A radioimmunoassay for the measurement of blood group Ii activities: Its application to glycoconjugates, oligosaccharides and intact cells

Ma/ecu/or Immunology. Vol 16. PP. 813-819. Pergamon Press Ltd 1979. Prmted tn Great Bnlam.

A RADIOIMMUNOASSAY FOR THE MEASUREMENT OF BLOOD GROUP Ii ACTIVITIES: ITS APPLICATION TO GLYCOCONJUGATES, OLIGOSACCHARIDES -AND INTACT CELLS* EDWIN

WOOD,

Division

JACQUELINE

of Communicable (First

received

LECOMTE,t

Diseases,

Clinical

12 Ocroher

ROBERT

Research

A. CHILDS

Centre,

197X; in revrsed./ivm

Harrow,

12 February

and

Middlesex,

TEN

FEIZIf

England

1979)

Abstract--A double antibody radioimmunoassay has been developed for the blood group Ii antigens usmg radioiodinated l-active sheep stomach glycoprotcins, an Ii-active extract from human meconium, and sera from patients with cold agglutinin disease. The radioimmunoassay has been used to study the expression of Ii antigens in human ovarian cyst glycoproteins with and without blood group A, B, H and Lewis activities, and it has revealed the presence of I and i activities in several glycoproteins not previously detected by quantitative precipitation assays. Precursor-like glycoproteins from individuals who were non-secretors of ABH antigens showed the highest I and i activities. Certain of the ABH active glycoproteins from secretors showed no I or i activities, while others showed weak or moderate 1and i activities. suggestmg that they contained unsubstituted precursorlike oligosaccharide chains. Hapten inhibition studies with anti-l Ma have shown that the radiolmmunoassay requires approximately 35 times less oligosaccharide inhibitors than inhibition of precipitation assay. The radioimmunoassay has been applied to the investigation of the I and i activities of very small quantities of glycosphingolipids. The assay has also been applied to the measurement of the Ii antigeris on intact human erythrocytes and on erythrocytes after treatment with pepsin or neuraminidase.

INTRODUCTION

blood group Ii antigens are defined by certain human monoclonal autoantibodies, which are cold agglutinins reacting preferentially with adult (anti-I) or cord (anti-i) erythrocytes (reviewed by Race & Sanger, 1975: Feizi, 1978). These antigens are also expressed on leucocytes (Lalezari & Murphy, 1967; Shumak et cd., 1971; Pruzanski et al., 1975) and macrophages (Pruzanski et al., 1977), and on watersoluble substances in secretions (Dzierzkowa-Borodej et al., 1970; Marsh et al., 1970; Cooper, 1970; Feizi et al., 197la, b; Feizi, 1978). The I antigen has been shown to 3, the major intrinsic be present on Band transmembrane protein of erythrocytes (Childs et al., 1978). The findings of Ii activities on ovarian cyst glycoproteins deficient in blood group A, B, H and The

* A preliminary report of this work has been presented at the 4th International Symposium on Glycoconjugates. Woods Hole, U.S.A., September 1977 (Wood et al., 1978). t Present address: Institut Armand Frappier, Boulevard des Prairies, Lava]-les-Rapides, Quebec, Canada. fAddress for correspondence: Dr. T. Feizi, Division of Communicable Diseases, Clinical Research Centre, Watford Road, Harrow, Middlesex HAI 3UJ, England. 9:Abbreviations used: PBS, 50mM sodium phosphate buffered isotonic saline. pH 7.4; PB, 50 mM phosphate buffer, pH 7.4: PB/BSA. PB containing 0.02”, (w/v) NaN, and I’?, (w/v) bovine serum albumin (BSA); D-Galp, D-galactopyranose; D-GpNAc; 2acetamido-2-deoxy-D-glucopyranose; L-Fucp, 6-deoxy-Lgalactopyranose; D-Gp, D-glucopyranose; Cer. ceramide. 813

