Immunochemical studies of conjugates of isomaltosyl oligosaccharides to lipid: Production and...

Molecular Immunology, Vol. 22, No. 9, pp. 1021-1037, 1985 Printed in Great Britain

0161m5890/85 $3.00 + 0.00 C 1985 Pergamon Press Ltd

IMMUNOCHEMICAL STUDIES OF CONJUGATES OF ISOMALTOSYL OLIGOSACCHARIDES TO LIPID:

PRODUCTION AND CHARACTERIZATION OF MOUSE HYBRIDOMA ANTIBODIES SPECIFIC FOR

STEARYL-ISOMALTOSYL OLIGOSACCHARIDES

ERIC LAI and ELVIN A. KABAT* Departments of Pharmacology, Microbiology, Human Genetics and Development, Neurology and the Cancer Center/Institute for Cancer Research, Columbia University College of Physicians and Surgeons,

New York, NY 10032, U.S.A.

(Received 5 December 1984; accepted 30 January 1985)

Abstract-Twelve C57BL/6J hybridoma clones, 9, 2 and 1 from mice immunized with stearyl- isomaltotetraose, stearyl-isomaltopentaose and stearyl-isomaltohexaose respectively were characterized. Seven produced IgA and 5 IgM. The specificities and sizes of their combining sites were determined by quantitative precipitin and precipitin inhibition assays. All 12 hybridoma antibodies precipitated with a l-6 dextran B512 and linear dextran LD,, indicating that they recognize an internal -Glca 1+6Glca 1+6Glc- determinant. This in contrast with the results with rabbit antisera obtained in response to the same immunogen which recognize the non-reducing terminal determinant Glcu1+6Glca 1+6Glc-. Of the 12 hybridoma antibodies, 1 has an antibody combining site complementary to 4 a l&6-linked glucoses while others have combining sites complementary to isomaltohexaose or isomaltoheptaose. The large combining-site sizes found in C57BL/6 hybridoma clones may be related to the pre-existing clonal repertoire in this strain. Binding constants of monomers of these antibodies for dextran B512 and isomaltoheptaose determined by affinity electrophoresis range from 1.4 x 10’ to 4.6 x 10Sml/g and from 1.2 x 10) to 3.5 x 10“ M-’ respectively, which is consistent with previous studies in the anti-dextran B512 system. The use of synthetic glycolipids as antigens enables us to study the gene control of antibody responses to glycolipids and to investigate the combining-site specificities of antibodies to a single antigenic determinant. Results so far show that all 12 hybridoma proteins are different despite the simplicity of the antigens. The findings provide further insight into the specificity of antibody combining sites.

INTRODUCTION

Early studies in this laboratory on the antibody response to TV l-+6 dextrans (homopolysaccharides consisting largely of al-t6-linked glucose) in man (Kabat, 1956, 1960) and the rabbit (Mage and Kabat, 1963) have shown that heterogeneous populations of antibody molecules were formed, despite the relative structural simplicity of these antigens. Attempts to restrict the antibody response in rabbits by immunizing with synthetic antigens, isomaltotrionic acid- or isomaltohexaonic acid-bovine serum albumin conjugates, also revealed heterogeneous antibody populations (Arakatsu et al., 1966; Outschoorn et al., 1974). Homogeneous dextran-binding myeloma proteins have been produced by pristane-induced plasma- cytomas (Potter and Boyce, 1962; Potter, 1972) and characterized immunochemically (Leon et al., 1970; Lundblad et al., 1972; Cisar et al., 1974). Hybridomas specific for dextran B512 with 96% al-6 linkages have been produced in both BALB/c and C57BL/6 mice immunized with dextran and immunochemical

*Correspondence should be addressed to: Dr Elvin A. Kabat, Department of Microbiology, Columbia Univer- sity, College of Physicians and Surgeons, 701 West 168th Street, New York, NY 10032, U.S.A.

characterization of the sizes and shapes of their combining sites has been reported (Sharon et al.,

1982~; Newman and Kabat, in press). It was shown that all the hybridoma antibodies had different combining sites; these studies have provided important insights into the size and nature of the repertoire of the antibody response.

Artificial glycolipid antigens of well-defined chemical structure have been prepared by coupling various isomaltose oligosaccharides to stearylamine (Wood and Kabat, 1981~); each stearyl-isomaltosyl oligosaccharide would be a homogeneous antigen as compared to the heterogeneous oligosaccharide-protein conjugates (Arakatsu et al., 1966; Outschoorn et al.,

1974). Antibodies to these synthetic glycolipids have been obtained in rabbits (Wood and Kabat, 1981b). These antibodies cross-reacted with dextrans; and had predominantly cavity-type combining sites (Wood and Kabat, 1981b), whereas hybridomas obtained by immunizing with native al-6 dextran B512 had groove-type combining sites (Sharon et al.,

1982a). The purpose of this study is to obtain hybridomas

directed to steary-isomaltosyl oligosaccharides and to attempt to evaluate the antibody repertoire to a synthetic glycolipid antigen. Twelve C57BL/6 hybrid-

1021

1022 ERIC LAI and ELVIN A. KABAT

oma antibodies have been obtained and characterized

immunochemically by quantitative precipitin and

precipitin inhibition assays and their binding con-

stants determined by affinity electrophoresis. The specificities, sizes and shapes of the antibody combining sites of these hybridoma antibodies as determined from their cross-reaction with various dextrans will be discussed. In addition, the specificities and the nature of the combining sites of these hybridoma antibodies are compared with those generated in C57BL/6 and BALB/c mice by immunization with native dextran B512 and with antisera to stearyl-isomaltosyl oligosaccharides raised in rabbits.

et al., 1979). Positive wells were subcloned in soft agarose over rat libroblasts as a feeder layer.

Passive hemagglutination

MATERIALS AND METHODS Mice

Mouse erythrocytes (MRBC) were obtained from normal C57BL/6 mice. Erythrocytes were coated with dextran according to the method of Ghanta et al. (1972) using partially periodate-oxidized BS 12 (Sanderson and Wilson, 1971). The coated erythrocytes were used as a 2% suspension in PBS. Hemag- glutination assays were performed in 96-well microtiter plates (Dynatech Laboratories Inc., Alexandria, VA) using a Takatsy microtitrator with a 2%~1 loop (Cooke Engineering Co., Alexandria, VA). The plates were read after 2 hr at room temp.

C57BL/6J and CBBFljJ (BALB/c P x C5731/6 3) were obtained from the Jackson Laboratory, Bar Harbor, ME. C57BL/6N and CB6Fl/N were received from Dr Michael Potter, National Institutes of Health, under NC1 contract NOl-CB-25584.

Replica immunoadsorption assay

Coupling of isomaltose oligosaccharides to stearylamine

Subcloning of hybridomas in soft agarose and detection of specific hybridomas by replica immunoadsorption were essentially as previously described (Sharon et al., 1979, 1981), except that a protective agarose overlay was not used to enhance the sensi- tivity of the assay.

The reaction was performed as described by Wood and Kabat (1981~) and was essentially that intro- duced by Gray (1978). Briefly, the reaction mixture containing oligosaccharides and a 5-fold excess of stearylamine and sodium cyanoborohydride was kept at 37°C for several weeks until free OIigosaccha~de was no longer detectable by paper chromatography. The products were then purified by using an LH-60 column (Pharmacia Fine Chemicals, Division of Pharmacia Inc., Piscataway, NJ) and repurified from methanol (Williams et ai., 1979).

Preparation of hybridoma anti-dextrans in ascites

CB6Fl mice 5 weeks of age or older were primed intraperitoneally with 0.5 ml of pristane (Aldrich Co., Milwaukee, WI). Seven-ten days later, l-5 x lo6 hybridoma cells were injected intraperitoneally in 0.2 ml of IMDM medium. Ascites from the same cell line were pooled and kept frozen in separate portions at -20°C. A working sample was kept at 4°C with 0.02% sodium azide.

Purr~cat~on qf hybridoma antibodies

Immunization

After a preimmunization bleeding, mice were immunized with 10 p g of glycolipid antigen followed by 5 pg bi-weekly for a total of 4 injections. The antigen was administered intraperitoneally in complete Freund’s adjuvant (CFA). After the fourth injection, the mice were rested for l month then injected intraperitoneally with 100 pg of glycolipid in CFA and 100 pg of glycolipid in saline intravenously. Fusions were performed 4 days after the final immunization.

