©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of the Antigenic Specificity of Four Different Anti-(28-Linked Polysialic Acid) Antibodies Using Lipid-conjugated Oligo/Polysialic Acids (*)

(Received for publication, May 9, 1995; and in revised form, May 30, 1995)

Chihiro Sato (1) Ken Kitajima (1)(§),   Sadako Inoue (2) Tatsunori Seki (3) Frederic A. Troy II (4)(§),   Yasuo Inoue (1)(§)

From the (1)Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Hongo-7, Tokyo 113, Japan, the (2)School of Pharmaceutical Sciences, Showa University, Hatanodai-1, Tokyo 142, Japan, the (3)Department of Anatomy, Juntendo University School of Medicine, Bunkyo-ku, Tokyo 113, Japan, and the (4)Department of Biological Chemistry, University of California School of Medicine, Davis, California 95616

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A rapid, sensitive, and facile method for screening and characterizing anti-polysialic acid (polySia) antibodies using lipid-conjugated oligo/polysialic acids (oligo/polySia) was developed, which is based on an enzyme-linked immunosorbent assay. Homooligo/polymers of alpha28-linked N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid, and 2-keto-3-deoxy-D-galacto-nononic acid (KDN) were conjugated with phosphatidylethanolamine dipalmitoyl (PE) by reductive amination to prepare neo-oligo/polysialoglycolipids (oligo/polySia-PE). Using this method, the anti-polySia equine antibody, H.46, bound to (8Neu5Acalpha2)-PE, where n = 9 or more residues, a result in confirmation of previous binding studies using radiolabeled oligo/polyNeu5Ac. The antigenic specificity and sensitivity of two monoclonal anti-poly/oligoNeu5Ac antibodies (mAb.12E3 and mAb.5A5) and one anti-oligoKDN antibody (mAb.kdn8kdn), were also determined. mAb.12E3 could detect as little as 25 pg/well of oligo/polyNeu5Ac-PE, while mAb.5A5 and polyclonal antibody H.46 required at least 0.4 ng/well of oligo/polyNeu5Ac-PE to be detected. mAb.kdn8kdn detected as little as 12 ng/well of oligoKDN-PE. Using a series of oligo/polySia-PE with defined degrees of polymerization (DP), the minimum chain length for immunoreactivity of the anti-polySia antibodies was determined to be: DP 5 for mAb.12E3; DP 3 for mAb.5A5; DP 2 for mAb.kdn8kdn; and DP 8 for H.46. Thus, mAb.12E3 and mAb.5A5 recognize shorter oligomers of Neu5Ac than H.46, a finding that is of practical value for identifying shorter oligoSia chains in glycoconjugates. Because mAb.12E3 and mAb.5A5 also recognize extended polySia chains, these antibodies cannot be used, however, to differentiate between short and long chains of polySia when both are expressed on the same molecule.


INTRODUCTION

The polysialic acid (polySia) (^1)glycotope represents a group of carbohydrate chains consisting of N-acetylneuraminic acid (Neu5Ac), N-glycolyneuraminic acid (Neu5Gc), and deaminoneuraminic acid (KDN; 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid) residues(1) . These sugar polymers have a remarkable structural diversity resulting from different modifications of the sialic acid (Sia) residues including different N-acyl substitutions at C-5 (Neu5Ac, Neu5Gc), deamination at C-5 (KDN), and the presence of O-acetyl or Olactyl substituents(1) . Our recent finding of a novel type of polyNeu5Gc structure consisting of (5-O-Neu5Gcalpha2) chains in a Sia-rich glycoprotein from the jelly coat of sea urchin eggs extends further the structural diversity of the polySia glycotope(2) . Homopolymeric structures of alpha28-linked Neu5Ac (polyNeu5Ac) chains are known to be expressed in certain fish egg polysialoglycoprotein (PSGP)(1) , on neural cell adhesion molecules (N-CAM) from a wide variety of animal species ranging from insects to humans(3, 4, 5) , on some postnatal tissues(6, 7) , and in a number of human tumors(8, 9, 10) . PolySia is also recognized as an oncodevelopmental antigen and is known to function as a regulator of cell adhesion and cell migration (11, 12, 13) . In neuropathogenic Escherichia coli K1 and Neisseria meningitidis serogroup B, the alpha28-polyNeu5Ac glycotope is a neurovirulent determinant(11) . A similar function has been proposed for the polySia glycotope that is expressed on several types of neuroinvasive human tumors, including malignant lymphoma(14) , acute myeloid leukemia(15) , and head and neck cancers(16) .

alpha28-Linked homooligomeric chains of KDN, oligoKDN, were first reported to occur in a KDN-rich glycoprotein (KDN-gp) of the vitelline envelope (17) and ovarian fluid (18) of rainbow trout. Because of the high affinities of these novel polyanions to bind Ca, we postulated that they may function to maintain calcium ion around eggs and developing embryos(19) . Recently, we developed a monoclonal antibody, mAb.kdn8kdn, that specifically recognizes the oligoKDN glycotope, although the chain length required for recognition remained unspecified. Using mAb.kdn8kdn, we discovered that KDN-containing glycoconjugates were expressed in rat tissues(20) , a finding that had not been previously described. The importance of this finding is that it demonstrates that oligoKDN structures are not restricted to fish egg glycoproteins but, in fact, are also expressed on mammalian cells.

