Biochimica et Biophysics Acta 835 (1985) 577-583

Elsevier

577

BBA 51982

Monoclonal antibodies raised against NeuAccw2-heolactotetraosylceramide

detect carcinoma-associated gangliosides

Olle Nilsson a, Leif Lindholm b, Jan Holmgren b and Lars Svennerholm a

a Department of Psychiatry and Neurochemistry, St. Jijrgens Hospital, S-422 03 Hisings Backa, and h Department of Medical Microbiology, Guldhedsgatan IO, S-41 3 46 Giiteborg (Sweden)

(Received March 4th. 1985)

Key words: Ganglioside antigen; Oncofetal antigen; Monoclonal antibody; Tumor-associated antigen

Monoclonal antibodies were obtained by the immunization of mice with 6’L,, (IV6NeuAc-nLcOse.Cer) adsorbed to SalmoneUa minnesota. The monoclonal antibodies showed a specificity for gangliosides with a terminal NeuAca26Gal substitution, which was demonstrated in solid-phase binding assay and in liposome inhibition assay. Gangliosides with a NeuAcaZiGal substitution were minor components of different normal tissues. However, these gangliosides were enriched in carcinomas of many tissues, and were particularly enriched in most colorectal carcinomas and in lung carcinomas. 6’L,, is a characteristic ganglioside in fetal intestinal mucosa (meconium). This ganglioside and other gangliosides with a terminal NeuAcaZ6Gal substitution might represent oncofetal antigens expressed in carcinomas owing to an activation of a ‘fetal’ sialyltransferase.

Introduction

Gangliosides (sialic acid-containing glycolipids) have recieved much attention during the last de- cade owing to their putative role in cell recognition phenomena [l]. Changes in the ganglioside com- position during normal cell maturation and differ- entiation as well as malignant transformation of cells have been documented in several studies (for review, see Ref. 1). The great complexity and low concentration of gangliosides in extra neural tis- sues has made the search for specific differentia- tion and tumour-associated gangliosides by chem- ical methods very tedious and complicated. The

Nomenclature: the ganglioside. nomenclature of Svennerholm was used [27]. 6’L,, and 3’L,, were used as short-hand designation for IV6NeuAc-nLcOse,Cer and IV’NeuAc- nLcOse.,Cer, respectively. 6’-Gangliosides were used as short hand designation for gangliosides with a terminal NeuAcaZ- 6Gal linkage.

use of monoclonal antibodies has therefore been of great practical importance for the identification and study of tumour-associated glycolipid and ganglioside antigens [2-lo].

In a previous paper, NeAca2-6-neolactotetra- sylceramide (6’LM,) was shown to be a major ganglioside in fetal intestine (meconium) [ll]. The present report describes the production of mono- clonal antibodies obtained after immunization with

6’L,, 1 identifying gangliosides with a terminal NeuAcaZ-6 galactose linkage, and demonstrates the accumulation of such gangliosides in human carcinomas of different tissue origin and histo- pathological type.

Materials

Horse-radish peroxidase-conjugated rabbit anti-mouse immunoglobulin and isotype-specific rabbit anti-mouse immunoglobulins were obtained

0005-2760/85/$03.30 0 1985 Blsevier Science Publishers B.V. (Biomedical Division)

578

from Dakopatt A/S, Copenhagen, Denmark, and

Meloy Laboratories, Inc., Springfield, VA, U.S.A. ‘251-anti-mouse F(ab’),- fra ment g was obtained

from The Radiochemic~ Centre, Amerham, U.K.

X-AR5 X-ray film used for the autoradiographic

detection of TLC-immunostained ganglioside anti-

gens were from Eastman-Kodak, Rochester. NY,

U.S.A. HPTLC-plates silica gel 60 were obtained

from Merck AG, Darmstadt, F.R.G. Sephadex G-25 Fine was obtained from Pharmacia Fine

Chemicals, Uppsala, Sweden. The anion exchange

resin Spherosil-DEAE-dextran, used for isolation and separation of gangliosides was a gift from the

Institute Merieux, Lyon, France [12]. Polystyrene beads (6.4 mm) were purchased from Precision Plastic Ball Co., Chicago, IL. U.S.A. Egg phos-

phatidylcholine was obtained from Sigma Chem-

ical Co., St. Louis, MO, U.S.A. All other chemicals were of analytical quality and used without further

purification.