Hybridoma antibodies were purified from ascites by enzymatic digestion of dextran-anti-dextran precipitates (Kabat, 1954). Specific antibodies were precipitated by dextran B512 as for a quantitative precipitin assay; precipitates were washed 5 times with chilled saline and completely digested at room temp overnight with 1 S-3 units of dextranase (Sigma Chemical Co.) (Su~ura ef al., 1973). Samples were then dialysed extensively against PBS. Dextranase was inactivated by placing samples at 56°C for 1 hr (Sharon et al., 1981).

Production of hybridomas Antisera

The procedure used was essentially that of Pearson et al. (1980) with minor modifications. Briefly, spleen celts were prepared from immunize mice and fused with P3-X63-Ag8.653 myeloma cells (Kearney et al., 1979) at a ratio of 10: 1 using polyethylene glycol 1000 (J. T. Baker Chemical Co., Phillipsburg, NJ). After fusion, cells were grown in hypoxanthine, amin- opterin, thymidine selection medium in 96-well plates. Culture supernatants were screened by passive hemagglutination with dextran-coated mouse red blood cells on days 10-14. One clone 37.1E5.2 was detected by replica immunoa~orption assay (Sharon

Rabbit antisera to mouse ~Fc, aFc, y,Fc, y2,Fc, yZDFc, y,Fc and A were obtained from Litton Bio- netics, Kensington, MD. Rabbit anti-mouse IC was the gift of Dr M. Potter, NIH.

Immunodtflusion

Cell Iysates or purified antibodies were isotyped by Ouchterlony double diffusion in 1% agar in PBS. Hybridoma cells were centrifuged at 1500 rpm for 10min and the resulting pellet was lysed in NP-40 buffer (1 O6 cells/ 10 ~1). Precipitin lines were examined after 24 hr at 4°C.

Hybridomas to isomaltosyl glycolipids 1023

Table I. Proportions of linkages in dextrans estimated from methylation and periodate-oxidation analysis

Tenninal a 1+5-Linked al-6 and non-reducing a1+6- al-r3- backbone with al*3-

Molecular end groups Linked Linked E I+2-linked linked Symbol Dextran Fraction wt c( 1+6-linked backbone backbone branches branches

A B 1299” S >2 x 10’6 :9.1c 26.0 34.9 m B1355’ S 6.9 46.9 35.0 11.2 0 B1424d 24.5 51.9 20.6 3.0

z B”% (N279) 4-10 4.2 x x 106 lo4 4.2 100.0 91.2 4.6

; N-l 50Ng 6x lo4 NRC Fr.18 1 x 104

0 NRC Fr.28 3.5 x 104 x NRC Fr.3* 5.1 x 104

$ NRC NRC Fr.4” Fr.6* 9.1 1.9 x x 104 105

: NRC D200W FI..~~ 4.1 2x x 105 106

“See Seymour ef al. (1977). “See Kobayashi et al. (1984). ‘Mole %. ‘+See Sevmour ef al. (19791. ‘See R&kc1 and Sch;ercd (1966). ‘See Van Clew ef al. (1956) and Lindberg and Svensson (1968). gDextran fractions made from partially hydrolyzed native dextran B512

Dextrans

The dextrans used are listed in Table 1. NRRL B512 (preparation N279) was obtained from Com- mercial Solvents Co. (Terre Haute, IN). Native dextrans B1355S (soluble fraction), B1299S (soluble fraction) and B1424 were obtained from Dr A. Jeanes of the Northern Regional Research Center (Peoria, IL). LD,, a completely linear t( l-*6 synthetic dextran was from Drs H. Ito and C. Schuerch (State University College of Forestry, Syracuse, NY). Dextran 2000, designated as D2000, was obtained from Pharmacia (Uppsala, Sweden). National Research Council (NRC) fractions 1-8 (NRC Fr. l-8) were prepared by Commercial Solvents Co. and obtained through the NRC (Kabat and Bezer, 1958).

Mono- and oligosaccharides

Maltose was obtained from Eastman Kodak Co. (Rochester, NY). IM2 and IM3 were from Dr A. Jeanes of the Northern Regional Research Center (Jeanes et al., 1953). IM6, IM7, IM8 and IM9 were obtained from Drs K. Granath and A. De Belder of Pharmacia (Uppsala, Sweden). Additional samples of IM2-IM9 were prepared in our laboratory. IM3-COOH-IM7-COOH were prepared from corresponding oligosaccharides by bromine oxidation and have been described previously (Arakatsu et al., 1966; Outschoorn et al., 1974). IM3-OH-IM7-OH were prepared from corresponding oligosaccharides by sodium borohydride reduction (Kabat, 1961).

Immunochemical methods

Quantitative precipitin and quantitative precipitin inhibition assays were performed with ascites containing hybridoma antibodies by the microprecipitin technique (Kabat, 1961) using 6-9pg of antibody nitrogen (AbN) per determination in a total vol of 400~1. In quantitative precipitin assays, the

antibody-dextran mixtures were incubated at 37°C for 1 hr and then kept at 4°C for 5 days with daily mixing. In precipitin inhibition assays, the antibody was incubated with the inhibitors at 37°C for 30 min before adding the dextran, the mixture incubated for another hour at 37°C and kept at 4°C for 5 days. The precipitates were washed twice with chilled saline and the total AbN in the washed precipitates was determined by the ninhydrin method (Schiffman et al., 1964) with 1Oml being the final vol used for A,,, measurements.

Fractionation of IgA hybridoma antibodies into monomer and poIymer

Hybridoma protein 42.4B12.2 was separated into polymer and monomer fractions on an 2.5 x 100 cm Bio-Gel P-300 (minus 400 mesh) (Bio-Rad Labora- tories, Richmond, CA) at room temp with PBS as solvent. Two distinct peaks were detected by measuring the 0.D.280. Tubes 88,91,95 and 111 were analyzed by 5% polyacrylamide gel electrophoresis and the contents of tubes 85-89, 9&93 and 94-100 were pooled to give polymer fractions. The second peak, tubes 106-118, was pooled to give the monomer fraction.

Ajinity electrophoresis and calculation of binding constants

Affinity electrophoresis was carried out using a modified gel system of Ornstein (1964) and Davis (1964) and was previously described for the determination of binding constants of various myeloma proteins and hybridoma antibodies (Take0 and Kabat, 1978; Sharon et al., 19826). Ascitic fluid containing approx. 0.4 pg AbN of specific hybridoma protein, as determined from quantitative precipitin assays, was applied to each tube or to each lane of a slab gel. All hybridoma antibodies were reduced to


monomer by treating with 0.15 M 2-mercapt~thanol for 1 hr at room temp (Sharon er al., 1982b).

For the determination of the association constants (K,) for dextran B512, varying amounts of B.512 were added to the separating gel and K0 calculated by the following equation (Take0 and Kabat, 1978):

where Rm, and Rm, are the relative migration in the absence and presence of dextran, respectively. When l/Rm, is plotted against c, a straight line is obtained which can be fitted by the least-squares method and whose intercept on the c-axis gives -l/I&

When IM7 was added to the separating gel containing dextran, the mobility of the antibody was restored. The association constant for IM’7 (Kh) can be obtained by the following equation (Take0 and Kabat, 1978):

where i is the concn of IM7 in the gel containing [c] amount of dextran and Rm, is the relative mobility of the antibody in the presence of both dextran and IM7. When Rm&Rm, - Rmi) is plotted against i for a constant concn of dextran, a straight line is obtained whose intercept on the i-axis gives - l/K;.

Least-squares curve fitting, SE of linear regression, binding constants and correlation coefficients were calculated with an HP55 calculator from Hewlett-Packard (Cupertino, CA).

RESULTS

Producrion of hybridomas

From a total of 62 hybridizations, 12 hybridoma cell lines have been established (Table 2). Nine hybridoma clones were derived from C57BL/6 mice immunized with ST-IM4, 2 clones from mice immunized with ST-IMS and I clone from a mouse immunized with ST-IM6. Eight hybridomas produced

IgAK and 4 produced IgMtc. Three IgM hybridomas, 42.2147.2, 42.9E5.2 and 59.1G2.2, and hybridoma 37.1E5.2 of the IgA isotype could be detected by the replica immunoadsorption technique and the remaining 8 hybridomas did not give any red blood cell spots. However, after transferring clones from soft agarose to microtiter plates, all hybridomas secreted antibodies into the culture supernatant which were detected by passive hemagglutination. No correlation was found between the serum titer of the mouse used for fusion and the successful isolation of anti- glycolipid hybridomas.