Lewis activities suggested that the Ii antigens might represent precursors of the major blood group antigens (Feizi et al., 19710) and this has now been confirmed (Feizi e/al., 1971h, 1978a, 1979; Niemannet al., 1978; Watanabe et al., 1979): the majority of anti-i antibodies (5 out of 6 tested) are specifically inhibited ‘by the straight ‘chain glycosphingolipid Iacto-n-norhexaosyl ceramide BDGalpI +4~~GpNAc1-+3~oGalpl+4~~GpNAcl+ 3pDGalpI +4fi~GpI + ICer and the majority of anti-1 antibodies (10 out of 11 tested) are inhibited by the branched glycosphingolipid lacto-N-iso-octaosyl ceramide @&alp 1+4/j’~GpNAc 1 1 $DGaIp l--t ~DGalpl+4~~GpNAcl 4fl~GpNAc I+ 3bDGalp 1+4b~Gp I + I Cer Both the straight chain and branched structures occur as the precursors of the blood group ABH-active glycosphingolipids of erythrocyte membranes (Hakomori et al., 1972; Watanabe et al., 1975). The monoclonal anti-1 and anti-i autoantibodies of different individuals differ in their fine specificities as shown by quantitative precipitin and haemagglutination assays (Feizi & Kabat, 1972; DzierzkowaBorodej et al., 1975). The differences among anti-1 antibodies have been shown to be due to the recognition of different domains on the precursor structures (Feizi & Kabat, 1974; Watanabe et al., 1979; Feizi et al., 1978a, 1979). This report describes a radioimmunoassay which was primarily designed to reduce the amount of hapten required (relative to inhibition of precipitation

814

EDWIN

assays) in studies of the I and i activities ot oligosaccharides. It has already proved invaluable in the measurement of I and i activities of erythrocyte glycosphingolipids which are available in very limited quantities (Feizi et al., 1978b, 1979; Niemann pt d., 1978; Watanabe et al., 1979. Furthermore, it has enabled a re-evaluation to be made of I and i activities of blood group A,B,H and Lewis active and inactive glycoproteins. An additional value of this assay has been its application to the study of I and i antigens on the surface of intact cells. MATERIALS

AND

METHODS

Antiseru The anti-l and anti-i sera were a11 obtained from patients with chronic cold agglutinin disease. and the majorit) have been described previously (Feizi ct N/.. 1971~. h; Feizi & Kabat. 1972: Lecomte & F&i, 1975). Serum Ver was a gift of Dr. John Clancy and has been reported IO have anti-1 specificity when tested at room temp (Pruranski catcl/., 1975). However, we have observed that at 4 C this antiserum haa unusually high titres with cord erythrocytes (titres with adult 01 cells, 640,000; with cord Oi cells X0.000-640.000). The anti-1 sera selected Include sevel-al types of I specificities (Feizi & Kabat. 1972).

Human ovarian cyst glycoproteins with and wlthout A, B. H and Lewis blood group activities were studied. The following were gifts of Dr. E. A. Kabat: blood group A (MSS). B (Beach), HLeb (JS). Le’ (two fractions: N-l phenol Insoluble and N-l lo’:,,). precursor substance OG (?o”,, 2X fraction) and the tirst stage Smith degradation ( 1stIO,) of MSS. Beach and JS. Detalled references to these antigens and an I-active substance from human milk (Fraction C) have been gt\en previously (Feizi cl crl., 1971~. h). Dr. W. Watkins kindly provided precursor substance FI (Morgan. 1960). blood group A (438/P2) and B (413iPl). and two ovarian cyst glycoproteins (484 and 502) lacking A, B, H and Lewla activities. With the exception of the latter four. the I and i activities of the above glycoproteins have been studied previously by quantitative precipitation with most of the sera used in this study (Feiri & Knbat, 1972). The glycoproteins OG. Fl. 484 and 502 were from patients who were nonsecretors of the blood group ABH antigens. and the remaining ovarian cyst glycoproteins from secretors. O/;~~o.scrc~c/lur;~~,.\ buGalp -r4~GpNAc +4~oGpNAcl+61,2,4,5,6 flDGalpl+~PDG~NAcI-6

