- Email: [email protected]
Immunochemical determination of forssman and blood group A-active glycolipids in human gastric mucosa by inhibition assay of liposome lysis
Journal of Immunological Methods, 53 (1982) 221-232 Elsevier Biomedical Press
221
Immunochemical Determination of Forssman and Blood Group A-Active Glycolipids in Human Gastric Mucosa by Inhibition Assay of Liposome Lysis Kei-ichi Uemura ~, Hiroshi Hattori, N o b u k o Kitazawa and Tamotsu Taketomi Department of Biochemistry, Institute of Adaptation Medicine. Shinshu University School of Medicine, Matsurnoto, Nagano 390, Japan
Received 9 November 1981, accepted 22 February 1982)
A simple liposome lmmunoassay, liposome immune-lysis inhibition (LILI) assay, is described for quantitative determination of individual glycolipid antigens. Liposomes containing fluorogenic marker, 4-methylumbelliferyl phosphate, were prepared from sphingomyelin, cholesterol, dicetylphosphate and standard glycolipid. Release of trapped markers from these liposomes by antibody and complement (liposome lysis) was inhibited by preincubating the antibody with test glycolipid incorporated into inhibitor liposomes. Based on the competitive inhibition, it was possible to quantitate each glycolipid antigen in less than picomolar amounts. The sensitivity and specificity of the assay were examined with purified glycolipid standards. LILI assay has been applied for the detection of Forssman glycolipid and blood group A-active glycolipid in human gastric mucosa and cancer tissues.
Key words: liposome lysis - - gl),colipid - - Forssman glycolipid - - blood group A -active glycolipid
Introduction Glycosphingolipids on cell surfaces may have functions in a number of biological recognition phenomena and represent a large variety of antigenic determinants (Yamakawa and Nagai, 1978). Various procedures have so far been developed for the sensitive and quantitative determination of glycolipids. The immunochemical methods are frequently used for glycolipid analyses and recently Young et al. (1979) devised a radioimmunoassay for quantitation of Forssman glycolipid. Such a highly sensitive technique is useful for detection of glycolipid antigens in limited amounts of tissues. Release of trapped markers from liposomes by antibody-complement i Correspondence address until the end of the year: Kei-ichi Uemura, Ph.D., Division of Communicable Diseases, Clinical Research Centre, Watford Road, Harrow, Middlesex, HA1 3U J, U.K. 0022-1759/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
222 (Kinsky, 1972) has been widely used for characterization of antibodies to glycolipids (Alving, 1977). I m m u n o a s s a y s based on the inhibition of liposome lysis have been devised for determination of antigens which compete for antibodies with amphipathic antigens incorporated into liposomes. Several different methods have been used for measurement of released markers, including spin labels and ESR (Wei et al., 1975; Hsia and Tan, 1978), fluorescent markers and fluorometry (Geiger and Smolarsky, 1977; Naito et al., 1980), and the use of special electrodes (Shiba et al., 1980; H a g a et al., 1980). However, application of these assays to determine substances in cells, tissues or biological fluid is still scarce, and a simple and flexible procedure should be developed. In this paper we report a liposome immunoassay for quantitative determination of individual glycolipids based on the competitive inhibition for antibodies in the liposome lysis reaction, which we have described previously as a sensitive fluorometric assay using a fluorogenic marker, 4-methylumbelliferyl phosphate (Six et al., 1974; U e m u r a et al., 1980a). The application of this assay for the detection of F o r s s m a n and blood group A-active glycolipids in h u m a n gastric mucosa and cancer tissues is described.
Materials and Methods Glycolipids *
Glycolipids were isolated from the following sources as previously described (Taketomi and Uemura, 1980): Forssman glycolipid from caprine erythrocytes; globoside from porcine and h u m a n erythrocytes; lactosylceramide, B-active galactosylparagloboside from bovine erythrocytes; galactosylceramide from h u m a n brain; globotriaosylceramide from urine of a F a b r y patient; asialo GM2 from guinea pig erythrocytes. Paragloboside and asialo GMi were prepared by mild acid hydrolysis of sialosylparagloboside and rabbit brain gangliosides, respectively, and were purified by c h r o m a t o g r a p h y on silicic acid. Blood group A-active glycolipid (A a) was isolated and purified from h u m a n erythrocytes. Total lipid extract was subjected to D E A E - S e p h a d e x A-25 chromatography, acetylatiom Florisil column chromatograp h y and deacetylation as described (Hattori et al., 1980). The neutral glycolipid fraction was fractionated on a Silica gel 40 (Merck) column and a crude fraction containing A a glycolipid was subjected to preparative T L C to isolate 'TLC-purified' A ~ glycolipid ( H a k o m o r i and Watanabe, 1976). * Forssman glycolipid, GalNAc(al ~ 3)GalNAc(fll ~ 3)Gal(al ~ 4)Gal(fll ~ 4)Glc(fll ~ l)ceramide; globoside, GalNAc(fll ~ 3)Gal(al ~ 4)Gal(fl 1~ 4)Glc(fl I -~ l)ceramide; globotriaosylceramide, Gal( al 4)Gal(fll~4)Glc(fll~l)ceramide; lactosylceramide, Gal(fll~4)Glc(fll~l)ceramide; galactosylceramide, Gal(fll ~ 1)ceramide; asialo GM2, GalNAc(J~l ~4)Gal(fll ~4)Glc(fll ~ l)ceramide; asialo GMI, Gal(fll~3)GalNAc(fll~4)Gal(fll~4)Glc(fll~l)ceramide; paragloboside, Gal(fll-.4)GlcNAc(fll~ 3)Gal(fll ~ 4)Glc(fll ~ 1)ceramide; sialosylparagloboside, NeuGc(et2 ~ 3)Gal(fll ~ 4)GlcNAc(fll 3)Gal(fll -. 4)Glc(fll ~ 1)ceramide; B-active galactosylparagloboside, Gal(al ~ 3)Gal(fl 1~ 4)GlcNAc(fll 3)Gal(fll ~ 4)Glc(fll ~ l)ceramide; A-active glycolipid (Aa), GalNAcal ~ 3Gal(2 ~- laFuc)fll 4GlcNAcfll ~ 3Galfll ~ 4Glcfll ~ lceramide.