Quantitative precipitin curves of the hybridoma antibodies to stearyl-isomaltosyl oligosaccharides with various dextrans are shown in Figs I (IgM) and 2 (IgA). To study the pattern of reactions for each hybridoma antibody, 4 dextrans, B512, B1424, B1299S and B1355S, that vary in their proportions of ~1-6 linkages, together with linear ~146 dextran, LD,, were used (Tabie 1). In addition, mol. wt fractions (N-ISON, D2000 and NRC Fr.l-8) obtained from partial hydrolysis of B512 were used to study the effect of mol. wt of dextrans on the quantitative precipitin curves. For each antibody the maximum amount precipitated by various dextrans and the relative amount required as compared to B512 are shown in Table 3.

For hybridoma antibodies of the IgM isotype, the maximum amounts precipitated by dextrans N-l 50N, LD,, D2000, B1424 and B1355S ranged from 80 to 110% of that precipitated by dextran B512 (Fig. 1 and Table 3). With hybridomas 42.2A7.2 and 42.985.2, LD, and N-15ON precipitated 110% of the AbN precipitated by B512 (Table 3). These 2 antibodies were studied further with the series of NRC dextran fractions (Fig. IB and D). All the NRC fractions, 1-8 with mol. wts ranging from 1 x lo4 to 4.1 x lo5 reacted equally well compared to B512 (Fig. 18 and D). With 58.2C10.3, NRC Fr.1 precipitated 42% of the AbN precipitated by B512. This clearly indicated that 58.2C10.3 differs from the other 3 IgM hybrid-

Table 2. Anti-stearyl-isomaltosyl oligosaccharide hybridoma clones

Antigen Hybridoma clone Isotype Supematant tit& A&tic Ruid titer” ___--_____- ._...___ ST--1M4 37.1E~Y2~ I&K: 29 2’6

42.2Al.2 42.4B12.2 42.5D4.2 42.7B3.2 42.7Cll.2 42.8E9.2 42.864.2 42.9E5.2

ST-IMS 58.2C10.3 59.1G2.2

IgMK IgAll I&+ IgAK IgAr IgAK IgAK IgMu 1aM~

2’6 2” 2’2 2’0 21’ 2’2 2” 2’6 2” 2’2

ST-tM6 62.3A6.2 26 2”

“Titers of culture supernatant and of ascites were determined by passive hemagglutination with dextran-coated mouse red blood cells. Titers of culture supernatant were done at a cell density of approx. 1 x 10’ce11s/m1.

‘Each hvbridoma clone is desinnated bv 3 sets of numbers reoresentine the fusion number fe.e. 371 and-the plate, row and col;mn reqkively (e.g. lE5) fro& which The hybridoma was obt&ed; and the last number (e.g. 2) refers to the number of times that the hybridoma has been subcloned.


A 42.2~7.2 i IO ,,I t IO 9 I 7

6 5 4 3 2

W”

‘SM

F 59.lG2.2 I 100 ui 1

Y 4

0 5 10 15 20 5 IO 15 20 0 50 100 150 200 t

: 50 100 150 200 A

MICROGRAMS DEXTRAN ADDED

Fig. 1. Precipitation of IgM hybridoma antibodies to stearyl-isomaltosyl oligosaccharides by various dextrans. Fifty to 100 ~1 of diluted ascites were used per determination. Dextran structure and symbols

used are indicated in Table 1. Lower scale for dextrans B13555 and B1299S.

oma antibodies with which NRC. Fr.1 precipitated over 90% of the AbN precipitated by I3512 Dextran Bl299S precipitate the same amount of AbN as dextran 3512 with hybridoma antibodies 58.2C10.3 and 59. I G2.2 but only reacted slightly with 42.2A7.2 and 42.9E5.2, again indicating that the hybridoma antibodies 42.2A7.2 and 42.9E5.2 differ from 58.2C10.3 and 59.1G2.2.

For the IgA hybridoma antibodies, dextrans N- 150N, LD,, D2000, B1424, B1355S and NRC Fr.1

precipitated 1 l-97% of the AbN precipitated by B512 (Fig. 2 and Table 3). IgA hybridoma antibodies are known to secrete monome~c and polymeric antibody molecules and the monomers have been shown to bind but not to precipitate with antigens (Eisen et al., 1968; Potter and Leon, 1968; Cisar et al., 1974). Thus, reduced precipitation may also be related to the monomer content of each hybridoma antibody. To study the influence of monomeric IgAs on precipitin curves, hybridoma antibody 42.4B 12.2 was purified

Table 3. Maximum precipitation of hybridoma antibodies by various dextrans

Protein B5 1 Z(N279) N-l SON LD, DZOOO B1424 B1355S 812995 NRC Fr.1

- 42.2A7.2 (IgM) 8.3 (1.0) 9.0(1.1) 9.5 (1.1) 8.3 (1.0) 8.5 (1.0) 7.9 (0.95) 0.7 (0.08) 8.3 (1.0) 42.9E5.2 (IgM) 8.5 (1.0) 9.0(1.1) 9.5(1.1) 7.8 (0.92) 8.5(1.0) 8.1 (0.95) 0.9(0.11) 7.5 (0.88) 58.2Ct0.3 (IgMf 5.9 (1.0) 5.0 (0.85) 5.0 (0.85) 5.3 (0.90) 4.7 (0.80) 5.1 (0.86) 5.6 (0.95) 2.5 (0.42) 59.162.2 (18Mf 7.1(1.0) 6.5 (0.92) 7.1(1.0) 7.4(1.0) 7.3 (1.0) 6.9 (0.97) 6.9 (0.97) 6.4 (0.90) 37.1E5.2 @A) 7.2 (1.0) 3.0 (0.42) 3.9 (0.54) 5.4 (0.76) 5.3 (0.74) 3.6 (0.50) 1.5 (0.21) 4.1 (0.57) 42.4B12.2 (18A) tl.S(l.0) 3.6 (0.53) 4.4 (0.65) 4.8 (0.71) 4.4 (0.65) 3.0 (0.44) 0.0 (0.0) 3.0 (0.44) Fr. lb 3.9 (1.0) 2.9 (0.74) 3.9 (1.0) 3.7 (0.95) 3.2 (0.82) 3.1 (0.80) 0.0 (0.0) 0.9 (0.23) Fr.2 4.8(1.0) 3.7 (0.77) 4.6 (0.96) 4.7 (0.98) 3.7 (0.77) 4.0 (0.83) 0.0 (0.0) 0.9 (0.23) Fr.3 3.0 (I .O) 1.1 (0.37) 1.3 (0.43) 2.2 (0.73) 0.8 (0.27) 1.8 (0.60) 0.0 (0.0) 0.2 (0.06) Fr.4 0 (0) 0 (0) 0 (0) 0 (0) 0 (fJ) 0 (0) o (0) 0 (0) 42.5D4.2 (I&4) S.O(l.0) 1.6 (0.20) 2.4 (0.30) 3.6 (0.45) 3.9 (0.49) 1.9 (0.24) 0.0 (0.0) 0.9 (0.11) 42.7B3.2 (IgA) 6.5(1.0) 5.3 (0.82) 5.5 (0.85) 6.3 (0.97) 5.8 (0.89) 6.2 (0.95) 0.0 (0.0) 3.5 (0.54) 42.7Cl I.2 &A) 7.9(1.0) 4.5 (0.58) 4.6 (0.59) 5.6 (0.72) 4.5 (0.58) 3.5 (0.45) 0.0 (0.0) 3.9 (0.50) 42.8E9.2 &A) 6.4(1.0) 2.8 (0.43) 3.2 (0.51) 4.1(0.64) 3.6 (0.56) 4.1 (0.64) 0.0 (0.0) 1.6 (0.25) 42.864.2 &A) ?.O(l.O) 2.8 (0.40) 3.3 (0.47) 4.6 (0.66) 3.6 (0.51) 3.3 (0.47) 0.0 (0.0) 1.5 (0.21) 62.3A6.2 &A) 7.0(1.0f 2.3 (0.33) 2.6 (0.37) 4.5 (0.64) 3.6 (0.51) 3.8 (0.54) 0.0 (0.0) 1.6 (0.23)

values represent maximum pg AbN precipitated; number in parentheses indicates normalized value relative to dextran B512. bFractions of 42.4B12.2 after separation by Bio-Gel P300 (see Figs 3-5).