As noted, the alpha28-linked polyNeu5Ac glycotope is a neurotropic factor in neuroinvasive E. coli K1 and N. meningitidis serogroup B, and many efforts have been made to develop poly- and monoclonal antibodies against this structure. Because of structural mimicry, the alpha28-linked polyNeu5Ac chains are poorly immunogenic in human and other animals(21, 22, 23, 24, 25) . Under special conditions, however, several anti-polySia have been developed, including (a) an equine polyclonal IgM, designated H.46(22, 26) , (b) a monoclonal antibody mAb.735(25) , (c) an IgM(27) , (d) a monoclonal antibody 2-2B (24, 28) , (e) monoclonal antibody mAb.12E3(13, 29) , and (f) monoclonal antibody mAb.5A5(30, 31) . H.46 (22, 26) and mAb.735 (25) are well characterized and have been shown to be highly specific for recognizing the oligo/polyNeu5Ac glycotope. These antibodies are not reactive with oligo/polyNeu5Gc, hybrid chains consisting of oligo/polyNeu5Ac and Neu5Gc residues or oligo/polyKDN chains(1) . The minimum chain length recognized by these two antibodies is about 8-10 Sia residues (DP 8-10) which is larger than the usual antibody binding sites(32, 33) . The longer polySia chain is required because H.46 and mAb.735 recognize an extended helical epitope consisting of about 10 Sia residues, the inner 6 of which comprise the helical domain(32, 33, 34, 35) .

Recently, we were successful in preparing another anti-polySia antibody, mAb.12E3, which has a high avidity for embryonic rat forebrain, but not adult forebrain(29) . This antibody appears to recognize the alpha28-linked polyNeu5Ac chains on N-CAM, because alpha28-linked sialyl oligomers (colominic acid) inhibited the immunoreaction on embryonic rat brain. The usefulness of this antibody in immunohistochemical studies in determining the expression and distribution of polysialylated N-CAM has already been verified(13, 29) , but the specificity and chain length recognized by this antibody has not been thoroughly studied.

A determination of the glycotope that is bound by these anti-polySia antibodies, particularly the minimum chain length that is recognized, is important because the length of the polyNeu5Ac chains on N-CAM changes markedly during development. The change in DP appears to be inversely correlated with the strength of the N-CAM-mediated homophilic binding of neural cells(36) . Binding and inhibition studies in solution, using radiolabeled free oligo/polyNeu5Ac, have been used to approximate the chain length specificity of H.46 and mAb.735(32, 33, 37) . These experiments usually require relatively large amounts of antibodies, free oligo/polySia chains, and can be time consuming and imprecise. Because of their hydrophilicity, oligo/polySia chains are difficult to non-covalently immobilize on plastic or membrane filters. Previous studies have shown that lipidation of free glycan chains can greatly facilitate their non-covalent immobilization on the solid surfaces(38, 39) . Immobilization of oligo/polySia chains on the solid surface was anticipated to permit us to immunodetect the oligosaccharide antigen rapidly and with high sensitivity.

In this study, we have developed a rapid, sensitive and facile method for detecting the oligo/polySia glycotope, using oligo/polySia chains that are conjugated to phosphatidylethanolamine dipalmitoyl. Using a series of lipidated oligoSia with defined DP has also allowed us to determine the minimum chain length required for binding of the two new anti-Neu5Ac monoclonal antibodies, mAb.12E3, mAb.5A5, and an anti-KDN antibody, mAb.kdn8kdn.


EXPERIMENTAL PROCEDURES

Materials

Colominic acid in sodium salt form, (8Neu5Acalpha2), was purchased from Sigma. Polysialoglycoproteins (PSGP) containing (8Neu5Gcalpha2)chains and KDN-glycoprotein (KDN-gp) having (8KDNalpha2)chains were prepared from unfertilized eggs and the ovarian fluid of rainbow trout, respectively, as described previously(1, 17) . Equine antiserum containing polyclonal IgM antibodies (H.46) (22, 26) specific to alpha28-linked polyNeu5Ac (32, 37) was kindly provided by Dr. J. B. Robbins (National Institutes of Health, Bethesda, MD). Monoclonal antibody 12E3 (IgM) was prepared as previously reported(29) . Monoclonal antibody mAb.5A5 (IgM) (30, 31) was generously provided by Dr. U. Rutishauser (Case Western Reserve University). Monoclonal antibody kdn8kdn (IgM) used was prepared as described previously(20) . Peroxidase-conjugated sheep anti-horse IgM was obtained from Bethyl Laboratories (Montgomery, TX). Peroxidase-conjugated goat anti-mouse (IgG + IgM) was purchased from American Qualex (La Mirada, CA).

Chemical Analyses

Sialic acids were quantitated by the thiobarbituric acid (40, 41) and the resorcinol methods(42) . KDN was quantitated by the thiobarbituric acid method(43) .