BDGalpl 1962), (Kabat, pentol(s) (N-l R, 0.71b) and 1,2,4,5,6 pentol(s) (N-l R, 0.7la)

I I r~Fucp (Lloyd et ul., 1968) were gifts of Dr. E. A. Kabat. /~.Galpl~(ioGpNAc was a gift of Dr. Adeline Gauhi. Erythrocytes Fresh adult and cord erythrocytes of blood group 0 were tested before and after treatment with bovine pancreatic trypsin (Difco) Vihrio and cholercre neuraminidase (Behringwerke). To 0.2 ml of washed, packed cells were added 0.6 ml of PBS containing 0.2”,, trypsin, or 0.2 ml PBS containing 50 I.U. neuraminidase; after Incubation with frequent shaking at 37C for 30 min the reaction tubes were cooled on ice. The cells were washed 5 times with 4 ml of icecold PBS. For inhibitlon of binding assays. serial IO-fold dilutions from packed cells were made in PBS. Pwparafion

qf labelled

mcrtcvirrls,forrcr~lioimmunou,s.sc~~~

An I-active glycoprotein from sheep gastric (abomasum) mucosal scrapings and an Ii-active substance from human

WOOD

(‘I oi.

meconlum be!-e cxtl-,rctcd by pep\ln digestion (Bendlch (,( U/ l+l6) followed h> c~hanol preclplratlon ‘,\ ouillncd by I‘u~I <‘r al. (1975). Several preparation\ of c;tch material were tested for I and I actl\ltles by qu;tntitatl\e prcclpltation assays (Fan cr t/i.. 197lh) ‘ind the most act~\e sheep glycoprotein (S2) and meconlum extract (Mcc) wcrc selected for I-adlo-lodinatioII. The I and I acti\ltlc\ of sheep stomach glycoprorelns and human meconium wltl bc described elae\+hcrc The mccomum c\tr;ict\ \\erc ;I pll’t 01‘ Mrs. Jean Pic;lrd. Divlalon of Surglcat Science\. .I, thl\ ln\tltute. The lwo antlgem S7 and Met 4crc I’llrther purllied on ;I Sephadex G200 column equltibrated in l ‘Idsor-ptlon (at 4 c‘) ;~nd elution (at 37 Cl ulth ‘in immuno~~dsorbent column made ulth the ;lntl-I cold :Igglutinin Step (Feizl & K,lbat. 1974). ‘The 37 C eluato were diluted 111PB-BSA to give appl-o4lm,ltely 10.000 counts, min per IO /It as counted on ;I Nuclear Enterprises 1600 gamma counter with ‘~1 efficlenck for “sI 01’ ;ipprox 45”,,. The Iabellcd cluatcs wcrc stored .I, -20 C’ until ubc. Their initl;ll content of free lodIne \%;!‘I5” (, ;IS dctermmed b) Seph;idc\ G50 chromatography. On stor~lge this Increased to IS,, o\elII pcrlod of IO ueehh. which w;~s the uacful norhlng Ilfc ofthc Iabetted mrltcri;lls.