All gangliosides and neutral glycolipids used for

immunization and determination of the reactivity

of the monoclonal antibodies were isolated at the

Department of Neurochemistry, University of

Goteborg.

Methods

Production of monoclonal antibodies

Balb/c mice, 6-8 weeks old, were immunized intravenously with 3 nmol of 6’L,, (IV’NeuAc-

nLcOse,Cer) isolated from meconium as described previously [ll]. The 6’L,, was adsorbed to ap-

prox. 50 pg of acid-treated Salmonella bacteria

[7,13] suspended in 100 ~1 of phosphate-buffered saline, pH 7.4. An identical booster dose was

administered 8-12 weeks after priming. The fusion

protocol of de St. Groth and Scheidegger was used

for the production of hybridomas [14]. Hybrids

were produced by fusion between Sp 2/O myeloma cells and spleen cells three days after the booster dose. Culture media were collected from each growing hybrid and tested for antibodies directed against 6’LM, in an ELISA system, with 25 pmol of 6/L,, adsorbed to the wells of microtiter plates. The preparation of 6’LM, used for immunization contained small amounts of sialosylfucosyllacto- tetraosylceramide (IV3NeuAc,II14Fuc-LcOse,- Cer); to exclude hybridomas producing antibodies

against this structure, the initially positive hy-

bridomas were tested against sialosylfucosyllacto-

tetraosylceramide isolated from COLO 205 cells [15]. Hybrids producing antibodies with specificity

for 6’L,, in both screening procedures were cloned

by limited dilution, and expanded. The isotype of the antibodies was determined by single radial

immunodiffusion in agarose containing isotype

specific anti-mouse immunoglobulins.

Determination of ganglioside reactivity of the mono-

clonal antibodies

Solid-phase binding assqv. The reactivity of the antibodies against various gangliosides and neutral

glycolipids was tested in solid-phase double anti- body radioimmunoassay with the glycolipids ad- sorbed to the wells of polyvinyl microtiter plates [7]. The wells containing different amounts of gly-

colipids were incubated with monoclonal antibody

medium for 4 h. Bound monoclonal antibody was detected by incubation with ‘251-anti-mouse

F(ab’)z fragment. The reactivity of the antibodies

was tested against 6’LM, [ll], 3’L,, (IV3NeuAc-

nLcOse,Cer) isolated from erythrocytes [16], L,, (nLcOse,Cer) obtained after sialidase or weak acid

hydrolyses of 6’L,, or 3’L,, and TLC purifi-

cation of the hydrolysed product, sialosylfucosyl- lactotetraosylceramide (IV 3NeuAc,-II14 Fuc- LcOse,Cer), a mixture of brain gangliosides (G M3,

G Ml’ GM,, Gn,, GDlar Gnu,, G,,,, Go,,) and a mixture of neutral glycolipids (GlcCer, LacCer, globotriaosylceramide, gIobotetraosyl~eramide, gangliotetraosylceramide).

Liposome inhibition assay. The reactivity of one of the monoclonal antibodies (LM-4) was also

tested in an inhibition assay. Liposomes contain- ing different gangliosides and glycolypids were

used as inhibitors of the binding of the LM-4

monoclonal antibody to 6’L,, immobilized on

polystyrene beads. The 6’L,, was adsorbed to the polystyrene beads by mixing the beads with 6’L,, dissolved in isopropanol/hexane/0.25% aq. KCl, 2 : 1 : 1 (10 pmol 6’L,,/O.25 ml per bead) and rotary evaporation of the solvent. The liposomes were prepared by dissolving 20 nmol of glycolipid and 100 nmol of egg phosphatidylcholine in chlo- roform/methanol/water (60 : 30 : 4.5, v/v). This was evaporated to dryness and dissolved in 1 ml of phosphate-buffered saline using ultrasonication.