Preparation of Oligo/PolySia and a Series of (Sia) with Defined DPs

Colominic acid (10 mg as Sia) was partially hydrolyzed in 50 mM sodium acetate buffer (pH 5.5) at 37 °C for 10 h (44) to obtain oligoNeu5Ac with an average DP (<DP>) of 8 (DPs ranged from 2 to 15). For preparation of oligoNeu5Gc, rainbow trout PSGP (10 mg as Sia) was partially hydrolyzed with 50 mM sodium acetate buffer (pH 4.8) at 37 °C for 6 h(44) , and subjected to Sephacryl S-200 chromatography (1.2 104 cm, 0.1 M NaCl) to separate the oligoNeu5Gc released from the glycoprotein. The <DP> of oligoNeu5Gc was 6 (range 2-13). OligoKDN was obtained by partial hydrolysis of KDN-gp (5.0 mg as KDN) in 50 mM sodium acetate buffer (pH 4.8) at 37 °C for 30 h (44) followed by Sephacryl S-200 chromatography. The <DP> of oligoKDN was 3 (range 1-7). These three oligoSia fractions were desalted by passage through a Sephadex G-25 column (1.2 104 cm, eluted with 5% ethanol) and lyophilized before use.

For preparation of the series of (8Neu5Acalpha2) oligomers with defined DPs, about 2.5 mg of colominic acid was applied on a Mono-Q HR5/5 anion-exchange column (0.5 5.0 cm, Pharmacia, Sweden) and separated on an Irika high performance liquid chromatography system. Sodium chloride gradients were generated using a Sigma 870 system controller. The sample was loaded on the column and eluted with 5 mM Tris-HCl (pH 8.0), followed by NaCl gradient (0-320 mM) in 5 mM Tris-HCl (pH 8.0). The flow rate was 500 µl/min, and fractions were collected every minute. The elution pattern was monitored by the resorcinol method. Oligomers with DPs ranging from 1 to 16 thus obtained were desalted by Sephadex G-25 chromatography (1.7 140 cm, 5% ethanol) and lyophilized. For preparation of the series of (8KDNalpha2) oligomers, about 30 mg of oligoKDN, obtained from KDN-gp (100 mg as KDN) by the procedure described above, were applied to a DEAE-Toyopearl 650 M column (1.4 22 cm) and eluted with a linear gradient of NaCl (0.0-0.3 M) in 10 mM Tris-HCl (pH 8.0). The elution profile was monitored by the thiobarbituric acid method. Oligomers with DPs ranging from 1 to 7 were desalted as described above and lyophilized.

Preparation of Lipid-conjugated Oligo/Polysialic Acids

Lipidation was carried out essentially according to the method of Stoll et al.(38, 39) , which was based on reductive amination. Fractions of oligoNeu5Ac, oligoNeu5Gc, and oligoKDN (100 µg as Sia) in 10 µl of water were added separately to phosphatidylethanolamine dipalmitoyl (PE, 450 µg) dissolved in 90 µl of chloroform/methanol (1: 2, v/v). After incubation at 60 °C for 2 h, 100 µg of sodium cyanoborohydride in 10 µl of methanol was added and incubation continued at 60 °C for 16 h(38, 39) . A series of homooligomers of Neu5Ac and KDN (0.02 µmol, 0.06 µmol as Sia, respectively) were dissolved separately in 10 µl of water and lipidated in the same way. Each lipidated sialyloligomer was tested for purity by silica gel thin layer chromatography (TLC) on a polygram silG sheet (Macherey-Nagel, Germany). The TLC sheet was developed in 1-propanol/25% NH(4)OH/water = 6:1:2.5 (v/v) for 12 h, and the lipid conjugated sialyloligomers were visualized with the resorcinol reagent(1, 44) , after washing the sheet with water for 1 h to remove unconjugated oligo/polySia chains. PE-conjugated oligoSia was designated oligoSia-PE. The structure of (8Neu5Acalpha2)-PE, for example, is shown in Structure I.

ELISA Analysis

The solid-phase ELISA method was carried out as follows. Fifty µl of lipidated sialyloligomers in ethanol were added to each well of a 96-well ELISA plate (E-plate, Sumitomo bakelite, Japan) and dried at 37 °C. Nonspecific protein binding was blocked by incubating the plates with 1% gelatin in phosphate-buffered saline (PBS) at 37 °C for 1 h. The concentration of antibodies was adjusted with 1% BSA/PBS to 0.5 mg/ml for H.46 and 31 µg/ml for mAb.12E3. For mAb.5A5, the saturated ammonium sulfate precipitant was diluted to 1:300 with 1% BSA/PBS. For mAb.kdn8kdn, the culture supernatant (Dulbecco's modified essential medium) of the mAb.kdn8kdn-producing hybridoma cell line containing 10% fetal bovine serum (Filtron) was used. Fifty µl of the antibody solution was added to each well and incubated at 37 °C for 2 h. After rinsing the wells four times with PBS containing 0.05% Tween 20 (TPBS), the secondary peroxidase-conjugated antibody was added and incubated at 37 °C for 1 h. Secondary antibodies used were peroxidase-conjugated sheep anti-horse IgM diluted to 1:2500 (0.4 µg/ml) with 1% BSA/PBS for H.46, and peroxidase-conjugated goat anti-mouse (IgG + IgM) diluted to 1:500 (2.0 µg/ml) with 1% BSA/PBS for mAb.12E3, mAb.5A5 and mAb.kdn8kdn. After rinsing the wells as described above, the peroxidase substrate o-phenylenediamine was added and incubated at 37 °C for 30-60 min. The reaction was stopped by the addition of 2 N H(2)SO(4) and absorbance at 492 nm was measured on an MTP-120 microplate reader (Corona Electric, Japan).