A 37.1E5.2 f 75rrlR

i”p[ E 42.7~11.2 ’ 33~11 IBA

‘“9 G 42.864.2 137”ll ‘BA a

B 42.4812.2 (25 rll ‘gA

0 50 100 150 200 A

‘;

E D 42.783.2 I 33~1 I I@

8

‘; F 42.BE9.2 175 pl1 IBA B 7

‘;

F

H 62.3A6.2 ,150 PII W’ 8

MICROGRAMS DEXTRAN ADDED

Fig. 2. Precipitation of IgA hybridoma antibodies to steary-isomaltosyl oligosaccharides by various dextrans. Fifty to 100 ~1 of diluted ascites were used per determination. Dextran structure and symbols

used are indicated in Table 1. Lower scale for dextrans B1355S and Bl299S.

from the ascitic fluid and separated on a Bio-Gel P300 column (Fig. 3). Two peaks were observed and tubes 88, 9 1, 95, 103 and 111 were analysed on 5% non-denaturing polyacrylamide gel (Fig. 3A). It can be seen that tube 88 consists of mainly polymeric IgA molecules while tube 111 consists solely of monomers. Two monomer-rich fractions [Fig. 3, lanes b (weak) and i], which were found to precipitate with dextran B512 (Cisar et al., 1974, 1975), were also included and these fractions were found to contain monomer and some polymeric IgA. Tubes 85-89, 9c-93, 94-100 and lo&l18 were pooled to give fractions 1,2, 3 and 4, respectively (Fig. 3B). Fraction 4 did not precipitate with any dextrans tested (Fig. 4D) and this is consistent with the finding by gel electrophoresis (Fig. 3B) that this fraction contained only IgA monomers. When fractions l-3 were compared to unfractionated 42.4B12.2 (Table 3), it can be seen that dextrans N-l 50N, LD,, D2000, B1424 and

Bl355S precipitated similar amounts as did B512 with the polymer-rich fractions 1 and 2, whereas decreased precipitation was seen with the dimer-rich fraction 3. This indicated that the reduced precipitation of the various dextrans with unfractionated 42.4B12.2 is due to the presence of IgA monomers. However, the nonreactivity of Bl299S with 42.4B12.2 is not due to difference in solubility of the antibody-dextran complexes or to the presence of IgA monomers but is related to the chemical structure of the dextran since the polymer-rich fractions also did not react with Bl299S.

Quantitatir;e precipitin inhibition studies

To determine the sizes and nature of the combining sites of these hybridoma antibodies, isomaltosyl oligosaccharides (IM2-IM9), reduced (IM3-OH-IM7- OH) and oxidized (IM3-COOH-IM7-COOH) isomaltosyl oligosaccharides were used to inhibit the


Tube No.

Fig. 3. Separation of purified IgA hybridoma antibody 42.4B12.2 into polymer and monomer fractions by chromatography on Bio-Gel P-300 (minus 400 mesh). Inset: 5% polyacrylamide slab gel electrophoresis of the 42.4B12.2 fractions. (A) Lanes a and j, purified myeloma protein W3 129; lane b, monomer fraction of W3129 [see Cisar er al. (1974)J; lane c, purified unfractionated 42.4B12.2; lane d, tube 88 in Fig. 3; lane e, tube 91; lane f, tube 95; lane g, tube 103; lane h, tube 111; lane i, monomer fraction of QUPC52 [see Cisar et al. (1975)]. (B) Combined fractions of 42.4B12.2: Fr.1, tubes 85-89; Fr.2, tubes 9&93; Fr.3, tubes 94-100; Fr.4; tubes 106-l 18. Gels were stained with Coomassie brilliant blue. The position of the IgA monomers in the gel was verified by using mildly reduced hybridoma antibodies as standards; the identity of the polymers was inferred from their positions in the gel and from previous studies that identified the

composition of each band (Sharon et al., 1981).

precipitation of the hybridoma antibodies by dextran B512.

It can be seen in Figs 5 and 6 that the inhibitory activities increase with oligosaccharides of increasing sizes until the antibody combining site is completely filled. Thus the size of the antibody combining site can be estimated as the size of the smallest oligosaccharide with the maximum inhibitory activity (Table 4). Two IgAs (42.7B3.2 and 42.7C11.2) have sites as big as 6 and 1 IgM (58.2C10.3) has a site complementary to 4 glucose residues, respectively. The remaining hybridoma antibodies have sites complementary to 7 glucose residues (Table 5).

The 12 hybridoma antibodies can be divided into 2 groups with respect to the sizes of their combining sites (Table 5). The first group is represented by hybridoma antibody 58.2C10.3 which had an antibody combining site complementary to the number of intact sugar rings present in the antigen. 58.2C10.3 was obtained from a mouse immunized with ST-IM5; thus, the antigen had 4 intact sugar rings plus the a-linked open sugar chain coupled to stearylamine. However, precipitation of 58.2C10.3 with dextran B512 was inhibited equally by IM4-IM9, indicating that the site is only as big as 4 glucoses. Thus, the size of the combining site of 58.2C10.3 is 1 glucose residue smaller than the carbohydrate determinant in the antigen. The second group consists of the remaining

hybridoma antibodies which have antibody combining sites bigger than the oligosaccharide determinant present in the immunizing antigens. The most striking examples are the hybridoma antibodies obtained from mice immunized with ST-IM4 which have sites as large as 7 sugars [e.g. 37.185.2 (Tables 2 and 5)].

Three distinct inhibition patterns were seen when the inhibition curves of the modified oligosaccharides were compared to those of the intact sugars. In- hibition pattern 1 consists of 7 hybridoma antibodies (Table 4) with which the oxidized oligosaccharide was equally active to the corresponding sugar but was more active than the reduced oligosaccharides. For hybridoma antibodies 42.2A7.2 (Fig. 5B), 42.4B12.2 (Fig. 5C), 42.8E9.2 (Fig. 6A), 42.864.2 (Fig. 6B), 42.9E5.2 (Fig. 6C) and 62.3A6.2 (Fig. 6F), the order of inhibition (Table 4) suggested that the combining sites of these hybridoma antibodies recognized either 7 intact sugars or 6 sugars plus the a-linked opened chain in its oxidized form. IM7-OH is no better as an inhibitor than IM6 with only 6 sugars, indicating that the opened chain in IM7-OH does not contribute binding energy. With 42.7B3.2 (Fig. 5E), since it has a site complementary to IM6, modification at the reducing end of IM7 did not affect the inhibitory activity of IM7.

Inhibition pattern 2 consists of hybridoma anti-


A Fr.l(Tubos 85 -89)

Fr.3(Tubes 94 -100)

;““” ,;,mU . 9

0 5 10 15 20 0 100 200 300 400 ;

MICROGRAMS DEXTRAN ADDED Fig. 4. Precipitation of hybridoma antibody 42.4B12.2 fractions by various dextrans. Dextran structure

and symbols used are indicated in Table 1. Lower scale for dextrans B1355S and B1299S.

body 42.7C11.2 (Fig. 6F). Since it also has a combining site complementary to 6 sugars, it is not surprising that the chemically modified IM7 has the same inhibitory activity as IM7 and IM6. When the reducing end of IM6 is chemically modified either by oxidation or reduction, the inhibitory activity decreases by a factor of 4, suggesting that the combining site of 42.7C11.2 is complementary to 6 intact sugars rings and not just to 5 sugars plus the a-linkage.

Inhibition pattern 3 consists of four hybridoma antibodies, 42.5D4.2 (Fig. 5D), 37.1E5.2 (Fig. 5A), 58.2C10.3 (Fig. 6D) and 59.162.2 (Fig. 6E). With this group of antibodies, the reduced oligosaccharides were better inhibitors than the oxidized oligosac-

charides, especially for the smaller oligosaccharides. With 42.5D4.2 (Fig. 5D), the COOH group in the IM7-COOH effectively decreased the interaction of the opened sugar ring with the site IM7-COOH being 1.6 times less inhibitory than IM7 and IM7-OH and was only as inhibitory as IM6. The inhibitory effect of the COOH group became more evident as the size of the inhibitors decreased since IM6-COOH was less inhibitory than IM6 and IM5. With hybridoma antibodies 37.1E5.2 (Fig. 5A) and 59.162.2 (Fig. 6E), the order of inhibition (Table 4) indicating that both hybridoma antibodies have combining sites complementary to 6 glycosyl residues plus the a-linked open chain structure, since the chemically modified terminal groups of IM7 did not affect the inhibitory

Hybridomas to isomaltosyl glycolipids

A 37.1ES.2 + 7.8 JJR DEXTRAN 6 42.2A7.2 + I.3 pg DEXTRAN lOOr 1OOr

1029

80

60 - .4!