579

The inhibition assay was performed by preincuba- tion of 100 ~1 of the LM-4 monoclonal antibody diluted in Tris-buffered saline, 1% bovine serum albumin (0.05 M Tris, 0.14 M NaCl, pH 7.8, 10 g bovine serum albumin/l, 100 mg merthiolate/l) to a concentration of approx. 0.5 pg/ml with 100 ~1 of liposomes diluted in Tris-buffered saline/ 1% bovine serum albumin in Minisorpm tubes 70 X 11 mm. One (i’L,,-coated bead was added after 45 min and the incubation was continued for a fur- ther 2 h. After washing with 3 X 2 ml of phos- phate-buffered saline the uninhibited monoclonal antibody bound to the polystyrene bead was de- tected by incubation overnight with 200 ~1 “‘I- anti-mouse F(ab’), fragment. The beads were then thoroughly washed with 3 X 2 ml of phosphate- buffered saline and counted in a gamma scintilla- tion counter. The inhibition assay was performed with liposomes containing 6’L,,, 3’L,, or L,, or with liposomes containing G Ml.

Determination of tissue distribution of gangliasides

with NeuAca2-6Gal linkage

The tissue distribution was determined by TLC-immunostaining of gangliosides isolated from different maIignant and normal tissues. The im- munostaining was performed essentially as de- scribed by Brockhaus et al. [17].

Total lipids were extracted from tissues with 2 X 20 vol. (w/v) of chloroform/methanol/water (4: 8 : 3, v/v) f18]. The total lipid extract was purified from low molecular conta~nants by chromatography on Sephadex G-25 1191. Ganglio- sides were isolated from the total lipid extract and separated into mono- and oligosialogangliosides by discontinuous gradient elution of gangliosides retained on the anion exchange resin Spherosil- DEAE-dextran [12].

A semiquantitative estimation of the concentra- tion of 6’-gangliosides in the different tissues was obtained by TLC-radioimmunostaining and com- parison of the staining intensity with known amounts of 6’L,,, run together with the samples on the TLC-plates.

Clinical material

Tumour and normal adult tissues were obtained at autopsy or at surgery. The tissues were frozen as soon as possible and stored at -20°C until

TABLE I

SURVEY OF THE CLINICAL MATERIAL

a Pooled material; b two cases analysed indi~duaIly, four cases analysed as a pool; ’ pooled from all ABH-blood groups; d isolated by gradient centrifugation from blood group A and

H; ’ pooled material [lo]; f Pooled from different types of leukemia; s a 20-week-old fetus aborted due to cerebroside-j?- galactosidase deficiency. care., carcinoma.

Tumour ?I Normal tissue n

Colorectal cart. Pancreatic cart. Gastric cart. Lung cart.

squamous cell care adeno cam. small cell care.

Kidney cart. Mammary cart. Uterus cam. Malignant meranoma Sarcoma Leukemia ’

24 I 6

Colon mucosa Pancreas Gastric mucosa Lung Spfeen Liver 4 Kidney Mammary gland Uterus endomet. Erythrocytes ’ Lymphocytes d Meconium Fetal lung, spleen,

and liver

6” 2

;;

3

6

4a

42 e

1s

analysed. The tumour tissue was dissected free from non-tumour and necrotic tissue, and only macroscopically obvious tumour tissue was analysed. Meconium was obtained and pooled from 42 full-term babies. Only the portion de- livered during the first 24 h after birth was col- lected [ll]. Fetal tissue was also obtained from a 20-week-old fetus aborted because of an inherited lysosomal storage disease (deficiency of cerebro- side+galactosidase), A survey of the clinical material analysed in given in Table I.