RESULTS

Preparation and Characterization of Lipid (PE)-conjugated OligoSia Chains

Partial acid hydrolysis of colominic acid, rainbow trout PSGP, and KDN-gp released alpha28-linked homooligomers of Neu5Ac, Neu5Gc, and KDN, which were designated as oligo Neu5Ac, oligoNeu5Gc, and oligoKDN, respectively. These homooligomer fractions were conjugated with PE as described under ``Experimental Procedures,'' and the resulting PE-conjugated oligoSia, designated oligoSia-PE, were analyzed by TLC (Fig.1). The lipidated sialyl oligomers (lanes2, 4, and 6) migrated about twice as fast as the corresponding free sialyl oligomers (lanes1, 3, and 5). Both the free and PE-conjugated oligoSia exhibited a ladder-shape pattern. OligoNeu5Ac-PE, oligoNeu5Gc-PE, and oligoKDN-PE had essentially the same chain length distribution as the corresponding parent sialyl oligomer fractions, although the yields of the conjugation reactions were low, as estimated from the apparent recoveries of Sia in the lipidated fractions. When colominic acid was conjugated with PE, the overall yield was about 15% after the free colominic acid was extracted from the reaction mixture with cold water. No further purification of each oligoSia-PE was necessary because unconjugated free oligoSia chains were readily removed by simply washing the wells of the ELISA plate or the TLC sheet with water.


Figure 1: Thin layer chromatography of free and lipid-conjugated oligo/polySia. The partial acid hydrolysates of colominic acid (oligoNeu5Ac), rainbow trout PSGP (oligoNeu5Gc), and rainbow trout KDN-gp (oligoKDN) were run in lanes 1, 3, and 5, respectively. In lanes 2, 4, and 6 are shown the lipid-conjugated oligoSia, i.e. oligoNeu5Ac-PE, oligoNeu5Gc-PE, and oligoKDN-PE, respectively. The numbers represent the corresponding n in (Sia)-PE for each sample.



A series of homooligomers of Neu5Ac and KDN, prepared by Mono-Q and DEAE-Toyopearl 650 M anion-exchange chromatographies of both colominic acid and the partial acid hydrolysates of KDN-gp, were conjugated separately with PE. Each of the lipidated oligoSia were examined for purity by TLC. The major component of each (8Siaalpha2)-PE fraction contained n-1 intact Sia residues originally present in the parent (8Siaalpha 2)chain (see Structure I). The higher oligoSia fractions, however, contained a small amount of lower oligoSia chains (results not shown). These data indicate that some partial hydrolysis of the oligoSia chains occurred during conjugation, probably because of being held at 60 °C for 16 h(44) . The contamination of such lipid-linked sialyl oligomers with smaller DPs did not affect the semi-quantitative analysis by ELISA, as described below.

Use of Lipidated Oligo/PolySia for Determining the Immunospecificity of H.46

The equine H.46 antibody was already shown to react only with (Neu5Acalpha2) chains with n geq 8(1, 32, 37) . The immunoreactivity using lipidated oligo/polySia was examined by the ELISA analysis with H.46. As shown in Fig.2a, H.46 reacted only with oligoNeu5Ac-PE (bullet). H.46 did not react with oligoNeu5Ac () because the free oligomers were not immobilized on the ELISA plate. No reaction was observed with oligoNeu5Gc-PE (black square), oligoKDN-PE (up triangle, filled), or phosphatidylethanolamine dipalmitoyl (). Immunoreactivity with oligoNeu5Ac required H.46, and no significant background color development was observed with normal horse serum (Fig.2b). H.46 detected oligoNeu5Ac-PE in amounts as little as 0.38 ng/well (as Neu5Ac) by this ELISA method. To determine the DP required for reaction with H.46, (8Neu5Acalpha2)-PE (where n = 1-16) were coated separately on the wells of an ELISA plate and tested for immunoreactivity. As shown in Fig.3a, (Neu5Ac)(1)-PE to (Neu5Ac)(8)-PE showed no immunoreactivity. Oligomers longer than (Neu5Ac)(9)-PE were reactive. Different concentrations of H.46 did not cause any difference in immunospecificity. We conclude from these results that H.46 is highly specific and sensitive for detecting PE-conjugated (Neu5Ac) oligomers where n is 9 or greater. Thus, H.46 recognizes alpha28-linked oligo/polyNeu5Ac chains with 8 or more Neu5Ac residues. These data are consistent with previous results obtained by inhibition studies(32, 37) .


Figure 2: ELISA of polyclonal H.46 binding to lipidated oligoSia-PE. Each well was coated with oligoNeu5Ac (), oligoNeu5Ac-PE (bullet), oligoNeu5GcPE (black square), oligoKDN-PE (up triangle, filled), and PE () at 0.4 ng-47 ng/well as Sia, or 11 ng-1.4 µg/well as PE, and determined for H.46 binding activity as described under ``Experimental Procedures.'' a, H.46 polyclonal antibodies were used as primary antibody. b, horse normal serum was used as control. c, analyses of chain length specificity of H.46 as a function of DP of oligo/polySia using lipidated oligo/polySia by the ELISA analysis. The plastic plate was coated with a series of lipidated (Neu5Ac), n = 1-16 (black square, 75 pmol/well; , 38 pmol/well; shaded square, 19 pmol/well).