A double antlbodq method w:lb used lor radlolnrnuno:ir\a)\ The an11-I and anll-I antlhodle\ which are of IgM clash (Fudenbcrg & Kunkel, 1957) were prccipltated in the presence ofa carrier, human IgM, by ;I rabbit anti-human IgM serum or its IgG fraction. The c;trrier IgM was laolated by Pevlkon block electrophoresis (Miiller-Eberhard & Kunkel, 1956) from a Waldenstriim macroglobulinaemia serum dc\oid of anti-l or antI- activity. The IgG-rich fraction of the rabbit antiseium was prepared by chromatography on a column of Whatman DE-52 cellulose. For bIndIng as~ys. 30 IIt of PB-BSA, 10 111 of cold agglutlnin serum diluted in PB-BSA and IO /II of labelled antigen were mixed in rimtcsb soda glass tubes, SO x 9 mm (GWS. England). After incubation for I hr (anti-l sera Ma. Step and Ver). 2 hr (anti-i sera) or 24 hr (anti-l scra Low. Da. Gra and Phi) at 4 C. IO 1’1 01‘ PB-BSA containing 2 /ig 01 carrier IgM were added. and the IgM proteins were precipitated with an excess (IO ktl) of rabbit anti-IgM herum or its IgG fraction. After incubation for a further 2 hr at 4 C. 0.5 ml ofcold isotonic saline was then added to each tube and the tubes centrifuged at 2000x for I5 min. and decanted. The preclpitatcs were mashed with 0.5 ml of cold isotonic saline. centrifuged and decanted ah above. Control tubes with (a) tahelled antigen alone, (b) labelled antigen and carrier IgM. (c) labelled antigen and anti-IgM and (d) labelled antigen. carrier IgM and anti-IgMvl. were included in all assays. When S2 was used as labetlcd antigen. the radioactIvity precipitated m the control tubes by rabbit anti-IgM whole serum was 2”,, 01 the added counts. However. when Met was used as Iabetled antigen. rabbit antI-IgM whole serum precipitated 50”,, ofthe added counts. When the IgG fraction of this antiserum was used. the counts m the control tubes were reduced to an ,icccptahlc level of < I (I”,, The counts in the control tubes (d) were subtracted from all counts in assays. All determmatlons were carried out 1n duplicate. For inhibition of bindmg assays. IO /II of unlabclled InhibItor and 20 111of PB-BSA were incubated with IO 111of a dilution of cold agglutinin serum at 4 C for I hr (anti-l Ma. Step and Ver: anti-i sera) or 4 hr (anti-1 Low. Da. Gra and Phi) prior to the addition of ‘“I-l~lbelled antigen.

A Radioimmunoassay

for Blood Group

Ii Antigens

815

% bmdmg

‘0°

1 a.

D~lutmn of cold

agglutlnlnVera

Fig. 1. Binding curves of anti-l sera and radioiodinated I-active S2 eluate [(a) and (b)] and anti-i sera and radioiodinated Ii-active Meceluate (c). Symbols: anti-l sera: @. Ma: 0. Ver: 0, Step: A. Low; 0, Da; A, Phi; n , Gra. Anti-sera: v. Den; v, Nit; +. Galli; 0, Tho.

Exploratory inhibition of binding assays were performed using unlabelled S2 or Met as inhibitor at various cold agglutinin serum dilutions that gave 15-75’:; of binding of the labelled antigen. Because of the substantially reduced requirement for inhibitors at the higher dilutions ofantibody, subsequent inhibition of binding assays were performed at dilutions giving 15-25”,, binding of labelled antigen.

RESULTS

Binding assays Initial binding assays with the ‘251-labelled sheep glycoprotein (S2) and meconium (Met) antigens prior to affinity chromatography gave negligible or no detectable binding with the cold agglutinin sera tested. Both S2 and Met gave binding curves with the anti-l sera after elution from the anti-l Step immunoadsorbent. The eluted Met (but not S2) gave binding curves with anti-i sera also. An initial investigation of incubation time required to give maximum or near maximum binding curves with the cold agglutinin sera was carried out. One or two hours incubation were required with the majority of antisera (Figs. la and c), but the anti-i sera Low, Da, Gra and Phi required 24 hr incubation (Fig. 1b). Inhibition oj’binding assays with glycoproteins

’