Results

Rea~tioity of the ant&S-L, monoclonal antibodies

The fusion between the Sp 2/O myeloma cells and spleen cells from Balb/c mice immunized with 6’L M1 yielded approx. 600 growing hybrids; four of these showed an apparent specificity for 6’L,, in the screening procedures and were cloned and used for the production of monoclonal an- tibodies. The monoclonal antibodies from the four clones reacted specificially with 6’L,,, without any detectable reactivity with the 3’-sialo deriva- tive or with the asialo derivative. Neither was any reactivity seen with the other gangliosides or neu-

580

tral glycolipids tested in the solid-phase radioim-

munoassay. As the four anti-6’L,, monoclonal antibodies all showed the same reactivity, specific-

ity results are illustrated only for one of them, the LM-4 monoclonal antibody (Fig. 1). The only liposomes which could inhibit the binding of the monoclonal antibodies to 6-L,, antigen were lipo-

somes containing 6’L,, , while the other liposomes had no effect on the binding (Fig. 2, this was only

tested with the LM-4 monoclonal antibody).

Figs. 3-5 show chemical staining with re-

sorcinol and immunostaining with LM-4 mono-

clonal antibody of monosialogangliosides isolated

from colorectal and lung carcinomas. The chem-

ical staining show a complex pattern of different

gangliosides, some of which comigrate with 6’-L,, . However, TLC migration should not be used alone

for identification of gangliosides as several gang- liosides may comigrate on TLC plates [15]. There-

fore, the identification of gangliosides based upon TLC migration should be confirmed by structural

analyses or, if available, immunostaining with specific monoclonal antibodies. The TLC- immunostaining with LM-4 antibodies of mono-

sialogangliosides isolated from carcinomas re- vealed at least three different gangliosides recog-

nized by the LM-4 antibodies in addition to 6’L,,

(Figs. 3-5). This was more clearly seen when the gangliosides were separated using an alkaline

Fig. 1. Solid-phase binding of LM-4 monoclonal antibody to

glycolipids. The assay was performed as described in Methods.

l . 6’L,,; n , 3’L,,; v, L,,; *, brain gangliosides (GM3, GM2,

G 1 GD,, GDI~~ GDlb- G,,b, GQlb); *, neutral glycoiipids

(G%er, LacCer, globutriaosylceramide, globotetraosylcera-

mide, gangliotetraosylceramide).

loo-

* 40 1

20/ 2 7.5 30 125 500

pmoi glycolipid

Fig. 2. Inhibition of LM-4 monoclonal antibody with glyco-

lipid-containing liposomes. The experimental procedure is de-

scribed in Methods. 0, liposomes containing 6’L,,; n , lipo-

somes containing 3’L,,; T, liposomes with LA,; A, liposomes

with GILI].

solvent system, which in some tumours separated

the 6’-ganglioside migrating as Gnlh into at least

two clearly distinguishable doublet bands. The TLC-immunostaining analyses also re-

vealed considerable differences in the ceramide

composition between 6’-gangliosides isolated from

carcinomas of different tissue origin. The 6’-gang- liosides in lung carcinomas migrated in all cases as doublet bands, owing to the differences in fatty acid chain length (Figs. 4 and 5). These doublets were also seen in normal lung tissue and fetal lung

tissue (Fig. 4). In colorectal carcinomas, the TLC-immunostaining indicated a much larger het-

erogeneity of the ceramide composition (Fig. 3). 6’L,, migrated in some cases as a single band, in

some as a double band, while in other cases the

6’Lh4, migrated in three to four close bands. This

complexity might depend upon the presence of both normal fatty acids and 2-OH fatty acids, as

well as phytosphingosines.