Figure 3: Determination of immunospecificity of mAb.12E3 by the ELISA procedure. a, the plastic wells were coated with various amounts of oligoNeu5Ac-PE (bullet), oligoNeu5Gc-PE (black square), oligoKDN-PE (up triangle, filled), and PE () (25 pg to 12 ng/well as Sia or 0.75 ng to 360 ng/well as PE). b, the plastic wells were coated with a set of lipidated (Neu5Ac), n = 1-16 (black square, 75 pmol/well; , 38 pmol/well; shaded square, 19 pmol/well).



Determination of the Immunospecificity of mAb.12E3 Using Lipidated Oligo/PolySia

mAb.12E3 was prepared from a hybridoma between mouse myeloma cells and splenocytes obtained from a mouse immunized with the forebrain of an 18-day-old Wister rat embryo (E18). mAb.12E3 showed immunohistochemical staining of frozen sections of E18 forebrain but not adult rat forebrain. Because the immunostaining of E18 rat brains was inhibited by oligoNeu5Ac (colominic acid), mAb.12E3 was presumed to be detecting the alpha28-linked polyNeu5Ac glycotope on the embryonic form of N-CAM(29) . The reactivity of this antibody toward different types of alpha28-linked polySia structures, e.g. polyNeu5Gc and polyKDN, and the dependence of the immunoreactivity upon the DP was not determined. Using lipidated oligo/polySia chains, the immunospecificity of mAb.12E3 was studied by the ELISA method. As shown in Fig.3a, mAb.12E3 reacted with oligo/polyNeu5Ac-PE (bullet), but not with oligo/polyNeu5Gc-PE (black square) nor oligoKDN-PE (up triangle, filled). This antibody could detect as little as 25 pg of oligoNeu5Ac-PE/well (as Neu5Ac). The chain length dependence study revealed that (8Neu5Acalpha2) with n = less than 5 did not bind to mAb.12E3 but that with n greater than 6 did bind (Fig.3b). (Neu5Ac)(6)-PE retains 5 Neu5Ac residues intact from the nonreducing terminus. Therefore, the minimum chain length required for mAb.12E3 binding is 5 Neu5Ac residues.

Determination of the Immunospecificity of mAb.5A5 Using Lipidated Oligo/PolySia

As shown in Fig.4a, mAb.5A5 reacted only with (8Neu5Acalpha2)-PE (bullet) and could detect amounts as low as 0.45 ng/well (as Neu5Ac). To determine the minimum number of Sia residues required for mAb.5A5 to bind to oligo/polyNeu5Ac, the ELISA wells were coated separately with (8Neu5Acalpha2)-PE (n = 1-10) and examined by the ELISA method. As shown in Fig.4b, this antibody required (Neu5Ac)(4)-PE, where trimeric Neu5Ac structure retains intact which needs for binding (see Structure I). Therefore, the minimum chain length required for mAb.5A5 to bind is a trimer.


Figure 4: Determination of immunospecificity of mAb.5A5 by the ELISA procedure. a, the plastic wells were coated with various amounts of oligoNeu5Ac-PE (bullet), oligoNeu5Gc-PE (black square), oligoKDN-PE (up triangle, filled), and PE () (0.45 ng to 190 ng/well as Sia or 14 ng to 5.7 µg/well as PE). b, the plastic wells were coated with a set of lipidated (Neu5Ac), n = 1-10 (black square, 75 pmol/well; , 38 pmol/well; shaded square, 19 pmol/well).



Determination of the Immunospecificity of mAb.kdn8kdn Using Lipidated Oligo/PolySia

mAb.kdn8kdn was obtained from the culture supernatant of a hybridoma cell line derived from immunizing a mouse with rainbow trout KDN-gp. The hybridoma produced an antibody that was reactive with KDN-gp but not with KDN-gp that had been deleted of KDN residues(20) . As shown in Fig.5a, mAb.kdn8kdn reacted only with oligoKDN-PE (up triangle, filled). No reaction was observed with oligoNeu5Ac-PE (bullet), oligoNeu5Gc-PE (black square), or phosphatidylethanolamine dipalmitoyl (). To determine the chain length dependence of mAb.kdn8kdn, (8KDNalpha2)-PE (n = 1-7) was coated separately on the wells of an ELISA plate and examined for immunoreactivity. As shown in Fig.5b, at least 2 residues were required because (KDN)(3)-PE was a minimum structure for binding. It is interesting to note that (KDN)-PE with n = 5, 6, and 7 were found to be less immunoreactive than the trimer. The efficiency of lipidation and immobilization of oligoKDN-PE was the same irrespective of the DP of oligoKDN. These results thus reveal that mAb.kdn8kdn is highly specific for detecting the (8KDNalpha2)(n) glycotope and that binding requires at least 2 KDN residues.


Figure 5: Determination of immunospecificity of mAb.kdn8kdn by the ELISA procedure. a, the plastic wells were coated with various amounts of oligoNeu5Ac-PE (bullet), oligoNeu5Gc-PE (black square), oligoKDN-PE (up triangle, filled), and PE (diao]) (6.0 ng to 750 ng/well as Sia or 180 ng to 23 µg/well as PE). b, the plastic wells were coated with a set of lipidated (KDN), n = 1-7 (black square, 28 pmol/well; , 14 pmol/well; shaded square, 7 pmol/well).