Figure 2 summarises the results of inhibition of binding assays with .the cold agglutinin sera and human blood group substances, including for reference unlabelled S2 and Met antigens. The substances with A,B,H and Lewis activities are shown to the left of the figure, and those lacking these activities (and which may be considered as precursors of the A,B,H and Lewis blood group antigens) are to the right. As has been demonstrated previously by haemagglutination inhibition and quantitative precipitation assays, the radioimmunoassays show that the anti-1 and anti-i antibodies of different patients differ in their relative reactivities with various glycoproteins. Of the 7 substances with A, B, HLeb and Lea activities, A(MSS), B (Beach) and HLeb (JS) showed no inhibitory activity at the highest concentration tested (1 mg/ml) with any of the anti-l or anti-i sera,

and the remaining four, A (438), B (413), Le’(N-I loo/, and N-l phenol insoluble) showed slight or moderate activity with certain of the antisera. The three substances that were inactive in the untreated state acquired some I activity after one stage of Smith degradation, which removes the terminal monosaccharides involved in the A,B,H and Lewis specificities (Lloyd & Kabat, 1968). The activities of A (MSS) 1st IO,, B (Beach) 1st 10, and Milk Fr C recognized by the anti-l sera Gra and Phi had not been detected previously by quantitative precipitation assays. Of the 4 ovarian cyst substances lacking A,B,H and Lewis activities, two (OG and Fl) showed moderate or strong activity with all the anti-1 and anti-i sera tested. The activity of Fl with the anti-1 sera Gra, Da and Phi and with the anti-i sera had not been detected previously by quantitative precipitation assays. Substance 484 was active with anti-1 Ma and Step, and with 3 of the 4 anti-i sera, and substance 502 was active exclusively with the anti-i sera. With the sheep glycoprotein S2, some inhibitory activity with the antii sera was observed, although no binding curve could be obtained with these sera and the 12sI-labelled S2 eluate. Inhibition of binding assays with oligosaccharides Figure 3 shows hapten inhibition assays with anti-1 Ma and L2SI-labelled S2. The oligosaccharides N-l R,0.7la & b containing the sequence fl~Galpl-+4~~GpNAc1+6 were the most active inhibitors ; the disaccharide N-acetyl lactosamine (pDGalpl+4~GpNAc) was approximately IO-fold less active. The data for N-l R,0.71b and N-acetyl lactosamine are in agreement with inhibition of precipitation assays (Feizi et al., 19716). However, 35 times less oligosaccharides were required in the radioimmunoassays. For inhibition of precipitation, 0.6 pmoles of N- I R,0.7 I b and 6.24 pmoles of N-acetyl lactosamine were required to give 50% inhib’ition, whereas by radioimmunoassay 17 and 170 nmoles, respectively, were required. Inhibition of binding assays with erythrocytes Figure 4 shows the results of inhibition of binding assays with 3 anti-l sera and one anti-i serum using

816

EDWIN WOOD 01 I//.

A B HLeb Lea rnnyl

m

Anti-i

Ma Step Low Gra Da Phi Ver

Anti-i

Tho Den Galli Nit

1st 104

n-n

ABHLe Inactive

Standards

FIN. 2. I and i actiwties of blood group A. B. H and Lewis act~vt‘and Inactl\e @qcoproteln\ ;L\evaluated b\ inhlbition of blnding assays wth seven antI- and four antI- acra. The results ale expl-ewxi a\ the concentration of glycoprotein added that gave 50”,, Inhibltlon of bIndIng of the antthodles to the radloiodinated I-active S2 eluatc (anti-l &era or to the radiolodinatcd II-actl~e I\lec eludtc 1,1nt1-I xr;, Symbols. n . l-10 lip ml. 8, 1~100 /lg;ml: a, 100 ltg m-l mg ml. q . 3k50”,, lnhlbltion ;II the hlghe\c concentration tested (I mg.ml~:U. Ic\\ than X”,, Inhlbltion at I mg ml. group 01 adult and group Oi cord erythrocytes as inhibitors. The inhibition curves clearly show that the 3 anti-1 sera differ in their relative reactions with cord :tnd adult erythrocytes. At one extreme, with anti-l Ma, 50”,, inhibition W;ISgiven by 1.3 x IO’ untreated adult erythrocytes and no inhibition by 7 x IO’ cord cells. At the other extreme, with anti-l Ver. the % Inhibition

100

difference between untreated adult and cord erythrocytes wus only 4-fold. With LInti- Step, there was a 60-fold difference between adult and cord erythrocytes. Trypsin or neuraminidase tre;ttmcnt produced 2140. fold increase in the inhibitor! activity of adult erythrocytes with anti-l Ma. but no change in the