223
A n tisera Antisera to Forssman glycolipid were prepared in rabbits as described earlier (Uemura et al., 1979) by a single injection of the glycolipid with Freund's complete adjuvant. Anti-globoside, anti-asialo G M2, anti-asialo G Mj and anti-paragloboside sera were prepared by injection into rabbits of a mixture of the respective glycolipid and bovine serum albumin in Freund's complete adjuvant (Uemura et al., 1980b). H u m a n anti-A blood group typing serum was obtained from Ortho Diagnostics (Raritan, N J). All the sera were heat-inactivated at 56°C for 30 min. A part of the anti-A serum (1.0 ml) was absorbed with Forssman glycolipid-containing liposomes (1.0/~mol of Forssman glycolipid) by incubating for 1 h at room temperature and then the mixture was centrifuged at 30,000 × g for 20 min (Uemura et al., 1980b). The supernatant was used for assay. Fractions containing IgG antibodies were separated by Sephadex G-200 gel filtration as described previously (Uemura et al., 1980b). Tissues and preparation of glycolipids Normal gastric mucosa and cancer tissues were obtained surgically or at autopsy from 9 individuals in Shinshu University and its associated hospitals (kindly provided by Drs. T. Katsuyama and K. Ono, Department of Pathology, Shinshu University School of Medicine). Total neutral glycolipid and ceramide pentasaccharide fractions were prepared as described previously (Hattori et al., 1980, 1981). Liposome immune-lysis inhibition assay (LILI assay) Bovine brain sphingomyelin, dicetylphosphate and 4-methylumbelliferyl phosphate were purchased from Sigma Chemical Co., St. Louis, MO; cholesterol from E. Merck, Darmstadt; alkaline phosphatase (EC 3.1.3.1) from Boehringer-ManheimYamanouchi, Tokyo. Liposomes containing entrapped fluorogenic marker, 4-methylumbelliferyl phosphate, were prepared essentially as described previously (Uemura et al., 1980b) with some modifications for microassay. Multicompartment liposomes were made from sphingomyelin, cholesterol, dicetylphosphate and glycolipid (100, 75, 10 and 5 nmol, respectively) by dispersing dried lipids in 100 /~1 of 100 mM 4-methylumbelliferyl phosphate solution adjusted to pH 7 with NaOH. The untrapped marker was removed by dialysis for 3 h against 4 changes of 200 ml of 75 mM NaC1/75 mM KCI and the final volume of the liposome preparation was adjusted to 1.25 ml with Tris-buffered saline (20 mM Tris-HC1, pH 8.0/150 mM NaC1/0.15 mM CaC12/0.5 m M MgCI2). The amount of 4-methylumbelliferyl phosphate trapped inside the liposomes was 0.150 -+ 0,032 (S.D.)/~mol and remained almost unchanged during the storage at 4°C for at least 2 days. The above preparation (marker liposome) was diluted further to 1 : 100 with Tris-buffered saline before use. Antibody titration was performed by incubating Tris-buffered saline (25 t,1), various amounts of antibody preparation (25 #1), liposomes (25/~1) and guinea pig serum diluted to 1:25 (25 #1) for 30 rain at room temperature. After incubation 0.5 ml of Tris-buffered saline containing 0.2 U of alkaline phosphatase was added to each assay tube and the extent of marker released from the liposomes was de-
224 termined fluorometrically as described previously (excitation wavelength, 365 nm: emission wavelength 448 nm) (Uemura et al., 1980a). The amount of antibody which caused 90% of the maximum marker release was determined for the use in the following inhibition assays, in that the antibody was limiting and the complement was in excess. A constant amount (75 nl) of rabbit anti-Forssman IgG, thus determined, gave 66.4 -+ 5.9 (S.D.) % lysis of Forssman liposome preparations made over a period of several months. The amount of guinea pig serum (240 260 C H s 0 / m l ) required for 50% marker release was 25 ~tl of 1:200 dilution. To prepare the glycolipid antigen for inhibition assay, the procedure of Young et al. (1979) was essentially followed. Each glycolipid as the standard or control (5 nmol), or each aliquot of glycolipid fractions prepared from the tissues (corresponding to 50-400 mg wet weight) was mixed with 100 nmol of sphingomyelin and 75 nmol of cholesterol in organic solvent. The lipids were dried under a nitrogen stream and then dissolved in 20/xl of methanol by warming, Liposomes were formed by the addition of 480 /~1 of hot Tris-buffered saline with vigorous mixing on a vortex mixer. Liposomes prepared in this manner have smaller sizes (closer to the unilamellar state) and can expose more antigens on their surface than the multilamellar liposomes made by dispersing the dried lipids directly with buffer (see below, Fig. 5). This preparation (inhibitor liposome) was further diluted with Trisbuffered saline to give appropriate glycolipid concentrations. L I L I assay was performed at room temperature in 10 m m × 75 m m glass tubes. The inhibitor liposomes of various dilutions (25 ~tl) and the antibody preparation (25 /zl) were incubated for 60 rain. The marker liposomes (25 /~1) and guinea pig serum diluted to 1:25 (25 /~1) were then added to each tube and incubation was continued for 30 min. The extent of marker released from the marker iiposomes was determined as described above. Results are given as the percentage inhibition of marker release and were calculated from duplicates according to the formula: % inhibition = 100 X (C - T ) C where C is the amount of marker released without inhibitor liposomes and T is the amount of marker released with inhibitor liposomes. The variation among duplicate or triplicate samples in an assay was not greater than 10%, usually being within 5%.
Results
Specificity of anti-Forssman and anti-A In complement-dependent liposome lysis, rabbit IgG antibodies against Forssman glycolipid produced specific marker release from liposomes containing Forssman glycolipid (Fig. 1), but did not show any cross-reactivity with liposomes containing blood group Aa glycolipid (Fig. 2). The IgM fraction of rabbit anti-Forssman serum reacted to some extent with globoside and asialo GM2 (Uemura et al., 1980b). Accordingly, the I g G fraction was used as a specific anti-Forssman reagent for LILI assay.
225 80 r
80' ///
_~4o: ~
lr
_
' ........
/
40?
.,
;
/
z .....
,
/ ./
i ',,r 0 ~ ......... 0
I00 200 Antiserum ( n I )
soo '?o5
0
I00
500 Antiserum
560 ( nl )
Fig. 1. lmmune-lysis of Forssman-liposomes with rabbit anti-Forssman lgG (O), human anti-A serum (unabsorbed) (Q), or human anti-A serum absorbed with Forssman-liposomes (x). Liposomes were prepared from sphingomyelin, cholesterol and dicetylphosphate (1:0.75:0.1, by tool) with Forssman glycolipid (0,05 mol/mol sphingomyelin). Marker release (% of the trapped marker) was determined after incubation of liposomes containing 1 pmol of the glycolipid with various amounts of antibodies in the presence of guinea pig complement. With heat-inactivated guinea pig serum the marker release did not exceed 2%. Fig. 2. Immune-lysis of Aa-liposomes with rabbit anti-Forssman IgG (O), human anti-A serum (unabsorbed) (O), or human anti-A serum absorbed with Forssman-liposomes (x). Liposomes were prepared from sphingomyelin, cholesterol and dicetylphosphate (1:0.75:0.1, by mol) with Aa glycolipid (0.03 mol/mol sphingomyelin). With heat-inactivated guinea pig serum the marker release did not exceed 1%.
O n the other hand, h u m a n a n t i - b l o o d g r o u p A serum reacted with l i p o s o m e s c o n t a i n i n g F o r s s m a n glycolipid (Fig. 1) as well as with l i p o s o m e s c o n t a i n i n g b l o o d g r o u p A a g l y c o l i p i d (Fig. 2). This a n t i - F o r s s m a n activity was c o m p l e t e l y r e m o v e d by a b s o r p t i o n of the a n t i - A serum with l i p o s o m e s c o n t a i n i n g F o r s s m a n glycolipid (Fig. 1). T h e a b s o r b e d serum c a u s e d specific m a r k e r release from l i p o s o m e s c o n t a i n ing A a glycolipid, a l t h o u g h the a n t i - A a glycolipid activity was r e d u c e d by this t r e a t m e n t (Fig. 2), i n d i c a t i n g that the h u m a n a n t i - A serum c o n t a i n e d some antib o d i e s reactive with b o t h F o r s s m a n a n d A ~ d e t e r m i n a n t s . T h e a b s o r b e d a n t i - A s e r u m was used as a specific a n t i - A r e a g e n t for L I L I assay.
Inhibition by purified glycolipids of liposome irnmune-lysis T h e i m m u n e - l y s i s of m a r k e r l i p o s o m e s c o n t a i n i n g F o r s s m a n glycolipid was i n h i b i t e d b y p r e i n c u b a t i n g a n t i - F o r s s m a n a n t i b o d y with various a m o u n t s of Forssm a n glycolipid in i n h i b i t o r l i p o s o m e s (Fig. 3). C o m p l e t e i n h i b i t i o n was o b t a i n e d with 30 p m o l of F o r s s m a n glycolipid, while 0.3 p m o l ( a b o u t 0.5 ng) was the lower limit of detection. I n h i b i t i o n b y A a g l y c o l i p i d was negligible; c r o s s - r e a c t i o n with a n t i - F o r s s m a n , if any, was less than 0.25%. N o i n h i b i t i o n was o b s e r v e d with o t h e r glycolipids tested. Fig. 4 i n d i c a t e s that the i m m u n e - l y s i s of m a r k e r l i p o s o m e s c o n t a i n i n g A a glycolipid was i n h i b i t e d d e p e n d i n g on the a m o u n t s of A a glycolipid a d d e d as i n h i b i t o r liposomes. A b o u t 0.6 ng of A a glycolipid gave 50% inhibition, whereas 100 ng of F o r s s m a n glycolipid, as well as o t h e r glycolipids, showed no i n h i b i t i o n at all. Similar assay systems were e x a m i n e d using r a b b i t I g G a n t i b o d i e s to globoside,
226
,oo[
100
y/
g
/
g *7-
.f
~_ 50
/
Z c
c
oh
-- ;
~o--
Glycolipid
( pmoi
,6~ )
5O
o:j
'l Glycolipid
J
-,o
T -1 i___
~oo
( ng )
Fig. 3. Inhibition of liposome immune-lysis by purified glycolipids (Forssman assay system). Rabbit anti-Forssman IgG (75 nl in 25 ~1 of Tris-buffered saline) was incubated at room temperature for 60 min with various amounts of inhibitor liposomes (25 #1) containing Forssman glycolipid (O), Aa glycolipid (0), or other glycolipids. The amount of each glycolipid added per assay is indicated on the abscissa. Incubation was continued for 30 min after the addition of the marker liposomes (25 ill, 1 pmol of Forssman glycolipid) and guinea pig serum (1:25 dilution, 25 #1). The bar indicates the range of % inhibition caused by inhibitor liposomes which contained the following glycolipids: galactosylceramide, lactosylceramide, globotriaosylceramide, asialo GM2, asialo GM~, globoside, paragloboside and B-active galactosylparagloboside. Fig. 4. Inhibition of liposome immune-lysis by purified glycolipids (Aa assay system). Human anti-A serum absorbed with Forssman glycolipid (500 nl in 25 /~1 of Tris-buffered saline) was incubated at room temperature for 60 min with various amounts of inhibitor liposomes (25/~1) containing Aa glycolipid (O), Forssman glycolipid (0), or other glycolipid. The amount of each glycolipid added per assay is indicated on the abscissa. Incubation was continued for 30 min after the addition of the marker liposomes (25 ~1, 0.6 pmol of Aa glycolipid) and guinea pig serum (1:25 dilution, 25 /~1). The bar indicates the range of % inhibition caused by other glycolipids (see the legend to Fig. 3).