DISCUSSION

Because the polySia glycotope is often expressed in minute amounts and is spatiotemporally restricted in development, it is often difficult to detect by chemical methods(1) . Anti-polySia antibodies have been powerful reagents for detecting, localizing, and elucidating the functional involvement of different polySia structures. As noted, the polySia-containing molecules occur in diverse molecular forms that appear to be species, organ, and developmentally specifically expressed in a wide variety of human and other animal species(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 45, 46, 47, 48, 49) . Furthering our knowledge of the possible physiological roles of the polySia glycotopes involves discovering new polysialylated structures with possibly different functions in divergent species. One approach to this analysis is to develop anti-polySia monoclonal antibodies specific for recognizing the different polySia structures. Although the development of immunological probes with defined specificities was anticipated to be essential to further our understanding of the occurrence, structure, localization, and function of various polySia glycotopes, several difficulties have had to be overcome. First, polySia has long been known to be poorly immunogenic(21, 22, 23, 24, 25) . Second, many of the anti-polySia antibodies are IgM, and generally show weak immunoreactivity(22, 27, 50) . Third, the failure to immobilize polySia on plastic or silica gel TLC plates has prevented the development of a highly sensitive detection method.

In this study, we examined four anti-polySia antibodies to determine sensitivity and specificity for identifying the component nonulosonate in different oligo/polySia structures, and their chain length specificity, using as antigens a series of lipid-conjugated oligo/polySia of known structure and DP. Using these neoglycolipids, we have developed a highly sensitive method for identifying the different polySia structures and to determine the chain length dependence of the antibodies. Lipid-conjugated oligo/polySia samples with increasing DPs were found to migrate in a ladder-like fashion on TLC. In contrast to the free sialyl oligomers, the lipid-conjugated oligo/polySia remained bound to the solid surfaces after washing with water, thus allowing immunodetection to be carried out directly on ELISA or TLC plates. The lowest limit of detection of oligo/polySia-PE was determined to be 0.4 ng/well (as Neu5Ac) for H.46 and mAb.5A5, 25 pg/well (as Neu5Ac) for the monoclonal antibody 12E3, and 12 ng/well (as KDN) for mAb.kdn8kdn. Because the background color in the ELISA method was low, this procedure is ideal for screening and characterizing anti-polySia antibodies. A further advantage is that the method is applicable for the immunodetection of any mono-, oligo-, or polysaccharide that can be lipid conjugated with retention of the immunological epitope.

Only 7-75 pmol/well of lipidated antigens (as Sia) were required to determine the minimum chain length that is recognized by these antibodies. In contrast, much larger amounts of free oligo/polySia chains are required to carry out inhibition studies to generate the same information.

A problem of particular importance has been to determine the minimum DP that can be recognized by the monoclonal antibodies, mAb.12E3, mAb.5A5, and mAb.kdn8kdn. The antigen specificities of four different antibodies characterized in this study are summarized in Table1. Evidence showing that antibodies can bind alpha28-linked oligoSia chains containing 5 and 3 Neu5Ac residues, and 2 KDN residues, respectively, has been provided by the present study. In contrast, H.46 and mAb.735 required about 8 Neu5Ac residues for binding. The high specificity of these antibodies and their ability to bind to shorter oligoNeu5Ac chains make them potentially useful reagents detecting shorter oligoSia chains that may be expressed on some glycoproteins and glycolipids. mAb.12E3 and mAb.5A5 cannot be used, however, to distinguish between short and long chains of oligo/polySia when both are expressed on the same glycoconjugate (e.g. N-CAM). This follows because antibodies also detect full-length polySia chains. Unlike antibodies against oligo/polyNeu5Ac, only 2 KDN residues are required to be bound by mAb.kdn8kdn. Maximal binding was thus attained for (8KDNalpha2)(3)-PE and then decreased with more KDN residues.



An increasing number of reports on the use of specific antibodies to detect the alpha28-linked polyNeu5Ac glycotope in a variety of animal species have appeared(4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16) . However, the occurrence of other polySia structures such as polyNeu5Gc and polyKDN has not yet been described except for our original finding of oligo/polyNeu5Gc and KDN chains in salmonid fish egg polysialoglycoproteins and KDN-glycoproteins(1, 45, 48) . Thus, the importance of the present findings is that it describes the development of a specific, sensitive, and facile method to identify new polySia structures, and to determine the minimum DP in their structures that is recognized by the different anti-polySia antibodies. The use of this new set of monoclonal antibodies with different specificities not only increases our experimental approaches to discover new polysialylated structures, but it also allows us to study how their structural diversity may relate to the myriad of functions that the polySia glycotopes have been implicated in.


FOOTNOTES

*
This work was supported in part by Grants-in-Aid 04044055 for International Scientific Research Program: Joint Research from the Ministry of Education, Science, and Culture of Japan (to Y. I.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Fax: +81-3-5684-2394 (to K. K. and Y. I.); +1-916-752-3516 (to F. A. T.).