40 -

1 20 /

c 42.4812.2 + 15.6 LIB DEXTRAN D 42.5D4.2 + 13 ,,R DEXTRAN loo- 100

00 60

60 I 60

40 - 40

20 - 20

0

100

L

E 42.783.2 + 5-2 “g DEXTRAN loo[ F 42.7C11.2 + 78 ,,R DEXTRAN

;I / ,,&f--_ 10 IO2 IO3 lo4

00

60

40

20

0

IO2 lo3 IO4

NANOMOLES INHIBITOR ADDED

Fig. 5. Inhibition by various oligosaccharides of precipitation of hybridoma antibodies by dextran B512 (N279). Symbols used were: 0, maltose; A, IM2; A, IM3; 0, IM4; 0, IM5; 0, IM6; 0, IM7; n , IM8; A, IM9; +, IM3-COOH; V, IMCCOOH; V, IMS-COOH; 0, IMBCOOH; ‘*, IM’I-COOH; ‘+,

IM3-OH; a, IM4-OH; 0, IMS-OH; 8, IM6-OH; j, IM7-OH.

A 42.BE9.2 + 10.4 ,,R DEXTRAN B 42.804.2 + IO.4 PO DEXlRAN lOOr

loor Y

,Oor E 59.1G2.2 + l-6 LIO DEXTRAN IOOr F 62 3A6.2 + 6 5 pB DEXTRAN

;I , ,,&L,,,i 0

10 IO2 IO3 lo4 10 ,02 IO3 IO4

NANOMOLES INHIBITOR ADDED

Fig. 6. Inhibition by various oligosaccharides of precipitation of hybridoma antibodies by dextran B512 (N279). Symbols used were those in Fig. 5.

Tab

le

4. N

anom

oles

of

ch

emic

ally

m

odif

ied

olig

osac

char

ide

requ

ired

fo

r 50

%

inhi

bitio

n of

pr

ecip

itatio

n of

hy

brid

oma

anti-

dext

rans

by

de

xtra

n B

512

Prot

ein

IM7

IM’I

-CO

OH

IM

’I-O

H

IM6

IM6-

CO

OH

IM

6-O

H

IM5

IMS-

CO

OH

IM

S-O

H

IM4

IMC

CO

OH

IM

CO

H

IM3

42.2

A7.

2 lO

O(1

.0)

100(

1.0)

19

0(1.

9)

175(

1.8)

30

0 (3

.0)

300

(3.0

) 40

00 (

40)

1850

’ 18

50”

42.4

B12

.2

220

(1.0

) 22

0(1.

0)

410(

1.9)

41

0(1.

9)

800

(3.6

) 80

0 (3

.6)

800”

30

00”

3000

” 42

.864

.2

62(1

.0)

62(1

.0)

lOO

(1.6

) lO

O(l

.6)

180

(2.9

) 18

0 (2

.9)

1200

(19

) 30

00 (

48)

3000

(48

) 42

.8E

9.2

53 (

1.0)

53

(1.0

) 10

2(1.

9)

102(

1.9)

10

2(1.

9)

155

(2.9

) 10

00 (

19)

2400

(45)

24

00 (

45)

42.9

E5.

2 12

0 (1

.0)

120(

1.0)

24

0 (2

.0)

240

(2.0

) 36

0 (3

.0)

360

(3.0

) 30

00 (

25)

1350

” 13

50”

62.3

A6.

2 58

(1.

0)

58(1

.0)

98 (

1.7)

98

(1.

7)

135

(2.3

) 16

5 (2

.8)

900

(16)

45

0”

45w

42

.7B

3.2

270

(1.0

) 27

0(1.

0)

270(

1.0)

27

0(1.

0)

270(

1.0)

60

0 (2

.2)

3200

(12

) 16

50”

1650

” 42

.7C

11.2

32

0 (1

.0)

320(

1.0)

32

0(1.

0)

320(

1.0)

13

00(4

) 13

00 (

4)

700”

24

00”

2400

” 42

.5D

4.2

56(1

.0)

90(1

.6)

56(1

.0)

90(1

.6)

200

(3.6

) 90

(1.6

) 13

0 (2

.3)

640(

11)

130(

2.3)

37

.1E

5.2

lOO

(l.0

) lO

O(l

.0)

lOO

(l.0

) 19

0(1.

9)

560

(5.6

) 19

0(1.

9)

380

(3.8

) 38

0”

560

(5.6

) 59

.162

.2

260

(1.0

) 26

0(1.

0)

260

(1 .O

) 60

0 (2

.3)

980

(3.8

) 60

0 (2

.3)

980

(3.8

) 98

0 (3

.8)

58.2

C10

.3

I10

(1.0

) llO

(1.0

) 11

0(1.

0)

llO(l

.0)

160(

1.5)

llO

(l.0

) llO

(1.0

) 16

0(1.

5)

llO(l

.0)

llO(l

.0)

340(

3.1)

llO

(l.0

) 30

0 (2

.7)

Inh

ibit

ion

p

att

ern

1.

Inh

ibito

ry

activ

ity

of

oxid

ized

ol

igos

acch

arid

es

> r

educ

ed

olig

osac

char

ides

.

(A)

IM7

= I

M’I

-CO

OH

>

IM

’I-O

H

= I

M6:

Pr

otei

ns:

42.2

A7.

2,

42.4

812.

2,

42.8

64.2

, 42

.8E

9.2,

42

.9E

5.2,

62

.3A

6.2.

(B)

IM7

= I

M7-

CO

OH

=

IM

I-O

H

= I

M6

= I

M6-

CO

OH

>

IM

6-O

H:

Prot

ein:

42

.7a3

.2.

inh

ibit

ion

p

atr

em

2. I

nhib

itory

ac

tivity

of

ox

idiz

ed

olig

osac

char

ides

=

red

uced

ol

igos

acch

arid

es.

(A)

IM7

= I

M7-

CO

OH

=

IM

’I-O

H

= I

M6

> I

M6-

CO

OH

=

IM

6-O

H:

Prot

ein:

42

.7C

11.2

. In

hib

irio

n

pa

tter

n

3. I

nhib

itory

ac

tivity

of

re

duce

d ol

igos

acch

arid

es

> o

xidi

zed

olig

osac

char

ides

.

(A)

IM7

= I

M’I

-OH

>

IM

’I-C

OO

H

= I

M6:

Pr

otei

n:

42.5

D4.

2.

(B)

IM7

= I

M’I

-OH

=

IM

’I-C

OO

H

> I

M6

= I

M6-

OH

>

IM

6-C

OO

H:

Prot

eins

: 37

.1E

5.2,

59

.1G

2.2.

(C)

IM7

= I

M6

= I

M5

= I

M4

= I

MC

OH

>

IM

6-C

OO

H

= I

MS-

CO

OH

: Pr

otei

n:

58.2

C10

.3.

*Tw

enty

-fiv

e pe

rcen

t in

hibi

tion

at

indi

cate

d am

ount

s,

valu

es

in

pare

nthe

ses

indi

cate

in

hibi

tory

ac

tivity

re

lativ

e to

IM

9.


Table 5. Nanomoles of oligo~~ha~de required for 50% inhibition of precipitation of hybridoma anti-dextrans by dextran BS12

Protein IM9 IM8 IM7 IM6 IM5 IM4 IM3 IM2 Site size ~.-

I8M 42.2A7.2 100(1.0t” lOql.0) 100(1.0) 175(1.8) 4000(40) 7b 42.9E5.2 120(1.0) 12q1.0) 12O(LOf 24of2.0) 3000(25) 7 58.2C10.3 i lO(l.0) 1 lO(1.0) 1 lO(1 .O) llO(I.0) 1 10( 1 .O) 1 lO(l.0) 300(2.7) looO(9) 4 59.162.2 260( 1 .O) 260(1.0) 260( 1 .O) 600(2.3) 980(3.8) 4000(15) 7

IgA 37.1E5.2 lOO(l.0) lOo(l.0) 100(1.0) 190(1.9) 380(3.8) 1050(11) 7 424812.2 220( 1 .O) 220( I .O) 220(1.0) 410(1.9) 800’ 7 42.5D4.2 56fl .O) 56(I -0) 56(1.0) 90(1.6) 130(2.3) 37016.6) 7 42.7B3.2 270(1.0) 270( 1 .O) 270( 1 .O) 270(1.0) 3200(12) 6 42.7Cll.2 320( 1 .O) 320( 1 .O) 320(1.0) 320( I .O) 700’ 6 42.8E9.2 53( 1 .O) S3(1.0) 53(1.0) 102(1.9) lOOO(19) 7 42.8G4.2 62( 1 .O) 62( 1 .O) 62(1.0) IOO(l.6) 1200(19) 7 62.3Ab.2 58( 1 .O) S8( 1 .O) 58( 1 .O) 98f 1.7) 9OWl6) 7

‘Values in parentheses indicate inhibitory activity relative to IM9. ‘Number of glucose residues. Twenty-five percent inhibition at indicated amounts,

activity of IM7-OH and IM7-COOH (e.g. the terminal groups of the opened sugar chains were not in contact with the site). Hybridoma antibody 58.2C10.3 was found to be inhibited equally by the reduced oligosaccharides as the corresponding sugars from IM7-OH to IMCOH, indicating that the reduced opened sugar chain contributes binding to the site. When the oxidized oligosaccharides were tested for their inhibitory abilities, IM7-COOH was as inhibitory as IM7 but, as the number of intact sugar rings decrease, the inhibitory power of the oxidized oligosaccharides decreased 1.5 times for IMdCOOH and IMS-COOH.

