN, r. U. KOSANEN and O. V. RENKONEN, Extraction of serum

inositides and other phosphatides. Ann. Med. exp. Biol. Fenniae (Helsinki), 41, 375 (1963). H. JATZK~W~TZ, Line neue Methode zur quantitativen Uhramikrobestimmung der Sphingolipoide aus Gehirn. Z. physiol. Chem. 326, 61 (1961). E . R . WEIBEL, G. S. KmTLER and W. F. SCH~.RLE, Practical stereological methods for morphometric cytology, or. Cell. Biol. 30, 23 (1966). B.D. SCHWARTZ,K. KATO,S. E. CULLENand S. G. NATttENSON,H-2 histocompatibility alloantigens. Some biochemical properties of the molecules solubilized by NP-40 detergent. Biochemistry 12, 2157 (1973).