asialo G M2, asialo G M1 or paragloboside a n d the respective marker liposomes. Each glycolipid a n t i g e n was d e t e r m i n e d with high sensitivity a n d specificity (Table I). In control experiments (Fig. 5), the highest sensitivity was achieved when inhibitor liposomes c o n t a i n i n g F o r s s m a n glycolipid were prepared with s p h i n g o m y e l i n a n d cholesterol by d i l u t i n g with buffer the lipids dissolved in hot m e t h a n o l ; less effective was the liposomes prepared by dispersing the dried lipids directly with buffer which were of m u l t i l a m e l l a r structures in heterogeneous sizes (Six et al., 1974); F o r s s m a n glycolipid alone in micellar form was f o u n d to be least effective as inhibitor. T h e i n h i b i t i o n curve was not affected by varying the c o n t e n t of F o r s s m a n glycolipid in i n h i b i t o r liposomes from 1 to 15 mol% of sphingomyelin. These observations were consistent with those reported previously ( Y o u n g et al., 1979; N a i t o et al., 1980). T o examine whether a large a m o u n t of glycolipids might disturb the assay by their a n t i c o m p l e m e n t a r y effect, liposomes were prepared from s p h i n g o m y e l i n (100 nmol), cholesterol (75 n m o l ) a n d total n e u t r a l glycolipids (52 fig) o b t a i n e d from n o r m a l h u m a n gastric mucosa. W h e n guinea pig serum was i n c u b a t e d with these liposomes u n d e r the i m m u n e - l y s i s assay condition, 1 n m o l of glycolipids (10 times as m u c h as in the s t a n d a r d assay) did not reduce the c o m p l e m e n t activity c o m p a r e d with the buffer control (175 C H s 0 / m l a n d 185 C H s 0 / m l , respectively).
TABLE I SENSITIVITY AND SPECIFICITY OF LILI ASSAY FOR INDIVIDUAL GLYCOLIPIDS T e s t gtycolipid in inhibitor l i p o s o m e
L I L I assay for Globoside
Galactosylceramide Lactosylceramide Globotriaosylceramide Globoside F o r s s m a n glycolipid
. .
Forssman .
-
Paragloboside
. . n.d.
Asialo G Mz
.
.
.
* 15.0 -
Asialo G uf2 Asialo GMj G a l ( a l ~ 3)paragloboside A a glycolipid
a
Asialo G Mi
. .
.
.
. -
.
.
.
.
-
-
5.35
-
.
-
0.56
. *
. -
.
0.30 .
.
A a
.
2.12
.
Paragloboside
. n.d.
. n.d.
-
0.37
N u m b e r s are the a m o u n t of glycolipid ( p m o l ) which g a v e 50% inhibition of l i p o s o m e lysis. --, less t h a n 10% inhibition at 100 p m o l ; *, less t h a n 15% inhibition at 100 p m o l ; n,d., not d e t e r m i n e d . A n t i b o d i e s used were r a b b i t l g G p r e p a r e d by i m m u n i z a t i o n with purified glycolipid except anti-A, w h i c h was h u m a n blood g r o u p t y p i n g s e r u m a b s o r b e d with F o r s s m a n - l i p o s o m e s .
228 IOC
I00
5O
5°t
/
! J
0.1
Forssman glycolipid
tO0
I
( pmol )
o ~,
o:, Tissue
_ ~
, wet weight
]
,o (rag)
Fig. 5. Effect of various types of inhibitor liposomes on the inhibition of liposome immune-lysis. Inhibition assay was performed as described in Fig. 3 with the following inhibitors: a, Forssman glycolipid alone in micellar form: b, liposome prepared by dispersing the dried lipids directly with buffer (sphingomyelin, cholesterol and Forssman glycolipid, 1 : 0.75 : 0.05, by mol); c, d, e, liposomes prepared by diluting with buffer the lipids dissolved in hot methanol (the standard method), the amount of Forssman glycolipid being varied, 0.15, 0.05 and 0.01 mol/mol sphingomyelimrespectively. Fig. 6. Determination of Forssman antigen in human gastric mucosa by inhibition of liposome immunelysis. Forssman assay system was used as described in the legend to Fig. 3. An aliquot of the neutral glycolipid fraction from patient no. N2 (corresponding to 65 mg wet tissue) was mixed with 50 nmol of sphingomyelin and 37.5 nmol of cholesterol to prepare inhibitor liposomes. The % inhibition of liposome immune-lysis was plotted against the tissue wet weight corresponding to the amount of glycolipidsin each assay tube. I n a n o t h e r control experiment, the presence of other glycolipids did not interfere very m u c h the antigenic activity of F o r s s m a n glycolipid. The i n h i b i t i o n by F o r s s m a n glycolipid of i m m u n e - l y s i s was, however, reduced by 5 - 8 % when the total neutral glycolipids of h u m a n gastric mucosa were i n c o r p o r a t e d into the same i n h i b i t o r liposomes (in a ratio of the n e u t r a l glycolipids to F o r s s m a n , 25:1 by mol). The n e u t r a l glycolipid fraction consisted m a i n l y of galabiosylceramide (25%), globoside (28%) a n d fucolipids c o n t a i n i n g more than 5 sugar residues (12%) (Hattori et al., 1980). The above result indicated that F o r s s m a n glycolipid could be d e t e r m i n e d in crude glycolipid fractions, though the c o n t e n t s measured were slightly underestimated.
Quantitative determination of Forssman antigen in tissues by L I L I assay By c o m p a r i s o n with the i n h i b i t i o n curve for purified F o r s s m a n glycolipid (Fig. 3), the a m o u n t of F o r s s m a n antigen in tissues could be q u a n t i t a t e d . Aliquots of the n e u t r a l glycolipid fractions prepared from the h u m a n gastric mucosa or cancer tissues were i n c o r p o r a t e d into i n h i b i t o r liposomes for LILI assay. Two p r e p a r a t i o n s (N1 a n d N2) out of 5 n o r m a l gastric m u c o s a showed F o r s s m a n activity (Table II). Fig. 6 shows the i n h i b i t i o n curve as expressed o n the basis of tissue wet weight. N o F o r s s m a n activity was detected in the n e u t r a l glycolipids or ceramide pentasaccharide fractions from cancer tissues.
Quantitative determination of blood group A antigen in tissues by L I L I assay The f u c o s e - c o n t a i n i n g glycolipids in h u m a n gastric mucosa were reported previ-
229
TABLE II F O R S S M A N - A C T I V E G L Y C O L I P I D IN THE N E U T R A L G L Y C O L I P I D F R A C T I O N OF H U M A N G A S T R I C MUCOSA D E T E R M I N E D BY LILI ASSAY F O R F O R S S M A N G L Y C O L I P I D Tissue
Normal Normal Normal Normal Normal
Patient no.
mucosa mucosa mucosa mucosa mucosa
Cancer tissue Cancer tissue Uninvolved tissue Early cancer
Blood group
N1 N2 N5 N6 N7
n.d. a n.d. B AB A
C3 C,J, C4 C10
O O O A
Forssman-active glycolipid ( n g / g wet wt.) b Total neutral glycolipids
Ceramide pentasaccharide
148 15070 -<53 <56 -< 17
130 n.d. < 10 < 10 -< 10
-< 23 -< 52 < 36 -<27
-< 20 < 23 -< 16 <20
a n.d., not determined. b Values determined by LILI assay and expressed on the basis of tissue wet weight.