The slopes of the precipitin inhibition curves (Figs 5 and 6) also vary from hybridoma to hybridoma, with 58.2C10.3 showing the steepest slopes, whereas slopes for all the remaining hybridomas are more shallow. An interesting pattern was found with 42.2A7.2; the slopes of IM7-OH, IM6-COOH and IM6-OH were distinctly different from those of the corresponding oligosaccharides. These differences in

slope indicate differences in the interactions of the various inhibitors with the site.

K, ofanti-glycolipid hybridoma proteins with dextran BS12 and IA47

The K, of anti-glycolipid hybridoma antibodies with dextran B512 and IM7 are shown in Table 6. Among the IgAs, hybridoma protein 37.1E5.2 has the highest association constant with B512. Its K,, 4.6 x lO*ml/g, is 300-fold higher than that of 42.7B3.2 which has the lowest binding constant, 1.4 x 10’ ml/g. 42.5D4.2 has the second highest binding constant, K, being 6.5 x 103. The remaining IgAs, the weak binders, have similar binding constants. Among the IgMs, 59.1G2.2 has the highest iu,, 5.0 x lo4 ml/g. The rU, of the remaining 3 IgM hybridoma antibodies ranged from 1.3 to 1.6 x lo3 ml/g. Thus, the IgM and IgA hybridoma antibodies have very similar association constants. 58.2C10.3 and 42.9E5.2 have identical X;, despite large differences in the sizes of their combining sites

Table 6. Association constants of hybridoma antibodies with d&ran B512 and 1M7

Association constant

Dextran B512 IM7

Protein Site size” (mIl8Y (iM_‘) ______...----

I%M 42.2A7.2 I 1.6kO.l x 10’ 42.9ES.2 7 1.4kO.1 x 10’

58.2C10.3 4 1.3iO.l x 10s 59.1G2.2 7 5.0 rt: 0.6 x IO4

IgA 37.1852 7 4.6+ 1.0 x 10’ 42.4B12.2 7 1.6kO.l x 10’ 42.5134.2 I 6.5 4 0.3 x 10s 42.7B3.2 6 1.4*0.1x 103 42.7C11.2 6 1.51_0.1 x 10’

42.889.2 I 1.6+0.1 x 10’ 42.864.2 7 3.1 * 1.0 x 10’ 62.3A6.2 7 2.1 zf: 0.1 x 10’

“Number of glucose residues (see Table 4). %ZorreIation coefficients ranged from 0.94 to 0.99. “Correlation coefficients ranged from 0.90 to 0.99.

4.8 + 0.5 x 10’ 2.1 f 0.1 x 10’ 2.7 f 0.2 x 10s 1.8*0.3x lo4

3.5 + 0.2 x lo4 1.5f0.2~ IO’ 1.5*0.1x lo4 1.5+0.1 x 103 1.2 f 0.2 x 10” 1.2 + 0.1 x 10’ 1.9kO.2 x lo3 1.3 f0.2 x 10s


(Tables 5 and 6). Association constants for IM7 (Kb) ranged from 3.5 x lo4 M-’ for 37.1E5.2 to 1.2 x lo3 M-’ for 42.8E9.2 and 42.7C11.2, the highest being 30 times the lowest. The relative order of antibody affinities for IM7 (Kb) corresponded roughly to their relative order with dextran B512 (K,) (Table 6).

DISCUSSION

This study provides the first data for evaluating the size and nature of the antibody repertoire to glycolipids and for comparing the antibody combining sites to isomaltosyl glycolipids with those of dextrans. The antigens used, stearyl-isomaltosyl oligosaccharides, are a set of relatively simple and chemically well-defined homogeneous glycolipids. Antisera to these synthetic glycolipids raised in rabbits (Wood and Kabat, 1981~) and characterized immunochemically (Wood and Kabat, 19816) were found to contain different populations of antibodies despite the simplicity of the antigenic determinant. Thus it was of interest to use the hybridoma technique (Kohler and Milstein, 1975) to isolate individual clones secreting various antibodies to stearyl-isomaltosyl oligosaccharides. Among the 12 hybridoma antibodies derived from C57BL/6 mice immunized with stearyl-isomaltosyl oligo-

saccharides, only u and p heavy chains were found, in contrast to the results with rabbit antisera in response to the same immunogen, in which an IgG response was seen (Wood and Kabat, 1981a-c). This might be related to species differences or to the screening methods used for the detection of hybridomas, or to a combination of these factors. The screening method used for the detection of hybridomas secreting anti-glycolipid antibodies may play an important role in selecting hybridomas which secrete certain isotypes. Passive hemagglutination and the replica immunoadsorption assays favor the detection of isotypes with high polymeric contents (i.e. IgA and IgM). Both assays are based on cross-linking of antibody and antigen and require multivalent antibody molecules. It is well known that IgM and IgA of high polymer contents give strong hemagglutination while IgG gives weak reactions.

When the hybridomas were screened by replica immunoadsorption, only 4 hybridomas, 42.2A7.2, 42.9E5.2, 59.162.2 and 37.1E5.2, could be detected. The failure of replica immunoadsorption to detect the remaining hybridomas may involve the following factors. (1) The isotype of the secreted antibodies: because IgM is pentameric and decavalent, it can be easily detected by methods that require cross-linking of antigens (i.e. passive hemagglutination and replica immunoadsorption). This is supported in that 3 out of 4 hybridoma antibodies detected by replica immunoadsorption were IgM. (2) The affinity of the antibody for dextran: 37.1E5.2 is the only IgA hybridoma detected by replica immunoadsorption and it has the highest affinity for dextran B512 (Table 6). Most of the IgA antibodies were not detected even though

they have higher binding constants for dextran B512 than IgM hybridoma antibodies 42.2A7.2 and 42.9E5.2. The second highest binder, 42.5D4.2, K, = 6.5 x 10’ ml/g, was not detected by the replica immunoadsorption, suggesting that there may be a threshold affinity constant for the assay, hybridoma antibodies with binding constants below the threshold not being detected. This is in accordance with the finding of Sharon et al. (1982b), in which all the IgA hybridoma antibodies were detected by replica immunoadsorption and the lowest binding constant for dextran B512 was 1.52 x 10’ ml/g. Thus the threshold affinity lies somewhere between 6.5 x IO3 and 1.5 x lo5 ml/g. (3) The quantities of antibody secreted by the hybridomas: 58.2C10.3 was the only IgM hybridoma not detected by the replica immunoadsorption assay even though it has a binding

constant for dextran B512 similar to that of 42.2A7.2 and 42.9E5.2 (Table 6). 58.2C10.3 secreted 8 times less antibody into the culture supernatant than did 42.2A7.2 and 42.9E5.2 as estimated by supernatant hemagglutination titers (Table 2).

Immunochemical studies of BALB/c myeloma proteins by Cisar et al. (1975) showed that antibody combining sites could be directed either toward the non-reducing terminal ends of chains or toward linear internal segments termed cavity-type and groove-type sites, respectively. The classification was based on the ability of the antibodies to precipitate with a synthetic linear dextran. Cavity-type sites were defined by myeloma protein W3129, which could bind only to the non-reducing end and did not precipitate with synthetic linear dextran. The groove- type site is represented by myeloma protein QUPC52, which binds to the internal chains with -Glccc 1-+6Glca: 1+6Glc- determinants, thus recognizing the linear dextran as multivalent and being precipitated with it. These findings were substantiated by Bennett and Glaudemans (1979) and Schalch et al. (1979).

Of the 20 hybridoma antibodies directed to dextran B512 previously obtained in this laboratory (Sharon et al., 1982~; Newman and Kabat, in press), all precipitated with linear dextran, indicating that they can bind to internal linear determinants on the dextran

and that they have groove-type sites. Since dextran B512 has only 4% branching (i.e. approx. 1 al+3 branch every 25 glucosyl residues), the majority of the antigenic determinants are linear and it is not surprising that hybridoma antibodies to this x(1-6) dextran have groove-type sites.