1

60 -

Nanomoles of oligosaccharide added FIB. 3. Inhibition of binding of anti-l Ma to radioiodlnated used: 0. /~nGalp-r4o~pNAc: A, /~oGalpl +hnGpNAc: pentol(s) (N-l R,0.7lb): +. /kA3alpl*4[bGpl\jA~l 3

r~Fucp

I-active S2 eluate. Oligosiccharldc

mhibltors

0. /~~Galpl+4~~11GpNAc I +6 I. 3. 4. 5. 6 -6

I. 2. 4. 5. 6 pcntnl(\)

(N-l

K,O 71~1)

A Radioimmunoassay

RI7

for Blood Group Ii Antigens

activity ofcord cells was detected. With anti-l Step and Ver, enzyme treatment resulted in a 4- to 20-fold increase in the inhibitory activities of both adult and cord erythrocytes. In the case of anti-l Ver, enzyme treated cord cells became almost as active as adult erythrocytes. With anti-i Den, 50”/, inhibition was given by 5 x IO’ untreated and 4 x IO6 enzyme treated cord cells, but no inhibition was given by 1.3 x lo* untreated or 7 x 10’ enzyme treated adult erythrocytes.

% inhibition 100

7

Anti-l Ma

100 DISCUSSION

Although the sheep stomach glycoprotein and the meconium extract used in these studies showed strong I and Ii antigenic activities, respectively, satisfactory binding of these labelled glycoproteins with anti-l and anti-i sera could only be demonstrated after elution from an anti-l immunoadsorbent. These findings suggest that the I and i antigens are carried on a subpopulation of these glycoproteins. Preparative scale affinity chromatography experiments have shown that only 15-25”, of an l-active sheep glycoprotein preparation was specifically retained by an anti-l immunoadsorbent (E. Wood and T. Feizi, to be described elsewhere). The 1251-labelled meconium antigen eluted from the immunoadsorbent was enriched for both I and i activities, indicating that there are macromolecules carrying both types of antigenic determinant. Such co-existence of I and i determinents has been previously demonstrated by affinity chromatography experiments on human amniotic fluid (Feizi rt al., 1975; Feizi, 1977). By radioimmunoassays some I and i activities were revealed that were not previously detected by haemagglutination inhibition or quantitative precipitation assays. The most striking example was the blood group precursor-like glycoprotein Fl; by quantitative precipitation assays this glycoprotein showed activity only with the anti-1 sera Ma and Step (Feizi & Kabat, 1972) whereas the radioimmunoassays showed it to be rich in the I and i determinants recognized by all the antisera tested. Among the blood group ABH active ovarian cyst glycoproteins tested, some were inactive as inhibitors of anti-l and anti-i sera, while others were moderately active with certain anti-l sera. The B active glycoprotein 413 in addition showed some i activity. These I and i activities suggest the presence of unsubstituted precursor chains on these glycoproteins. Two types of glycoprotein lacking blood group A,B,H and Lewis activities were studied: those naturally lacking these antigens and those obtained by one stage of Smith degradation (1st IO,) of ABH active glycoproteins. Both the naturally inactive and the 1st IO, glycoproteins showed varying activities with the several anti-l sera tested. A notable difference between the two types of glycoprotein was the presence of moderate or marked i activity in the naturally ABHinactive glycoproteins and the lack of such activity in the 1st IO, glycoproteins, indicating that the naturally occurring ABH-inactive glycoproteins contain unbranched carbohydrate chains lacking or hidden in the ABH 1st IO, glycoproteins. The differences among the monoclonal anti-l or