TABLE III BLOOD G R O U P A ACTIVITY IN T H E C O M P L E X N E U T R A L G L Y C O L I P I D F R A C T I O N S OF T H E G A S T R I C C A N C E R TISSUES D E T E R M I N E D BY LILI ASSAY Patient no.
Tissue
TLC fraction of glycolipid ~
A-active glycolipid (as ng A a / g wet weight) b
Forssman-active glycolipid (as ng F o r s s m a n / g wet weight) b
Gastric cancer C3 (Blood group O)
Cancer
FI F2 F3 F4
280 168 131 -< 10
< 20 -<20 -<20 -<20
Gastric cancer C4 (Blood group O)
Cancer
F5 F6 F7
< 12 -< 12 -< 12
`< 23 -<23 -<23
Uninvolved
F8 F9 F10
-< 8 -<8 <8
< 16 -< 16 -< 16
a The major glycolipids in these fractions were previously reported (H a nori et al., 1981) to be Lea-active pentaglycosylceramide (F1, F5 and FS), Leb-active hexaglycosylceramide (F2 and F9), Lea-active heptaglycosylceramide (F3 and F6), Leb-active octalglycosylceramide (F10), or more complex fucolipids (F4 and F7). b Values determined by LILI assay and expressed on the basis of tissue wet weight.
230 ously to have Le a o r L e b activity (Hattori et al., 1980). The complex neutral glycolipids obtained from gastric cancer tissues were also Lewis blood group-active, and in addition some fractions separated by TLC showed weak blood group A activity detectable by hemagglutination inhibition tests (Hattori et al., 1981). These fractions were examined by LILI assay and results shown in Table III indicate that blood group A activity was detected in the cancer tissue of a patient of blood group O. These A-activities were distinct from structurally related Forssman antigen, and were present in the fractions of glycolipids with 5-8 sugars at the concentration equivalent to 130-280 ng of A a glycolipid/g wet tissue.
Discussion
The interaction of various glycolipid antigens with their respective antibodies has been investigated conveniently by fmmune-lysis of liposomes into which the glycolipid is incorporated. The immune-lysis assay used in the present study (Figs. 1 and 2) requires only 1 pmol of glycolipid antigen for each assay, which is 1000 times more sensitive than the method reported previously (Uemura et al., 1980b). Liposomes sensitive to immune-lysis can be usually made with 0.5 to 0.05 mol% of glycolipid antigen relative to phospholipids (Taketomi and Uemura, 1980), and thus as little as 0.1 to 0.01 pmol of glycolipid is now detectable by this assay. A simple liposome immunoassay (LILI assay) has been developed for quantitative determination of picomolar amounts of individual glycolipids. The major advantage of this technique is its simplicity and convenience so that no special equipments or reagents are required. With the purified standard glycolipids and appropriate specific antibodies, assay systems have been easily set up for various glycolipid antigens. A disadvantage may come from the fact that the immune-lysis of liposomes is a secondary manifestation of antigen-antibody reaction and, therefore, one should be careful not to introduce a false inhibition caused by any anticomplementary effects. In the present experiments, however, the glycolipid preparations obtained from tissues by conventional procedures did not show such adverse effects. Nonspecific inhibition by auxiliary lipids in liposomes (phospholipid and cholesterol) was not found, either, in contrast to the observation by others (Geiger and Smolarsky, 1977). Another problem intrinsic to amphipathic glycolipid antigens is how to make the inhibitor preparation in which the glycolipid antigens are most effectively available to react with antibodies. For this the inhibitor liposomes were prepared by dissolving the glycolipid and auxiliary lipids (sphingomyelin and cholesterol) in hot methanol and by diluting the solution with buffer (Rapport and Graf, 1969). As discussed by Young et al. (1979), this method proved to be simple and satisfactory. Hakomori et al. (1977) reported that Forssman activity could not be detected in the total neutral glycolipid fractions of the gastrointestinal mucosa and tumor tissues but became strong after purification of the ceramide pentasaccharide fraction. This 'masking effect' was not so pronounced in our study and Forssman activity was detectable in the total neutral glycolipid fractions of some gastric mucosa, though the standard
231
curve was affected in the presence of the total neutral glycolipids which caused a slight reduction of inhibitory activity of Forssman glycolipid. Since the observed reduction by 5-8% would result in an underestimate of the amount of Forssman glycolipid, the effect of a large amount of other glycolipids was not completely negligible. Therefore, to determine glycolipid antigens more accurately, it is desirable to purify the glycolipid as far as possible. In spite of the above, the sensitivity and specificity of LILI assay proved to be useful for detection of a small amount of glycolipid antigens in tissues. A similar liposome immunoassay system, using carboxyfluorescein as a marker, has been developed by Naito et al. (1980). Results of the present study indicate that human anti-A serum showed marked cross-reaction in liposome immune-lysis with Forssman glycolipid in contrast to the specificity of rabbit anti-Forssman IgG antibodies. The antibodies specific for N' glycolipid were obtained after absorption of human anti-A serum with Forssman glycolipid-containing liposomes. Using these specific antibodies, Forssman-active glycolipid was detected in 2 out of 5 normal mucosa, and blood group A-active glycolipids were found in the cancer tissue of a patient of blood group O. Forssman glycolipid has been recently detected in certain human tissues such as a metastatic tumor of biliary adenocarcinoma in liver (Kawanami, 1972), gastric and colonic mucosa and tumor tissues (Hakomori et al., 1977), and normal lung and lung carcinoma (Yoda et al., 1980). The present investigation did not permit a conclusion about the distribution of these glycolipids in normal and tumor states because of the limited number of specimens examined. However, the liposome immune-lysis inhibition assay should prove useful for the analysis of individual glycolipid antigens, especially quantitative changes associated with oncogenic transformation and cell differentiation.
Acknowledgement This study was supported in part by a grant from the Ministry of Education, Science and Culture of Japan.
References Alving, C.R., 1977, in: The Antigens, Vol. 4, ed. M. Sela (Academic Press, New York) p. 1. Geiger, B. and M. Smolarsky, 1977, J. Immunol. Methods 17, 7. Haga, M., H. Itagaki, S. Sugawara and T. Okano, 1980, Biochem. Biophys. Res. Commun. 95, 187. Hakomori, S. and K. Watanabe, 1976, in: Glycolipid Methodology, ed. L.A. Witting (American Oil Chemists' Society, Champaign, IL) p. 13. Hakomori, S., S.-M. Wang and W.W. Young, Jr., 1977, Proc. Natl. Acad. Sci. U.S.A. 74, 3023, Hattori, H., K. Uemura and T. Taketomi, 1980, Jpn. J. Exp. Med. 50, 145. Hattori, H., K. Uemura and T. Taketomi, 1981, Biochim. Biophys. Acta 666, 361. Hsia, J.C. and C.T. Tan, 1978, Ann. N.Y. Acad. Sci. 308, 139. Kawanami, J., 1972, J. Biochem. 72, 783. Kinsky, S.C., 1972, Biochim. Biophys. Acta 265, 1. Naito, Y., J. Ueno, T. Yasuda, T. Tsumita and T. Tadakuma, 1980, Proc. Jpn. Soc. Immunol. 10, 681.
232 Rapport, M.M. and L. Graf, 1969, Prog. Allergy 13, 273. Shiba, K., Y. Umezawa, T. Watanabe, S. Ogawa and S. Fujiwara, 1980, Anal. Chem. 52, 1610. Six, H.R., W.W. Young, Jr., K. Uemura and S.C. Kinsky, 1974, Biochemistry 13, 4050. Taketorni, T. and K. Uemura, 1980, Adv. Exp. Med. Biol. 125, 349. Uemura, K., M. Yuzawa and T. Taketomi, 1979, Jpn. J. Exp. Med. 49, 1. Uemura, K., M. Yuzawa-Watanabe, N. Kitazawa and T. Taketomi, 1980a, J. Biochem. 87, 1221. Uemura, K., M. Yuzawa-Watanabe, N. Kitazawa and T. Taketomi, 1980b, J. Biochem. 87, 1641. Wei, R,, C.R. Alving, R.L. Richards and E.S. Copeland, 1975, J. Immunol. Methods 9, 165. Yamakawa, T. and Y. Nagai, 1978, Trends Biochem. Sci. 3, 128. Yoda, Y., T. Ishibashi and A. Makita, 1980, J. Biochem. 88, 1887. Young, W.W., Jr., J.W. Regimbal and S. Hakomori, 1979, J. Immunol. Methods 28, 59.