Tissue d~~t~~but~~n of the 6’-gang~i5side antigens The LM-4 monoclonal antibody of the IgG 2a

subtype was chosen for further studies of the tissue distribution of the corresponding antigens. The 6’-ganglioside antigens (i.e., gangliosides with a terminal NeuAccuZCtGal linkage) were detected in small amounts in various normal adult tissues; spleen, liver, lung, kidney, erythrocytes, lympho- cytes and uterus (i.e., the concentration was 0.2-2

581

- ,_ -.._-

Ref a I 2 3 4 5 6 7 8 9 10

GM3 1

GM2 -

GM1 - mm

GDla -

GDlb -

GTlb - m Origin_ _ _ . . . . _ . . . -

a 1 2 3 4 5 6 7 a 9 10

nmol/g tissue), but were not detected in normal adult colon mucosa, gastric mucosa, pancreas, brain, mammary gland, thyroid or thymus (i.e., the concentration was less than 0.1 nmol/g tissue). The antigens were also detected in high concentra- tions in fetal intestine (meconium) (Fig. 3) and were present in fetal lung (fig. 4), spleen and liver (not shown).

The 6’-gangliosides were highly enriched in most

Fig. 3. Monosialoganghosides isolated from

normal colon and coloncarcinomas. Upper

plate: staining with resorcinol. The references

contain, from above, G,,, G,,, G,,, Go,,

G Dla, GDW GT,, as indicated in the margin.

Lower plate staining with LM-4 monoclonal

antibody. Upper and lower plates: lane a,

6’Lhlt isolated from meconium Ill]. 2 nmol

upper plate, 20 pm01 lower plate; lane 1,

meconium 10 mg of tissue; lane 2, total gang-

liosides from normal adult colon mucosa, up-

per plate 50 mg tissue, lower plate 10 mg

tissue; lanes 3-8, colon carcinomas 2-5 mg

tissue, (lanes 3 and 6-8, primary tumour;

lanes 4 and 5, metastatic tumour); lanes 9

and 10, rectal carcinoma, 3 and 4 mg tissue.

The gangliosides were separated on HPTLC-

plates using chloroform/methanol/0.25% aq.

KCI 50 : 40 : 10 as solvent.

colorectal carcinomas (19/24), with a concentra- tion of 3-150 nmol/g tissue in comparison to less than 0.1 nmol/g in normal adult column mucosa (Fig. 3). The 6’-gangliosides were detected in lung carcinomas of different histopathological types (Figs. 4 and 5). They were elevated in 9/10 squamous epithelial lung carcinomas (concentra- tion 2-30 nmol/g) and in 5/8 small cell lung carcinomas (concentration l-8 nmol/g) as com-

234507 59 10 11 12 13 14 Fig. 4. Monosialogangliosides isolated from

normal lung and squamous epithelial cell lung QM3 - I carcinomas. Upper plate staining with re-

Qt.42 - * sorcinol. The references are as in Fig. 3. Lower

plate immunostaining with LM-4 monoclonal

antibody. Lane a, 2 nmol6’L,t [ll]. Lane 1,

normal adult lung, 25 mg tissue; lane 2, fetal lung, 25 mg tissue, upper plate, 10 mg tissue

lower plate; lanes 3-13, squamous epithelial

cell lung carcinomas, 3-5 mg tissue; lane 14,

(jr,o,” _ . . ..I . . . . ,... *I adenocarcinoma, 5 mg tissue. Lane 9 upper

plate x artefact band. Gangliosides were sep- 123456750 10 11 12 13 14 arated as in Fig. 3.

Fief b

GM3 = GM2 -

GM1 -

GDla-

GDlb-

GTlb- GOlb-

oripill.

a 1 pp 2m 3 4p 4m 5~ 5m 6 7~ i’m 6 Fig. 5. ~onosialog~gliosides isolated from

small cell lung carcinomas. Upper piate: re-

sorcinol staining, the references are as in Figs.

3 and 4: lane b, Fuc-GM, 2 nmol isolated

from case 1 [28]. Lower plate: immunostain-

ing with LM-4 monoclonal antibody. Lane a,

2 nmol 6’L,, upper plate, 50 pm01 lower

plate [II]: lanes l-8, small cell lung

carcinomas. 5-10 mg tissue. cases 1-X. re-

spectively. p. primary tumour: m. metastasis.