80 60 40 20 0 100 80 60 40

100

AntI-i Den .+

80

Number of cells added

Fig. 4. Inhibition of binding of anti-l sera Ma, Step and Ver to radioiodinated I-active S2 eluate, and of anti-i Den to radioiodinated Ii-active Met eluate, using untreated and enzyme treated human erythrocytes as inhibitors. Symbols, adult blood group 01 cells (solid lines): l , untreated; n, pepsin treated; A, neurdminidase treated. Cord blood group Oi cells (dotted lines): 0, untreated; 0, pepsin treated; A, neuraminidase treated. ‘_

anti-i antibodies of individual patients in their reactions with the I and i antigens expressed on different glycoproteins are greater than the differences that are usually observed among conventionally elicited antibodies against one antigen. Different antiI antibodies have previously been shown to recognize different domains on the branched lacto-N-isooctaosyl structure (Watanabe et al., 1979; Feizi et al., 1979). Presumably different anti-i antibodies similarly react with different determinants on the lacto-N- norhexaosyl structure. The varying reactions of anti-l and anti-i antibodies with the various glycoproteins may well reflect differences in the micro-environment of the two types of carbohydrate sequence in each glycoprotein, which result in varying accessibility of the determinants recognized by each individual antibody.

EDWIN

818

WOOD

The radioimmunoassay was designed primarily to reduce the amounts of oligosaccharides required for hapten inhibition assays. Results with anti-1 Ma showed that this assay is 35 times more sensitive than the previously used technique of inhibition of precipitation. This level of sensitivity allows a positive approach to the isolation of Ii active oligosaccharides by detection of antigenically active oligosaccharides at an early stage of their purification from complex mixtures (E. Wood and T. Feizi, unpublished observations). The radioimmunoassay has been successfully applied to the screening of I and i activities of chemically synthesized oligosaccharides related to the precursors of the A. B. H and Lewis antigens (Feizi et ul., 1978~). The anti-1 antibody from patient Woj was shown to resemble that of Ma being specifically synthetic oligosaccharide inhibited by the ~DGalpl-t4~~GpNAcl +hr,Galp. radioimmunoassay for The use of the glycosphingolipids has been described elsewhere (Feizi et al., 19786; Niemann et al., 1978; Wood et ul.. 1978). As with oligosaccharides, the small requirements for inhibitors in radioimmunoassay have enabled the screening of crude glycosphingolipid fractions and monitoring of Ii activities during purification. into glycosphingolipids Incorporation of cholesterol/lecithin liposomes to form polyvalent haptens allows antigenic analyses with many anti-1 and anti-i antibodies to be performed with pg quantities of glycosphingolipids. The advantages of radioimmunoassay over haemagglutination inhibition and gel precipitation assays for glycosphingolipids have been discussed in detail by Feizi et ul. (1978h). The application of radioimmunoassay to the measurement of I and i antigens on intact cells is 01 value not only because of the increased accuracy relative to haemagglutination assays, but also because it enables the antigenic analysis of non-agglutinable cells such as bovine erythrocytes (Wiener et cd., 1956) to be performed. The changes in expression of the Ii antigens detected after enzyme treatment of erythrocytes indicate that the assay has potential use for measuring the changes in expression of the Ii antigens on lymphocytes during different stages of the cell cycle (Thomas, 1974) and for comparison of the antigens on normal and neoplastic cells (Picard et al., 1978).

Acknowledgement.spJ. L. was holder of a Quebec Government Fellowship and E.W. was holder of a Medical Research Council (U.K.) Research Studentship.

REFERENCES Bendich

A., Kabat E. A. & Bezer A. E. (1946) Immunochemical studies on blood groups. III. Properties of purified blood group A substances from individual hog stomach linings. J. exp. Med. 83, 485-497. Childs R. A., Feizi T., Fukuda M. & Hakomori S. (1978) Blood group 1 activity associated with Band 3, the major intrinsic transmembrane protein of human erythrocytes. Biochem. J. 173, 333-336. Cooper A. G. (1970) Soluble blood group I substance in human amniotic fluid. Nature.Land.227, 508-509.

(“I cl/.