221
Immunochemical Determination of Forssman and Blood Group A-Active Glycolipids in Human Gastric Mucosa by Inhibition Assay of Liposome Lysis Kei-ichi Uemura ~, Hiroshi Hattori, N o b u k o Kitazawa and Tamotsu Taketomi Department of Biochemistry, Institute of Adaptation Medicine. Shinshu University School of Medicine, Matsurnoto, Nagano 390, Japan
Received 9 November 1981, accepted 22 February 1982)
A simple liposome lmmunoassay, liposome immune-lysis inhibition (LILI) assay, is described for quantitative determination of individual glycolipid antigens. Liposomes containing fluorogenic marker, 4-methylumbelliferyl phosphate, were prepared from sphingomyelin, cholesterol, dicetylphosphate and standard glycolipid. Release of trapped markers from these liposomes by antibody and complement (liposome lysis) was inhibited by preincubating the antibody with test glycolipid incorporated into inhibitor liposomes. Based on the competitive inhibition, it was possible to quantitate each glycolipid antigen in less than picomolar amounts. The sensitivity and specificity of the assay were examined with purified glycolipid standards. LILI assay has been applied for the detection of Forssman glycolipid and blood group A-active glycolipid in human gastric mucosa and cancer tissues.
Key words: liposome lysis - - gl),colipid - - Forssman glycolipid - - blood group A -active glycolipid
Introduction Glycosphingolipids on cell surfaces may have functions in a number of biological recognition phenomena and represent a large variety of antigenic determinants (Yamakawa and Nagai, 1978). Various procedures have so far been developed for the sensitive and quantitative determination of glycolipids. The immunochemical methods are frequently used for glycolipid analyses and recently Young et al. (1979) devised a radioimmunoassay for quantitation of Forssman glycolipid. Such a highly sensitive technique is useful for detection of glycolipid antigens in limited amounts of tissues. Release of trapped markers from liposomes by antibody-complement i Correspondence address until the end of the year: Kei-ichi Uemura, Ph.D., Division of Communicable Diseases, Clinical Research Centre, Watford Road, Harrow, Middlesex, HA1 3U J, U.K. 0022-1759/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
222 (Kinsky, 1972) has been widely used for characterization of antibodies to glycolipids (Alving, 1977). I m m u n o a s s a y s based on the inhibition of liposome lysis have been devised for determination of antigens which compete for antibodies with amphipathic antigens incorporated into liposomes. Several different methods have been used for measurement of released markers, including spin labels and ESR (Wei et al., 1975; Hsia and Tan, 1978), fluorescent markers and fluorometry (Geiger and Smolarsky, 1977; Naito et al., 1980), and the use of special electrodes (Shiba et al., 1980; H a g a et al., 1980). However, application of these assays to determine substances in cells, tissues or biological fluid is still scarce, and a simple and flexible procedure should be developed. In this paper we report a liposome immunoassay for quantitative determination of individual glycolipids based on the competitive inhibition for antibodies in the liposome lysis reaction, which we have described previously as a sensitive fluorometric assay using a fluorogenic marker, 4-methylumbelliferyl phosphate (Six et al., 1974; U e m u r a et al., 1980a). The application of this assay for the detection of F o r s s m a n and blood group A-active glycolipids in h u m a n gastric mucosa and cancer tissues is described.
Materials and Methods Glycolipids *
Glycolipids were isolated from the following sources as previously described (Taketomi and Uemura, 1980): Forssman glycolipid from caprine erythrocytes; globoside from porcine and h u m a n erythrocytes; lactosylceramide, B-active galactosylparagloboside from bovine erythrocytes; galactosylceramide from h u m a n brain; globotriaosylceramide from urine of a F a b r y patient; asialo GM2 from guinea pig erythrocytes. Paragloboside and asialo GMi were prepared by mild acid hydrolysis of sialosylparagloboside and rabbit brain gangliosides, respectively, and were purified by c h r o m a t o g r a p h y on silicic acid. Blood group A-active glycolipid (A a) was isolated and purified from h u m a n erythrocytes. Total lipid extract was subjected to D E A E - S e p h a d e x A-25 chromatography, acetylatiom Florisil column chromatograp h y and deacetylation as described (Hattori et al., 1980). The neutral glycolipid fraction was fractionated on a Silica gel 40 (Merck) column and a crude fraction containing A a glycolipid was subjected to preparative T L C to isolate 'TLC-purified' A ~ glycolipid ( H a k o m o r i and Watanabe, 1976). * Forssman glycolipid, GalNAc(al ~ 3)GalNAc(fll ~ 3)Gal(al ~ 4)Gal(fll ~ 4)Glc(fll ~ l)ceramide; globoside, GalNAc(fll ~ 3)Gal(al ~ 4)Gal(fl 1~ 4)Glc(fl I -~ l)ceramide; globotriaosylceramide, Gal( al 4)Gal(fll~4)Glc(fll~l)ceramide; lactosylceramide, Gal(fll~4)Glc(fll~l)ceramide; galactosylceramide, Gal(fll ~ 1)ceramide; asialo GM2, GalNAc(J~l ~4)Gal(fll ~4)Glc(fll ~ l)ceramide; asialo GMI, Gal(fll~3)GalNAc(fll~4)Gal(fll~4)Glc(fll~l)ceramide; paragloboside, Gal(fll-.4)GlcNAc(fll~ 3)Gal(fll ~ 4)Glc(fll ~ 1)ceramide; sialosylparagloboside, NeuGc(et2 ~ 3)Gal(fll ~ 4)GlcNAc(fll 3)Gal(fll -. 4)Glc(fll ~ 1)ceramide; B-active galactosylparagloboside, Gal(al ~ 3)Gal(fl 1~ 4)GlcNAc(fll 3)Gal(fll ~ 4)Glc(fll ~ l)ceramide; A-active glycolipid (Aa), GalNAcal ~ 3Gal(2 ~- laFuc)fll 4GlcNAcfll ~ 3Galfll ~ 4Glcfll ~ lceramide.