^1
The abbreviations used are: polySia, polysialic acid; Sia, sialic acid; Neu5Ac, N-acetylneuraminic acid; Neu5Gc, N-glycolylneuraminic acid; KDN, 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid or naturally occurring deaminoneuraminic acid; oligo/polySia, alpha28-linked oligo/polysialic acid; oligo/polyNeu5Ac, alpha28-linked homooligo/polymer of Neu5Ac or (8Neu5Acalpha2) or (Neu5Ac); oligo/polyNeu5Gc, alpha28-linked homooligo/polymer of Neu5Gc or (8Neu5Gcalpha2); oligo/polyKDN, alpha28-linked homooligo/polymer of KDN or (8KDNalpha2) or (KDN); PE, phosphatidylethanolamine dipalmitoyl; oligoSia-PE or (Sia)-PE, PE-conjugated oligoSia; DP, degree of polymerization; PSGP, polysialoglycoprotein isolated from salmonid fish eggs; KDN-gp, KDN-rich glycoprotein isolated from vitelline envelope and/or ovarian fluid of rainbow trout; N-CAM, neural cell adhesion molecule; H.46, equine polyclonal IgM antibodies specific for detecting alpha28-linked polyNeu5Ac; mAb.12E3 and mAb.5A5, monoclonal IgM antibodies specific for detecting alpha28-linked oligo/polyNeu5Ac; mAb.kdn8kdn, monoclonal IgM antibody specific for detecting alpha28-linked oligoKDN; NaBH(3)CN, sodium cyanoborohydride; TLC, thin layer chromatography; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline.


ACKNOWLEDGEMENTS

We are grateful to Dr. U. Rutishauser and Dr. J. B. Robbins for providing mAb.5A5 and H.46 antibody, respectively.