Gangliosides were separated as m Fig. 3. a 1 2p 2m 3 4~ 4171 5p 5m 6

pared to normal adult lung (approx. 0.5 nmol/g) (Fig. I ). 6’-Gangliosides were detected in

carcinomas originating from all tissues analysed.

They were elevated at least twice compared to the corresponding normal concentrations in 6/6

gastric carcinomas, 4/7 pancreatic carcinomas, 5/g kidney carcinomas, 2/‘4 mammary carcinomas

and Z/2 uterus carcinomas, with an approximate concentration ranging from OS-5 nmol/g tissue.

In contrast, these gangliosides were not detected in

the non-carcinoma tumours analysed (i.e.. the con- centration was less than 0.1 nmol/g).

Discussion

The monoclonal antibodies obtained after im- munization with 6/L,, showed an absolute specificity for gangliosides with a NeuAccu2-6Gal

linkage, which was demonstrated both in the

solid-phase binding assay and in the liposome

inhibition assay. Monoclonal antibodies with the same specificity have previously been described by Hakomori et al. [8,21]. The NeuAccu2-6 linkage of the sialic acid in gangliosides has thus far only been described in a terminal position in gangho- sides based upon the neolactotetraose structure [8,11,21-231, and monoclonal antibodies against the NeuAccu2-6Gal linkage of the sialic acid should be of value for the structural characterisation of such gangliosides. In addition to 6’L,, , which was

7p 7m 6

the dominating 6’-ganglioside in a11 tissues analysed, at least three other gangfiosides reacting with the LM-4 monoclonai antibody were detected

in carcinomas (Figs. 3 and 4). These gangliosides most likely represent N-acetyllactosaminyl chain

elongations of the neolactotetraose structure with a terminal N~uAc~2-6Gal-linkage 18,211.

The 6’-gangliosides were found in carcinomas

of all organs and were found in different histo-

pathological types of carcinomas, e.g., small cell

lung carcinomas (Fig. 5) and squamous cell lung carcinomas (Fig. 4). These gangliosides seem thus

to represent ganglioside antigens with a general carcinoma association. The distribution of the 6’- gangliosides was similar, but not identical, to the CA-50 ganglioside antigen, e.g., in colorectal carcinomas the antigens were complementary (Ref. 24, and Nilsson et al, unpublished data). In com- parison to the CA-50 ganglioside antigen the 6’- gangliosides were in most cases (except in pan-

creas carcinomas) the quantitatively dominating antigen (Ref. 24, and Nilsson et al., unpublished

data).

6’L,, is a minor component of normal adult tissues (Refs. 22, 23, and this study) and the ratio between 3’L,, and 6’L,, is greater than 10. In fetal intestine (meconium), 6’L,, has been shown to be the dominant ganglioside based upon neolactotetraose, while 3’L,U, was a minor compo- nent [11,25]. This indicates that the activity and/or

substrate specificity of sialyltransferases are differ-

ent during the fetal development, as compared to adult tissues. Malignant transformation has been suggested to lead to a retrogenetic expression of fetal glycosyltransferases. The accumulation of 6’- gangliosides in carcinomas might be interpreted as the result of an activation of a ‘fetal’ NeuAccr2-

6Gal sialyltransferase normally depressed in adult tissues.

Human carcinomas have a very complex gang-

lioside composition [26], and different carcinoma-

associated gangliosides have been characterized

[2,8-10,151. The 6’-gangliosides, although present in small amounts in normal tissues, may also be

useful markers of carcinomas.

Acknowledgements

This study was supported by grants from the Swedish Cancer Research Council, the Swedish Technical Research Council (STU), Stena Di-

agnostics AR, Goteborg, Sweden, the Swedish Medical Research Council, and the Medical Fa-

culty, University of Goteborg. The expert techni- cal assistance of Ms. Anne Nilsson is gratefully

acknowledged.

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