223
A n tisera Antisera to Forssman glycolipid were prepared in rabbits as described earlier (Uemura et al., 1979) by a single injection of the glycolipid with Freund's complete adjuvant. Anti-globoside, anti-asialo G M2, anti-asialo G Mj and anti-paragloboside sera were prepared by injection into rabbits of a mixture of the respective glycolipid and bovine serum albumin in Freund's complete adjuvant (Uemura et al., 1980b). H u m a n anti-A blood group typing serum was obtained from Ortho Diagnostics (Raritan, N J). All the sera were heat-inactivated at 56°C for 30 min. A part of the anti-A serum (1.0 ml) was absorbed with Forssman glycolipid-containing liposomes (1.0/~mol of Forssman glycolipid) by incubating for 1 h at room temperature and then the mixture was centrifuged at 30,000 × g for 20 min (Uemura et al., 1980b). The supernatant was used for assay. Fractions containing IgG antibodies were separated by Sephadex G-200 gel filtration as described previously (Uemura et al., 1980b). Tissues and preparation of glycolipids Normal gastric mucosa and cancer tissues were obtained surgically or at autopsy from 9 individuals in Shinshu University and its associated hospitals (kindly provided by Drs. T. Katsuyama and K. Ono, Department of Pathology, Shinshu University School of Medicine). Total neutral glycolipid and ceramide pentasaccharide fractions were prepared as described previously (Hattori et al., 1980, 1981). Liposome immune-lysis inhibition assay (LILI assay) Bovine brain sphingomyelin, dicetylphosphate and 4-methylumbelliferyl phosphate were purchased from Sigma Chemical Co., St. Louis, MO; cholesterol from E. Merck, Darmstadt; alkaline phosphatase (EC 3.1.3.1) from Boehringer-ManheimYamanouchi, Tokyo. Liposomes containing entrapped fluorogenic marker, 4-methylumbelliferyl phosphate, were prepared essentially as described previously (Uemura et al., 1980b) with some modifications for microassay. Multicompartment liposomes were made from sphingomyelin, cholesterol, dicetylphosphate and glycolipid (100, 75, 10 and 5 nmol, respectively) by dispersing dried lipids in 100 /~1 of 100 mM 4-methylumbelliferyl phosphate solution adjusted to pH 7 with NaOH. The untrapped marker was removed by dialysis for 3 h against 4 changes of 200 ml of 75 mM NaC1/75 mM KCI and the final volume of the liposome preparation was adjusted to 1.25 ml with Tris-buffered saline (20 mM Tris-HC1, pH 8.0/150 mM NaC1/0.15 mM CaC12/0.5 m M MgCI2). The amount of 4-methylumbelliferyl phosphate trapped inside the liposomes was 0.150 -+ 0,032 (S.D.)/~mol and remained almost unchanged during the storage at 4°C for at least 2 days. The above preparation (marker liposome) was diluted further to 1 : 100 with Tris-buffered saline before use. Antibody titration was performed by incubating Tris-buffered saline (25 t,1), various amounts of antibody preparation (25 #1), liposomes (25/~1) and guinea pig serum diluted to 1:25 (25 #1) for 30 rain at room temperature. After incubation 0.5 ml of Tris-buffered saline containing 0.2 U of alkaline phosphatase was added to each assay tube and the extent of marker released from the liposomes was de-
224 termined fluorometrically as described previously (excitation wavelength, 365 nm: emission wavelength 448 nm) (Uemura et al., 1980a). The amount of antibody which caused 90% of the maximum marker release was determined for the use in the following inhibition assays, in that the antibody was limiting and the complement was in excess. A constant amount (75 nl) of rabbit anti-Forssman IgG, thus determined, gave 66.4 -+ 5.9 (S.D.) % lysis of Forssman liposome preparations made over a period of several months. The amount of guinea pig serum (240 260 C H s 0 / m l ) required for 50% marker release was 25 ~tl of 1:200 dilution. To prepare the glycolipid antigen for inhibition assay, the procedure of Young et al. (1979) was essentially followed. Each glycolipid as the standard or control (5 nmol), or each aliquot of glycolipid fractions prepared from the tissues (corresponding to 50-400 mg wet weight) was mixed with 100 nmol of sphingomyelin and 75 nmol of cholesterol in organic solvent. The lipids were dried under a nitrogen stream and then dissolved in 20/xl of methanol by warming, Liposomes were formed by the addition of 480 /~1 of hot Tris-buffered saline with vigorous mixing on a vortex mixer. Liposomes prepared in this manner have smaller sizes (closer to the unilamellar state) and can expose more antigens on their surface than the multilamellar liposomes made by dispersing the dried lipids directly with buffer (see below, Fig. 5). This preparation (inhibitor liposome) was further diluted with Trisbuffered saline to give appropriate glycolipid concentrations. L I L I assay was performed at room temperature in 10 m m × 75 m m glass tubes. The inhibitor liposomes of various dilutions (25 ~tl) and the antibody preparation (25 /zl) were incubated for 60 rain. The marker liposomes (25 /~1) and guinea pig serum diluted to 1:25 (25 /~1) were then added to each tube and incubation was continued for 30 min. The extent of marker released from the marker iiposomes was determined as described above. Results are given as the percentage inhibition of marker release and were calculated from duplicates according to the formula: % inhibition = 100 X (C - T ) C where C is the amount of marker released without inhibitor liposomes and T is the amount of marker released with inhibitor liposomes. The variation among duplicate or triplicate samples in an assay was not greater than 10%, usually being within 5%.
Results
Specificity of anti-Forssman and anti-A In complement-dependent liposome lysis, rabbit IgG antibodies against Forssman glycolipid produced specific marker release from liposomes containing Forssman glycolipid (Fig. 1), but did not show any cross-reactivity with liposomes containing blood group Aa glycolipid (Fig. 2). The IgM fraction of rabbit anti-Forssman serum reacted to some extent with globoside and asialo GM2 (Uemura et al., 1980b). Accordingly, the I g G fraction was used as a specific anti-Forssman reagent for LILI assay.
225 80 r
80' ///
_~4o: ~
lr
_
' ........
/
40?
.,
;
/
z .....
,
/ ./
i ',,r 0 ~ ......... 0
I00 200 Antiserum ( n I )
soo '?o5
0
I00
500 Antiserum
560 ( nl )
Fig. 1. lmmune-lysis of Forssman-liposomes with rabbit anti-Forssman lgG (O), human anti-A serum (unabsorbed) (Q), or human anti-A serum absorbed with Forssman-liposomes (x). Liposomes were prepared from sphingomyelin, cholesterol and dicetylphosphate (1:0.75:0.1, by tool) with Forssman glycolipid (0,05 mol/mol sphingomyelin). Marker release (% of the trapped marker) was determined after incubation of liposomes containing 1 pmol of the glycolipid with various amounts of antibodies in the presence of guinea pig complement. With heat-inactivated guinea pig serum the marker release did not exceed 2%. Fig. 2. Immune-lysis of Aa-liposomes with rabbit anti-Forssman IgG (O), human anti-A serum (unabsorbed) (O), or human anti-A serum absorbed with Forssman-liposomes (x). Liposomes were prepared from sphingomyelin, cholesterol and dicetylphosphate (1:0.75:0.1, by mol) with Aa glycolipid (0.03 mol/mol sphingomyelin). With heat-inactivated guinea pig serum the marker release did not exceed 1%.
O n the other hand, h u m a n a n t i - b l o o d g r o u p A serum reacted with l i p o s o m e s c o n t a i n i n g F o r s s m a n glycolipid (Fig. 1) as well as with l i p o s o m e s c o n t a i n i n g b l o o d g r o u p A a g l y c o l i p i d (Fig. 2). This a n t i - F o r s s m a n activity was c o m p l e t e l y r e m o v e d by a b s o r p t i o n of the a n t i - A serum with l i p o s o m e s c o n t a i n i n g F o r s s m a n glycolipid (Fig. 1). T h e a b s o r b e d serum c a u s e d specific m a r k e r release from l i p o s o m e s c o n t a i n ing A a glycolipid, a l t h o u g h the a n t i - A a glycolipid activity was r e d u c e d by this t r e a t m e n t (Fig. 2), i n d i c a t i n g that the h u m a n a n t i - A serum c o n t a i n e d some antib o d i e s reactive with b o t h F o r s s m a n a n d A ~ d e t e r m i n a n t s . T h e a b s o r b e d a n t i - A s e r u m was used as a specific a n t i - A r e a g e n t for L I L I assay.
Inhibition by purified glycolipids of liposome irnmune-lysis T h e i m m u n e - l y s i s of m a r k e r l i p o s o m e s c o n t a i n i n g F o r s s m a n glycolipid was i n h i b i t e d b y p r e i n c u b a t i n g a n t i - F o r s s m a n a n t i b o d y with various a m o u n t s of Forssm a n glycolipid in i n h i b i t o r l i p o s o m e s (Fig. 3). C o m p l e t e i n h i b i t i o n was o b t a i n e d with 30 p m o l of F o r s s m a n glycolipid, while 0.3 p m o l ( a b o u t 0.5 ng) was the lower limit of detection. I n h i b i t i o n b y A a g l y c o l i p i d was negligible; c r o s s - r e a c t i o n with a n t i - F o r s s m a n , if any, was less than 0.25%. N o i n h i b i t i o n was o b s e r v e d with o t h e r glycolipids tested. Fig. 4 i n d i c a t e s that the i m m u n e - l y s i s of m a r k e r l i p o s o m e s c o n t a i n i n g A a glycolipid was i n h i b i t e d d e p e n d i n g on the a m o u n t s of A a glycolipid a d d e d as i n h i b i t o r liposomes. A b o u t 0.6 ng of A a glycolipid gave 50% inhibition, whereas 100 ng of F o r s s m a n glycolipid, as well as o t h e r glycolipids, showed no i n h i b i t i o n at all. Similar assay systems were e x a m i n e d using r a b b i t I g G a n t i b o d i e s to globoside,
226
,oo[
100
y/
g
/
g *7-
.f
~_ 50
/
Z c
c
oh
-- ;
~o--
Glycolipid
( pmoi
,6~ )
5O
o:j
'l Glycolipid
J
-,o
T -1 i___
~oo
( ng )
Fig. 3. Inhibition of liposome immune-lysis by purified glycolipids (Forssman assay system). Rabbit anti-Forssman IgG (75 nl in 25 ~1 of Tris-buffered saline) was incubated at room temperature for 60 min with various amounts of inhibitor liposomes (25 #1) containing Forssman glycolipid (O), Aa glycolipid (0), or other glycolipids. The amount of each glycolipid added per assay is indicated on the abscissa. Incubation was continued for 30 min after the addition of the marker liposomes (25 ill, 1 pmol of Forssman glycolipid) and guinea pig serum (1:25 dilution, 25 #1). The bar indicates the range of % inhibition caused by inhibitor liposomes which contained the following glycolipids: galactosylceramide, lactosylceramide, globotriaosylceramide, asialo GM2, asialo GM~, globoside, paragloboside and B-active galactosylparagloboside. Fig. 4. Inhibition of liposome immune-lysis by purified glycolipids (Aa assay system). Human anti-A serum absorbed with Forssman glycolipid (500 nl in 25 /~1 of Tris-buffered saline) was incubated at room temperature for 60 min with various amounts of inhibitor liposomes (25/~1) containing Aa glycolipid (O), Forssman glycolipid (0), or other glycolipid. The amount of each glycolipid added per assay is indicated on the abscissa. Incubation was continued for 30 min after the addition of the marker liposomes (25 ~1, 0.6 pmol of Aa glycolipid) and guinea pig serum (1:25 dilution, 25 /~1). The bar indicates the range of % inhibition caused by other glycolipids (see the legend to Fig. 3).