Dzierrkowa-BorodeJ W.. Seyfricd H.. Nichols M.. Reid M. & Marsh W. L. (1970) The rccognitlon 01‘ uatcr+olublc I blood group substance. lo.\- S&x. 18, Z-234. Dzierkowa-BorodeJ W.. Seyfried H. & l.isonska E. t lY7C) The serological cla~slticatlon of anti-I \cr;i. I.r)\ Srrrl,~. 48, 110-121. Felri T.. Kabat E. A.. Vicar1 G.. Anderson B. 6, Marsh W. I.. (1971rr) lmmunochemical studieson blood groups. XLVII. The I antigen complex-precursors in the A, B. H. Les ,tnd Leh blood group systems haemagglutin;~tlon InhIbition studies. J. ~‘.xp. Mccl. 133, 39-52. FeiIl T.. Kabat E. A.. Vicar! G., Anderson B. Ki Marhh W. L. (19711~) Immunochem~cal studies on blood groups. XLIX. The I antigen complex: Specificirq dlfferenccs among antIscra revealed by quantltatlvc prccipitin studies. p,lrtlal structure ofthe I determinant specific for one antl-I serum. J. Invwr~. 106, 1578-l 592. Feiri T. & Kabat E. A. (lY72) Immunochemlcal studies on blood groups. LIV. Classilication of antI- and antl-i sera into groups bahed on reactivity patterns with burtous antigen\ related lo the blood group A. B. ti. L c’. L.cb xnd precursor substances. .1. (‘y,/,. M&. 135, 1247-I 258. Feizl T. & Kabat E. A. (lY74) Immunochemlcal sludlca on blood groups. LVI. Purllication of glycoproteins with different 1 determinents from hydatld cqsl fluid and from human milk on Insoluble anti-l immllnoadsorhe~~ts. J. fmmun. 112, 145-l 50. Fei7i T.. Ccderqvlst L. L. & Childs R. A. (1975) The blood group 1and I antigens of amniotic fluid I. Assoclallon of I and i antigens with blood group A. B and H antlgens. Hr. J. Hamrut. 30, 489-497. Feiri T. (1977) Immunochcmlstrq of the blood group Ii antigens. In HWKUI Blood Gwup\. .Sth 1111. C‘OII~OC~. Iwwmmo/.. Buf/!fa/o. .V I’. 1976 (Edited hy Mohn J F.. Plunkett R. W.. Cunningham R. K. & Lambert R. M.l 1,. 164. Karger. Basel. Feizi T. (1978) The 1 and I antigens on cermln normal and pathological tissues. !+vtr<, F. Trtrrl\fu.tio,~ /rn,illrrro/ic;rlltrrologic 21, 165-174. Felzi T.. Wood E.. Augk C.. David S. Nr Veyrieres A. (lY780) The blood group I activities of synthetic oligosaccharides assessed by radioimmunoassay. fntmunoc~h~,mi.t/r,~ 15, 733-736. Fcizi T., Childs R. A.. Hakomorl S. & Powell M. E. (IY /8h) Blood group 11 active ganghosides of human erythrocqte membranes. Biochcm. J. 173, 245-254 Feizi T.. Chllds R. A., Watanabe K. & Hakomori S. (lY79) Three types of blood group I specificity amongmonoclonal anti-1 autoantibodies revealed by analogues of a branched crythrocqle glycollpld. ./. r’\,, .%‘c
A Radioimmunoassay

for Blood Group

Immunol. 20, 287-302. Lloyd K. O., Kabat E. A. & Licetio E. (1968) Immunochemical studies on blood groups. XXXVIII. Structures and activities of oligosaccharides produced by alkaline degradation of blood group Lewis’ substance. Proposed structure of the carbohydrate chains of human blood group A. B. H, Le’ and Leb substances. Bioch~,miv/r,~ 7, 2976-2990. Lowry 0. H.. Rosebrough N. J., Farr A. L. & Randall R. J. (1951) Protein measurement with the folin phenol reagent. J. biol. Chem. 193, 256-265.

agglutinins

819

Ii Antigens on human

Immunopathol.

B and T lymphocytes.

Clin. Immun.

4, 248-257.

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