REFERENCES

  1. Sato, C., Kitajima, K., Tazawa, I., Inoue, Y., Inoue, S., and Troy, F. A. (1993) J. Biol. Chem. 268,23675-23684 [Abstract/Free Full Text]
  2. Kitazume, S., Kitajima, K., Inoue, S., Troy, F. A., Cho, J-W., Lennartz, W. J., and Inoue, Y. (1994) J. Biol. Chem. 269,22712-22718 [Abstract/Free Full Text]
  3. Finne, J. (1982) J. Biol. Chem. 257,11966-11970 [Abstract/Free Full Text]
  4. Vimr, E. R., McCoy, R. D., Vollger, H. F., Wilkison, N. C., and Troy, F. A. (1984) Proc. Natl. Acad. Sci. U. S. A. 81,1971-1975 [Abstract]
  5. Roth, J., Kempf, A., Reuter, G., Schauer, R., and Gehring, W. J. (1992) Science 256,673-675 [Medline] [Order article via Infotrieve]
  6. Roth, J., Taatjets, D. J., Bitter-Suermann, D., and Finne, J. (1987) Proc. Natl. Acad. Sci. U. S. A. 84,1969-1973 [Abstract]
  7. Finne, J., Bitter-Suermann, D., Goridis, C., and Finne, U. (1987) J. Immunol. 138,4402-4407 [Abstract/Free Full Text]
  8. Roth, J., Zuber, C., Wagner, P., Taatjes, D. J., Weisgerber, C., Heitz, P. U., Goridis, C., and Bitter-Suermann, D. (1988) Proc. Natl. Acad. Sci. U. S. A. 85,2999-3003 [Abstract]
  9. Livingston, B. D., Jacobs, J. L., Glick, M. C., and Troy, F. A. (1988) J. Biol. Chem. 263,9443-9448 [Abstract/Free Full Text]
  10. Roth, J., Zuber, C., Komminoth, P., Scheidegger, E. P., Warhol, M. J., Bitter-Suermann, D., and Heitz, P. U. (1993) in Polysialic Acid : From Microbes to Man (Roth, J., Rutishauser, U., and Troy, F. A., eds) pp. 335-348, Birkh ä user Verlag, Basel
  11. Troy, F. A. (1992) Glycobiology 2,5-23 [Medline] [Order article via Infotrieve]
  12. Rutishauser, U., Acheson, A., Hall, A. K., Mann, D. M., and Sunshine, J. (1988) Science 240,53-57 [Medline] [Order article via Infotrieve]
  13. Seki, T., and Arai, Y. (1993) Neurosci. Res. 17,265-290 [CrossRef][Medline] [Order article via Infotrieve]
  14. Grogan, T., Guptil, J., Mullen, J., Ye, J., Hanneman, E., Vela, L., Frutiger, Y., Miller, T., and Troy, F. A. (1994) Lab. Invest. 70,110A (Abstr. 637)
  15. Scott, A. A., Kopecky, K. J., Grogan, T. M., Head, D. R., Troy, F. A., Mullen, J., Ye, J., Appelbaum, F. R., Theil, K. S., and Willman, C. L. (1994) Lab. Invest. 70,120A (Abstr. 695)
  16. Gandour-Edwards, R., Deckard-Janatpour, K., Ye, J., Donald, P. J., and Troy, F. A. (1995) Mod. Path. 8,101A
  17. Kanamori, A., Inoue, S., Iwasaki, M., Kitajima, K., Kawai, G., Yokoyama, S., and Inoue, Y. (1990) J. Biol. Chem. 265,21811-21819 [Abstract/Free Full Text]
  18. Kanamori, A., Kitajima, K., Inoue, S., and Inoue, Y. (1989) Biochem. Biophys. Res. Commun. 164,744-749 [Medline] [Order article via Infotrieve]
  19. Shimoda, Y., Kitajima, K., Inoue, S., and Inoue, Y. (1994) Biochemistry 33,1202-1208 [Medline] [Order article via Infotrieve]
  20. Kanamori, A., Inoue, S., Xulei, Z., Zuber, C., Roth, J., Kitajima, K., Ye, J., Troy, F. A., and Inoue, Y. (1994) Histochemistry 101,333-340 [Medline] [Order article via Infotrieve]
  21. Wyle, F. A., Artenstein, M. S., Brandt, B. L., Tramont, D. L., Kasper, D. L., Altieri, P., Berman, S. L., and Lowenthal, J. P. (1972) J. Infect. Dis. 126,514-521 [Medline] [Order article via Infotrieve]
  22. Sarff, L. D., McCracken, G. H., Schiffer, M. S., Glode, M. P., Robbins, J. B., Prskov, I., and Prskov, F. (1975) Lancet i,1099-1104
  23. Jennings, H. J., and Lugowski, C. (1981) J. Immunol. 127,1011-1018 [Abstract/Free Full Text]
  24. Mandrell, R. E., and Zollinger, W. D. (1982) J. Immunol. 129,2172-2178 [Free Full Text]
  25. Frosch, M., Görgen, I., Boulnois, G. J., Timmis, K. N., and Bitter-Suermann, D. (1985) Proc. Natl. Acad. Sci. U. S. A. 82,1194-1198 [Abstract]
  26. Allen, P. Z., Glode, M., Schneerson, R., and Robbins, J. B. (1982) J. Clin. Microbiol. 15,324-329 [Medline] [Order article via Infotrieve]
  27. Kabat, E. A., Nickerson, K. G., Liao, J., Grossbard, L., Osserman, E. F., Glickman, E., Chess, L., Robbins, J. B., Schneerson, R., and Yang, Y. (1986) J. Exp. Med. 164,642-654 [Abstract]
  28. Rougon, G., Dubois, C., Buckley, N., Magnani, J. L., and Zollinger, W. (1986) J. Cell Biol. 103,2429-2437 [Abstract]
  29. Seki, T., and Arai, Y. (1991) Anat. Embryol. 184,395-401 [Medline] [Order article via Infotrieve]
  30. Dodd, J., Morton, S. B., Karagogeos, D., Yamamoto, M., and Jessel, T. M. (1988) Neuron 1,105-116 [Medline] [Order article via Infotrieve]
  31. Acheson, A., Sunshine, J. L., and Rutishauser, U. (1991) J. Cell Biol. 114,143-153 [Abstract]
  32. Jennings, H. J., Roy, R., and Michon, F. (1985) J. Immunol. 134,2651-2657 [Abstract/Free Full Text]
  33. Häyrinen, J., Bitter-Suerman, D., and Finne, J. (1989) Mol. Immun. 26,523-529
  34. Michon, F., Brisson, J. R., and Jennings, H. J. (1987) Biochemistry 26,8399-8405 [Medline] [Order article via Infotrieve]
  35. Brisson, J. R., Baumann, H., Imberty, A., Pérez, S., and Jennings, H. J. (1992) Biochemistry 31,4996-5004 [Medline] [Order article via Infotrieve]
  36. Yang, P., Major, D., and Rutishauser, U. (1994) J. Biol. Chem. 269,23039-23044 [Abstract/Free Full Text]
  37. Finne, J., and Mäkelä, P. H. (1985) J. Biol. Chem. 260,1265-1270 [Abstract/Free Full Text]
  38. Stoll, M. S., Mizuochi, T., Childs, A., and Feizi, T. (1988) Biochem. J. 256,661-664 [Medline] [Order article via Infotrieve]
  39. Feizi, T., Stoll, M. S., Yuen, C.-T., Chai, W., and Lawson, A. M. (1994) Methods Enzymol. 230,484-519 [Medline] [Order article via Infotrieve]
  40. Aminoff, D. (1961) Biochem. J. 81,384-392
  41. Uchida, Y., Tsukada, Y., and Sugimori, T. (1977) J. Biochem. (Tokyo) 82,1425-1433 [Abstract]
  42. Svennerholm, L. (1957) Biochim. Biophys. Acta 24,604-611 [CrossRef]
  43. Kitajima, K., Inoue, S., Kitazume, S., and Inoue, Y. (1992) Anal. Biochem. 205,244-250 [Medline] [Order article via Infotrieve]
  44. Kitazume, S., Kitajima, K., Inoue, S., and Inoue, Y. (1992) Anal. Biochem. 202,25-34 [Medline] [Order article via Infotrieve]
  45. Inoue, S., and Iwasaki, M. (1978) Biochem. Biophys. Res. Commun. 83,1018-1023 [Medline] [Order article via Infotrieve]
  46. James, W. M., and Agnew, W. S. (1987) Biochem. Biophys. Res. Commun. 148,817-826 [Medline] [Order article via Infotrieve]
  47. Zuber, C., Lackie, P. M., Catterall, W. A., and Roth, J. (1992) J. Biol. Chem. 267,9965-9971 [Abstract/Free Full Text]
  48. Inoue, Y. (1993) in Polysialic Acid: From Microbes to Man (Roth, J., Rutishauser, U., and Troy, F. A., eds) pp. 171-181, Birkh ä user Verlag, Basel
  49. Landmesser, L., Dahm, L., Tang, J., and Rutishauser, U. (1990) Neuron 4,655-667 [Medline] [Order article via Infotrieve]
  50. Devi, S. J. N., Robbins, J. B., and Schneerson, R. (1991) Proc. Natl. Acad. Sci. U. S. A. 88,7175-7179 [Abstract]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.