asialo G M2, asialo G M1 or paragloboside a n d the respective marker liposomes. Each glycolipid a n t i g e n was d e t e r m i n e d with high sensitivity a n d specificity (Table I). In control experiments (Fig. 5), the highest sensitivity was achieved when inhibitor liposomes c o n t a i n i n g F o r s s m a n glycolipid were prepared with s p h i n g o m y e l i n a n d cholesterol by d i l u t i n g with buffer the lipids dissolved in hot m e t h a n o l ; less effective was the liposomes prepared by dispersing the dried lipids directly with buffer which were of m u l t i l a m e l l a r structures in heterogeneous sizes (Six et al., 1974); F o r s s m a n glycolipid alone in micellar form was f o u n d to be least effective as inhibitor. T h e i n h i b i t i o n curve was not affected by varying the c o n t e n t of F o r s s m a n glycolipid in i n h i b i t o r liposomes from 1 to 15 mol% of sphingomyelin. These observations were consistent with those reported previously ( Y o u n g et al., 1979; N a i t o et al., 1980). T o examine whether a large a m o u n t of glycolipids might disturb the assay by their a n t i c o m p l e m e n t a r y effect, liposomes were prepared from s p h i n g o m y e l i n (100 nmol), cholesterol (75 n m o l ) a n d total n e u t r a l glycolipids (52 fig) o b t a i n e d from n o r m a l h u m a n gastric mucosa. W h e n guinea pig serum was i n c u b a t e d with these liposomes u n d e r the i m m u n e - l y s i s assay condition, 1 n m o l of glycolipids (10 times as m u c h as in the s t a n d a r d assay) did not reduce the c o m p l e m e n t activity c o m p a r e d with the buffer control (175 C H s 0 / m l a n d 185 C H s 0 / m l , respectively).
TABLE I SENSITIVITY AND SPECIFICITY OF LILI ASSAY FOR INDIVIDUAL GLYCOLIPIDS T e s t gtycolipid in inhibitor l i p o s o m e
L I L I assay for Globoside
Galactosylceramide Lactosylceramide Globotriaosylceramide Globoside F o r s s m a n glycolipid
. .
Forssman .
-
Paragloboside
. . n.d.
Asialo G Mz
.
.
.
* 15.0 -
Asialo G uf2 Asialo GMj G a l ( a l ~ 3)paragloboside A a glycolipid
a
Asialo G Mi
. .
.
.
. -
.
.
.
.
-
-
5.35
-
.
-
0.56
. *
. -
.
0.30 .
.
A a
.
2.12
.
Paragloboside
. n.d.
. n.d.
-
0.37
N u m b e r s are the a m o u n t of glycolipid ( p m o l ) which g a v e 50% inhibition of l i p o s o m e lysis. --, less t h a n 10% inhibition at 100 p m o l ; *, less t h a n 15% inhibition at 100 p m o l ; n,d., not d e t e r m i n e d . A n t i b o d i e s used were r a b b i t l g G p r e p a r e d by i m m u n i z a t i o n with purified glycolipid except anti-A, w h i c h was h u m a n blood g r o u p t y p i n g s e r u m a b s o r b e d with F o r s s m a n - l i p o s o m e s .
228 IOC
I00
5O
5°t
/
! J
0.1
Forssman glycolipid
tO0
I
( pmol )
o ~,
o:, Tissue
_ ~
, wet weight
]
,o (rag)
Fig. 5. Effect of various types of inhibitor liposomes on the inhibition of liposome immune-lysis. Inhibition assay was performed as described in Fig. 3 with the following inhibitors: a, Forssman glycolipid alone in micellar form: b, liposome prepared by dispersing the dried lipids directly with buffer (sphingomyelin, cholesterol and Forssman glycolipid, 1 : 0.75 : 0.05, by mol); c, d, e, liposomes prepared by diluting with buffer the lipids dissolved in hot methanol (the standard method), the amount of Forssman glycolipid being varied, 0.15, 0.05 and 0.01 mol/mol sphingomyelimrespectively. Fig. 6. Determination of Forssman antigen in human gastric mucosa by inhibition of liposome immunelysis. Forssman assay system was used as described in the legend to Fig. 3. An aliquot of the neutral glycolipid fraction from patient no. N2 (corresponding to 65 mg wet tissue) was mixed with 50 nmol of sphingomyelin and 37.5 nmol of cholesterol to prepare inhibitor liposomes. The % inhibition of liposome immune-lysis was plotted against the tissue wet weight corresponding to the amount of glycolipidsin each assay tube. I n a n o t h e r control experiment, the presence of other glycolipids did not interfere very m u c h the antigenic activity of F o r s s m a n glycolipid. The i n h i b i t i o n by F o r s s m a n glycolipid of i m m u n e - l y s i s was, however, reduced by 5 - 8 % when the total neutral glycolipids of h u m a n gastric mucosa were i n c o r p o r a t e d into the same i n h i b i t o r liposomes (in a ratio of the n e u t r a l glycolipids to F o r s s m a n , 25:1 by mol). The n e u t r a l glycolipid fraction consisted m a i n l y of galabiosylceramide (25%), globoside (28%) a n d fucolipids c o n t a i n i n g more than 5 sugar residues (12%) (Hattori et al., 1980). The above result indicated that F o r s s m a n glycolipid could be d e t e r m i n e d in crude glycolipid fractions, though the c o n t e n t s measured were slightly underestimated.
Quantitative determination of Forssman antigen in tissues by L I L I assay By c o m p a r i s o n with the i n h i b i t i o n curve for purified F o r s s m a n glycolipid (Fig. 3), the a m o u n t of F o r s s m a n antigen in tissues could be q u a n t i t a t e d . Aliquots of the n e u t r a l glycolipid fractions prepared from the h u m a n gastric mucosa or cancer tissues were i n c o r p o r a t e d into i n h i b i t o r liposomes for LILI assay. Two p r e p a r a t i o n s (N1 a n d N2) out of 5 n o r m a l gastric m u c o s a showed F o r s s m a n activity (Table II). Fig. 6 shows the i n h i b i t i o n curve as expressed o n the basis of tissue wet weight. N o F o r s s m a n activity was detected in the n e u t r a l glycolipids or ceramide pentasaccharide fractions from cancer tissues.
Quantitative determination of blood group A antigen in tissues by L I L I assay The f u c o s e - c o n t a i n i n g glycolipids in h u m a n gastric mucosa were reported previ-
229
TABLE II F O R S S M A N - A C T I V E G L Y C O L I P I D IN THE N E U T R A L G L Y C O L I P I D F R A C T I O N OF H U M A N G A S T R I C MUCOSA D E T E R M I N E D BY LILI ASSAY F O R F O R S S M A N G L Y C O L I P I D Tissue
Normal Normal Normal Normal Normal
Patient no.
mucosa mucosa mucosa mucosa mucosa
Cancer tissue Cancer tissue Uninvolved tissue Early cancer
Blood group
N1 N2 N5 N6 N7
n.d. a n.d. B AB A
C3 C,J, C4 C10
O O O A
Forssman-active glycolipid ( n g / g wet wt.) b Total neutral glycolipids
Ceramide pentasaccharide
148 15070 -<53 <56 -< 17
130 n.d. < 10 < 10 -< 10
-< 23 -< 52 < 36 -<27
-< 20 < 23 -< 16 <20
a n.d., not determined. b Values determined by LILI assay and expressed on the basis of tissue wet weight.