All 12 hybridoma antibodies to the stearyl-isomaltosyl oligosaccharides precipitated with synthetic ~1-6 linear dextran, LD,, indicating that they bind to the internal determinants on the dextran. In this respect, they are similar to the anti-dextran hybridoma antibodies. This is an un- expected finding since the majority of the rabbit antisera obtained in response to the same immu- nogens recognize the terminal non-reducing end.


Rabbit antibodies recognizing internal determinants were also present in response to these glycolipids since 3 out of 15 rabbit antisera precipitated with linear dextran (Wood and Kabat, 19816). In addition, rabbit antibodies recognizing internal determinants on the dextran were also found in response to isomaltotrionic- and isomaltohexaonic acid-BSA conjugates, since linear dextran precipitated 10-50x of the antibodies present (Cisar ef al., 1975). Thus it seems that there may be species differences in the recognition of various carbohydrate conformations. Our findings are supported by the studies of Zopf et al. (1982) and Lundblad et af. (1984) who showed that, when the same tetrasaccharide (Glca 1-+6Glcct 1+4Glcct 1+4Glc) coupled to KLH was used as immunogen in the rabbit and mouse, the rabbit antisera recognized the non-reducing sequence, Glca 1+6Glca l&4-, while 2 BALB/c mouse hybridoma antibodies recognized the internal sequence -Glccr 1+4Glccc 1+4Glc-. It will be of importance to obtain hybridomas from different mouse strains to stearyl-isomaltosyl oligosaccharides to study strain differences in the recognition of carbohydrate se- quences.

Of the 12 hybridoma antibodies, only 3 (37.1E5.2, 59.1G2.2 and 58.2C10.3) reacted significantly with dextran B1299S. Such a large difference in reactivity with dextran B1299S was not expected since the antigens used to generate these hybridoma antibodies were structurally very similar except for the chain length of the oligosaccharides. The chain length in these antigens does not seem to play a role since 37.1E5.2 was from a mouse immunized with ST-IM4, and 58.2C10.3 and 59.1G2.2 were from mice immunized with ST-IM5. The difference in

reactivity with B1299S was also not related to the size of the antibody combining sites, since 58.2C10.3 is complementary to 4 sugars while 37.1E5.2 and 59.1G2.2 are complementary to 7 sugars. B1299S is a highly branched dextran with a structure consisting of an eel-*6-linked D-ghCOSy1 backbone that bears primarily single D-ghCOpyranOSy1 groups linked c( 142 to alternate backbone residues (Seymour et al., 1977, 1980; Watanabe et a1.,1980). It has been shown that these residues make linear determinants (CL l-+6) unavailable and account for the inability of B1299S to precipitate most of the LY l&+6-specific hybridoma antibodies (Newman and Kabat, in press). It is also known that B1299S has a few side chains which have terminal nigerosyl (~11-3) groups (Bourne et al., 1976; Kobayashi et al., 1984). It is possible that those hybridoma antibodies that do react with B1299S might have sites that could accommodate non-u l-r6 linkages. This is supported by the finding that dextran B1355S with alternating ctl+3, ~1 l-+6 linkages in the backbone and some terminal nigerosyl groups in the side chains is 10 times more reactive with 58.2C10.3 and 59.162.2 than with the other hybridoma antibodies when the amount of dextran required for 50% precipitation is compared.

With the 4 IgM hybridoma antibodies, similar amounts of AbN were precipitated by dextrans B512,

D2000, B1424 and B1355S. The low mol. wt dextrans N-150N and LD, precipitated more AbN than did dextran B512 with hybridoma antibodies 42.2A7.2 and 42.9E5.2. This was surprising since low mol. wt dextrans usually precipitated less AbN than native dextrans because of the lower number of antigenic determinants per molecule and the higher solubility of their antibody-antigen complexes (Kabat and Bezer, 1958). Hybridoma antibodies 42.2A7.2 and 42.9E5.2 were then studied further with the series of

NRC dextran fractions of graded mol. wts prepared by partial hydrolysis of dextran B512. Since NRC

dextran fractions have the same chemical composition as dextran B512, the differential reactivity between dextran B512 and the NRC dextran fractions will be due to differences in solubilities of the antibodydextran complex in relation to the mol. wt of the dextrans. All the NRC fractions, 1-8, precipitated a similar amount of AbN as did dextran B512

with 42.2A7.2 (Fig. 1B) and 42.9E5.2 (Fig. 1D). This indicated that there were no significant differences in the solubilities of dextran-antibody complexes with dextrans of mol. wt 1 x lo4 (NRC Fr.l)-1 x 10’ (B512). A similar result was observed with 59.1G2.2 where NRC Fr. 1 precipitated 90% of the AbN precipi-

tated by B512. This has never been observed in any of the anti-dextran sera (Kabat and Bezer, 1958) myeloma (Cisar et al., 1975) and hybridoma antibodies. Thus, these hybridoma antibodies might have physical properties different from those of the other anti-dextran hybridoma antibodies (e.g. solubility of the antibodydextran complexes). With 58.2C10.3, NRC Fr. 1 precipitated 42% of the AbN precipitated by B512 and thus is similar to human anti-dextran sera (Kabat and Bezer, 1958).

The quantitative precipitin curves of the IgA hybridoma antibodies were quite different from those of the IgM hybridoma antibodies. Dextran B5 12 precipitated best with all the IgA antibodies and dextrans N-150N, LD,, D2000, B1424, B1355S and NRC Fr.1 precipitated 1 l-97% of the AbN precipitated by B512 (Table 3). These differences might theoretically result from differential solubility of different

dextran-antibody complexes, from differential reactivity of the antibodies with the various dextrans, from differences in the degree of polymerization of the antibodies (Potter and Leon, 1968; Cisar et al., 1974), or from a combination of all these factors. To resolve this question IgA hybridoma antibody 42.4B12.2 was fractionated into polymer and monomer fractions to study the effect of polymerization on the precipitin curves. Fraction 4 (monomers) did not precipitate with any of the dextrans tested, consistent with the findings of Potter and Leon (1968) and Wood and Kabat (1981~) that IgA monomers and IgG are capable of binding antigen but do not precipitate. When the precipitin curves of fractions l-3 were compared to that of unfractionated


42.4B12.2 (Table 3) the following conclusions can be drawn. (1) The relative amounts of AbN precipitated by dextrans N-150N, LD,, D2000, B1424 and B1355S compared to B512 are higher with polymer-rich fractions 1 and 2 than with unfractionated 42.4B12.2, indicating that the IgA monomers reduced the precipitation of dextrans N-150N, LD,, D2000, B1424 and B1355S compared to B512. (2) The ability of monomers competitively to inhibit precipitation by polymer was much greater with low mol. wt dextran N-150N than with dextrans of higher mol. wts (i.e. D2000 and B512). It can be seen from Table 3 that with the unfractionated 42.4B12.2, D2000 and N- 150N precipitated 71 and 53x, respectively, of the AbN precipitated by B512. Since dextrans N-150N, B512 and D2000 differ only in mol. wt, it appears that the IgA monomer acts more effectively in inhibiting precipitation with antigens having fewer determinants per molecule. (3) The reduced precipitation of dextrans compared to B512 in the polymer-rich fractions 1 and 2 is probably due to differential reactivity of the antibody with various dextrans and not to differential solubility of different

dextran-antibody complexes. This is suggested by the finding that D2000 and LD,, which are both derived from dextran B512 and have the same chemical composition, precipitated the same amount of AbN as did B512 with fractions 1 and 2 (Table 3). An effect of solubility was observed only with NRC Fr. 1, mol. wt 1 x 104.

With the IgA hybridoma antibodies, clinical dextran N- 150N, perhaps due to its greater polydispersity,

is generally poorer in precipitating power than synthetic linear dextran, LD,, although it has a higher av. mol. wt (Kabat and Bezer, 1958).

Quantitative precipitin inhibition assays were used to determine the sizes of the antibody combining sites. Fifty per cent inhibition was chosen for com- parison of the relative potency of the inhibitors (Kabat, 1956).

Precipitin inhibition studies showed that 2 IgA hybridoma antibodies have sites as big as 6 and 1 IgM hybridoma antibody has a site complementary to 4 c( l&6-linked glucose residues. The remaining 9 hybridoma antibodies have sites complementary to 7 ctl+6 glucose residues. 58.2C10.3 was the only hybridoma antibody which had an antibody combining site smaller than the size of the carbohydrate determinant in the antigen. Nine out of 15 rabbit antisera raised against the same glycolipid antigens were also shown to have antibody combining sites smaller than the size of the carbohydrate determinants in the glycolipids (Wood and Kabat, 19816). All 20 hybridoma antibodies directed to native dextran B512 (Sharon et al., 1982a; Newman and Kabat, in press) had groove-type combining sites complementary to either 6 or 7 glucose residues; 58.2C10.3 has the only groove-type site as small as 4 glucose residues. It will be of importance to compare 58.2C10.3 with the anti-dextran hybridoma antibodies by DNA se-

quencing and X-ray crystallography to determine the factors contributing to the differences in the sizes and shapes of the combining sites.