TABLE III BLOOD G R O U P A ACTIVITY IN T H E C O M P L E X N E U T R A L G L Y C O L I P I D F R A C T I O N S OF T H E G A S T R I C C A N C E R TISSUES D E T E R M I N E D BY LILI ASSAY Patient no.
Tissue
TLC fraction of glycolipid ~
A-active glycolipid (as ng A a / g wet weight) b
Forssman-active glycolipid (as ng F o r s s m a n / g wet weight) b
Gastric cancer C3 (Blood group O)
Cancer
FI F2 F3 F4
280 168 131 -< 10
< 20 -<20 -<20 -<20
Gastric cancer C4 (Blood group O)
Cancer
F5 F6 F7
< 12 -< 12 -< 12
`< 23 -<23 -<23
Uninvolved
F8 F9 F10
-< 8 -<8 <8
< 16 -< 16 -< 16
a The major glycolipids in these fractions were previously reported (H a nori et al., 1981) to be Lea-active pentaglycosylceramide (F1, F5 and FS), Leb-active hexaglycosylceramide (F2 and F9), Lea-active heptaglycosylceramide (F3 and F6), Leb-active octalglycosylceramide (F10), or more complex fucolipids (F4 and F7). b Values determined by LILI assay and expressed on the basis of tissue wet weight.
230 ously to have Le a o r L e b activity (Hattori et al., 1980). The complex neutral glycolipids obtained from gastric cancer tissues were also Lewis blood group-active, and in addition some fractions separated by TLC showed weak blood group A activity detectable by hemagglutination inhibition tests (Hattori et al., 1981). These fractions were examined by LILI assay and results shown in Table III indicate that blood group A activity was detected in the cancer tissue of a patient of blood group O. These A-activities were distinct from structurally related Forssman antigen, and were present in the fractions of glycolipids with 5-8 sugars at the concentration equivalent to 130-280 ng of A a glycolipid/g wet tissue.
Discussion
The interaction of various glycolipid antigens with their respective antibodies has been investigated conveniently by fmmune-lysis of liposomes into which the glycolipid is incorporated. The immune-lysis assay used in the present study (Figs. 1 and 2) requires only 1 pmol of glycolipid antigen for each assay, which is 1000 times more sensitive than the method reported previously (Uemura et al., 1980b). Liposomes sensitive to immune-lysis can be usually made with 0.5 to 0.05 mol% of glycolipid antigen relative to phospholipids (Taketomi and Uemura, 1980), and thus as little as 0.1 to 0.01 pmol of glycolipid is now detectable by this assay. A simple liposome immunoassay (LILI assay) has been developed for quantitative determination of picomolar amounts of individual glycolipids. The major advantage of this technique is its simplicity and convenience so that no special equipments or reagents are required. With the purified standard glycolipids and appropriate specific antibodies, assay systems have been easily set up for various glycolipid antigens. A disadvantage may come from the fact that the immune-lysis of liposomes is a secondary manifestation of antigen-antibody reaction and, therefore, one should be careful not to introduce a false inhibition caused by any anticomplementary effects. In the present experiments, however, the glycolipid preparations obtained from tissues by conventional procedures did not show such adverse effects. Nonspecific inhibition by auxiliary lipids in liposomes (phospholipid and cholesterol) was not found, either, in contrast to the observation by others (Geiger and Smolarsky, 1977). Another problem intrinsic to amphipathic glycolipid antigens is how to make the inhibitor preparation in which the glycolipid antigens are most effectively available to react with antibodies. For this the inhibitor liposomes were prepared by dissolving the glycolipid and auxiliary lipids (sphingomyelin and cholesterol) in hot methanol and by diluting the solution with buffer (Rapport and Graf, 1969). As discussed by Young et al. (1979), this method proved to be simple and satisfactory. Hakomori et al. (1977) reported that Forssman activity could not be detected in the total neutral glycolipid fractions of the gastrointestinal mucosa and tumor tissues but became strong after purification of the ceramide pentasaccharide fraction. This 'masking effect' was not so pronounced in our study and Forssman activity was detectable in the total neutral glycolipid fractions of some gastric mucosa, though the standard
231
curve was affected in the presence of the total neutral glycolipids which caused a slight reduction of inhibitory activity of Forssman glycolipid. Since the observed reduction by 5-8% would result in an underestimate of the amount of Forssman glycolipid, the effect of a large amount of other glycolipids was not completely negligible. Therefore, to determine glycolipid antigens more accurately, it is desirable to purify the glycolipid as far as possible. In spite of the above, the sensitivity and specificity of LILI assay proved to be useful for detection of a small amount of glycolipid antigens in tissues. A similar liposome immunoassay system, using carboxyfluorescein as a marker, has been developed by Naito et al. (1980). Results of the present study indicate that human anti-A serum showed marked cross-reaction in liposome immune-lysis with Forssman glycolipid in contrast to the specificity of rabbit anti-Forssman IgG antibodies. The antibodies specific for N' glycolipid were obtained after absorption of human anti-A serum with Forssman glycolipid-containing liposomes. Using these specific antibodies, Forssman-active glycolipid was detected in 2 out of 5 normal mucosa, and blood group A-active glycolipids were found in the cancer tissue of a patient of blood group O. Forssman glycolipid has been recently detected in certain human tissues such as a metastatic tumor of biliary adenocarcinoma in liver (Kawanami, 1972), gastric and colonic mucosa and tumor tissues (Hakomori et al., 1977), and normal lung and lung carcinoma (Yoda et al., 1980). The present investigation did not permit a conclusion about the distribution of these glycolipids in normal and tumor states because of the limited number of specimens examined. However, the liposome immune-lysis inhibition assay should prove useful for the analysis of individual glycolipid antigens, especially quantitative changes associated with oncogenic transformation and cell differentiation.
Acknowledgement This study was supported in part by a grant from the Ministry of Education, Science and Culture of Japan.
References Alving, C.R., 1977, in: The Antigens, Vol. 4, ed. M. Sela (Academic Press, New York) p. 1. Geiger, B. and M. Smolarsky, 1977, J. Immunol. Methods 17, 7. Haga, M., H. Itagaki, S. Sugawara and T. Okano, 1980, Biochem. Biophys. Res. Commun. 95, 187. Hakomori, S. and K. Watanabe, 1976, in: Glycolipid Methodology, ed. L.A. Witting (American Oil Chemists' Society, Champaign, IL) p. 13. Hakomori, S., S.-M. Wang and W.W. Young, Jr., 1977, Proc. Natl. Acad. Sci. U.S.A. 74, 3023, Hattori, H., K. Uemura and T. Taketomi, 1980, Jpn. J. Exp. Med. 50, 145. Hattori, H., K. Uemura and T. Taketomi, 1981, Biochim. Biophys. Acta 666, 361. Hsia, J.C. and C.T. Tan, 1978, Ann. N.Y. Acad. Sci. 308, 139. Kawanami, J., 1972, J. Biochem. 72, 783. Kinsky, S.C., 1972, Biochim. Biophys. Acta 265, 1. Naito, Y., J. Ueno, T. Yasuda, T. Tsumita and T. Tadakuma, 1980, Proc. Jpn. Soc. Immunol. 10, 681.
232 Rapport, M.M. and L. Graf, 1969, Prog. Allergy 13, 273. Shiba, K., Y. Umezawa, T. Watanabe, S. Ogawa and S. Fujiwara, 1980, Anal. Chem. 52, 1610. Six, H.R., W.W. Young, Jr., K. Uemura and S.C. Kinsky, 1974, Biochemistry 13, 4050. Taketorni, T. and K. Uemura, 1980, Adv. Exp. Med. Biol. 125, 349. Uemura, K., M. Yuzawa and T. Taketomi, 1979, Jpn. J. Exp. Med. 49, 1. Uemura, K., M. Yuzawa-Watanabe, N. Kitazawa and T. Taketomi, 1980a, J. Biochem. 87, 1221. Uemura, K., M. Yuzawa-Watanabe, N. Kitazawa and T. Taketomi, 1980b, J. Biochem. 87, 1641. Wei, R,, C.R. Alving, R.L. Richards and E.S. Copeland, 1975, J. Immunol. Methods 9, 165. Yamakawa, T. and Y. Nagai, 1978, Trends Biochem. Sci. 3, 128. Yoda, Y., T. Ishibashi and A. Makita, 1980, J. Biochem. 88, 1887. Young, W.W., Jr., J.W. Regimbal and S. Hakomori, 1979, J. Immunol. Methods 28, 59.