The remaining 11 hybridoma antibodies had combining sites larger than the sugar determinant in the antigens. This is an unusual finding but has been reported in the literature. Arakatsu et al. (1966) showed that, of the 6 rabbit antisera directed to isomaltotrionic acid-BSA, 2 had combining sites as

big as 4 sugars and 1 had a site complementary to 5 sugars. In addition, 3 out 15 rabbit antisera raised against the same glycolipids used in this study also

had combining sites larger than expected (Wood and Kabat, 1981b). These findings could be explained in 2 ways. First, since all the hybridoma antibodies have been shown to precipitate with the linear dextran, LD,, their combining sites are specific for the internal linear -Glccc(l+6)-Glc- sequence. Antibody specific for such an internal sequence may continue to show an increase in inhibitory power with oligosaccharides larger than those that fit best into the site because such larger size inhibitors could present several possi-

bilities for attachment of the antibody. For example, IM6 would have 3 times as many internal determinants as IM4 if the site is complementary to 4 sugars. Indeed Arnon et al. (1965) showed that when rabbit antibodies to polylysyl rabbit serum albumin were inhibited by oligolysines of increasing chain length, inhibiting power of oligolysines increased with increasing size up to nonalysine and decalysine. The data were interpreted as the site being complementary to a pentalysine and the increased inhibiting power up to the nonalysine might be related to the larger oligomers of lysine presenting more combinations of 5 lysines to the antibody (Arnon et al., 1965). This effect might be expected to give a

sudden sharp increase in inhibiting power per mole of oligomer when it appeared. Since large increments in the inhibitory power of the oligosaccharides were not observed in any inhibition curves of the hybridoma antibodies, the estimate of the size of the combining site of these hybridoma antibodies is probably cor- rect.

The second possibility for explaining the findings involves preexisting clones in the mice which were specific for native dextran and were triggered by the cross-reacting glycolipid antigens. Native dextrans are produced by bacteria in the gastrointestinal tract and antidextran antibodies have been found in humaa, dog, rat, bull, horse and rabbit sera without deliberate immunization (Kabat and Berg, 1953; Grabar, 1955; Maurer, 1953; Wood and Kabat, 1981a). Indeed all the C57BL/6 mice were found to have significant amounts of anti-dextran before immunization with glycolipids (Lai et al., 1985). Thus, the cross-reacting antigens stearyl-isomaltosyl oh- gosaccharides might have triggered the proliferation of preexisting B-lymphocytes specific for dextrans. Hybridomas specific for glycolipids should be produced using mouse strains that do not have a


pre-immune titer to the carbohydrate moiety to elim- inate the possibility of triggering cross-reacting clones. US/J mice have been found to be good responders to stearyl-isomaltotetraose but have very little or no pre-immune titer to dextran B512 (Lai et

al., 1985); thus this strain might be ideal to study hybridoma antibodies to stearyl-isomaltosyl oligosaccharides.

The nature of the antibody combining sites was further studied using chemically modified oligosaccharides with their reducing ends either oxidized to acids or reduced to alcohols. Such a series of modified oligosaccharides allowed fine mapping of the combining regions and evaluation of the contribution of the glucopyranosyl ring at the reducing end. Three distinct inhibition patterns were seen among the 12 hybridoma antibodies. Seven hybridoma antibodies were inhibited equally by the intact sugar and the oxidized oligosaccharides but the reduced oligosaccharides were less active (Table 4), suggesting that the combining sites of these antibodies recognized oligosaccharides with glucopyranosyl rings at the reducing ends either intact or opened but in the oxidized forms. The COOH group in the oxidized oligosaccharides might contribute additional binding energy to the opened sugar ring if there are anionic amino acid residues located inside or near the combining site. In addition, the carboxyl group is not blocked by substitution and aldonic acids in aq. solutions may lactonize by condensation with the hydroxyl group on C-4 or C-5 to generate the 1,4- or 1 $lactone respectively, thus preserving a ring structure (Theanor, 1980). Since the acid and lactone forms exist in equilibrium in solution it is not possible to assess the relative binding contribution of each form. The OH group in the reduced oligosaccharides might prevent optimal contact of the opened-chain structure with the combining sites of this group of hybridoma antibodies as suggested by the finding that IM7-OH was not a better inhibitor than IM6, indicating that the opened chain in IM’I-OH did not contribute binding energy. Anionic amino acid residues which favor binding of the oxidized oligosaccharides might be located near the site rather than in the combining site of hybridoma antibodies 42.4B12.2, 42.864.2, 42.9E5.2 and 42.2A7.2. This is suggested by the studies showing that when the size of the inhibitor decreases the oxidized oligosaccharides were less inhibitory than the corresponding oligosaccharides. With 42.7B3.2, even though the OH group did not contribute as much binding energy as the COOH group, it did not prevent the binding of the opened sugar chain to the site since IM6-OH was less inhibitory than IM6 but more active than IM5.

42.7C11.2 is the only hybridoma antibody (Table 4) in which the chemically modified isomaltohexaose was less inhibitory than IM6, clearly indicating that the site is complementary to 6 sugar residues with the reducing end sugar ring intact.

The remaining 4 hybridoma antibodies were inhibited equally by the intact oligosaccharides and the reduced oligosaccharides, but the oxidized oligosaccharides were less inhibitory. The charged COOH grouping might be inhibiting optimum contact in the site if there are acidic amino acids located in or near the combining site.

Since the glycolipids were prepared by coupling of the aldehyde groups at the reducing ends of the isomaltose oligosaccharides to the amino groups of the stearylamine, secondary amino groups were formed by the linking of the carbohydrate determinant and the lipid moiety. Further fine mapping of the antibody combining sites might be provided by using inhibitors prepared by reductive amination of isomaltose oligosaccharides with methylamine; such a series of inhibitors might permit evaluation of the contribution of the secondary amine relative to the stearyl groups to the antigenic determinant of the glycolipids.

The association constants of the hybridoma antibodies specific for stearyl-isomaltosyl oligosaccharides have the same range as the association constants of mouse anti-dextran myeloma and hybridoma antibodies (Take0 and Kabat, 1978; Sharon et al., 19826). No correlation is found between the site size of the hybridoma antibodies and their association constants for dextran B512. Hybridoma antibody 58.2C10.3 with a site complementary to 4 glucose residues has a K, similar to 42.9E5.2, which has a site as large as 7 glucose residues (Table 6), further establishing that larger sites do not necessarily result in higher binding constants (Cisar et al., 1975). The binding constant would have to depend on the actual area of contact between the site and the dextran, which, in turn, depends on the exact shape of each site. The association constants of the hybridoma antibodies with IM7 were also in the same ranges as anti-dextran myeloma and hybridoma antibodies (Sharon et al., 19826). The relative order of the antibody affinities for IM7 corresponded roughly to their order with dextran B512.

Of the 12 hybridoma antibodies to stearyl- isomaltosyl oligosaccharides, most of the antibodies differed one from another either in their size, shape, nature or binding constant. 42.2A7.2 and 42.9E5.2 were the only antibodies that showed similar results in all the assays, except for a slight difference in the inhibition curves. With 42.9E5.2, the inhibition curves of the chemically modified oligosaccharides had the same slopes as the corresponding sugars, whereas, with 42.2A7.2, the slopes of IM’I-OH, IM6-COOH and IM6-OH were different from those of the corresponding oligosaccharides. Since all of these antibodies were raised to a single antigenic determinant, it is not surprising that they are similar. Further studies by DNA sequencing and X-ray crystallography should elucidate their primary and 3-dimensional structures; together with the immunochemical data they promise important insights


into the fine specificity and structure of antibody combining sites.

Acknowledgements-Work in this laboratory is supported by grants from the National Science Foundation, NSF- PCM-81-02321, and the National Institute of Allergy and Infectious Diseases, lRO1 AI-19042 to E.A.K., NIH Train- ing Grant 07182 to the Department of Pharmacology, and Cancer Center Support Grant CA 13696 to the Cancer Center, Columbia University. Cancer Center Support Grant CA 34196 to the Jackson Laboratory is also acknowledged.

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