Specificity of human anti-NOR antibodies, a distinct species of "natural" anti-{alpha}-galactosyl antibodies

Maria Duk2, Ulrika Westerlind3, Thomas Norberg3, Galina Pazynina4, Nicolai N. Bovin4 and Elwira Lisowska1,2

2 Department of Immunochemistry, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Rudolf Weigl St. 12, 53-114 Wroclaw, Poland
3 Department of Chemistry, Swedish University of Agricultural Sciences, S-750 07 Uppsala, Sweden
4 Laboratory of Carbohydrate Chemistry, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, v-437 Moscow, Russian Federation

Received on August 26, 2002; revised on November 27, 2002; accepted on November 28, 2002


    Abstract
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Natural anti-NOR antibodies are common in human sera and agglutinate human erythrocytes of a rare NOR phenotype. The NOR phenotype-related antigens are unique neutral glycosphingolipids recognized by these antibodies and Griffonia simplicifolia IB4 isolectin (GSL-IB4). The oligosaccharide chains of NOR glycolipids are terminated by Gal{alpha}1-4GalNAcß1-3Gal{alpha} units. To characterize the specificity of anti-NOR antibodies and compare it with specificities of GSL-IB4 and known anti-Gal{alpha}1,3Gal antibodies, {alpha}-galactosylated saccharides and saccharide-polyacrylamide conjugates were used. New synthetic oligosaccharides, corresponding to the terminal di- and trisaccharide sequence of NOR glycolipids and the conjugate of the NOR-tri with HSA were included. These compounds were tested by microtiter plate ELISA and hemagglutination inhibition. Anti-NOR antibodies reacted most strongly with Gal{alpha}1-4GalNAcß1-3Gal (NOR-tri), and over 100 times less strongly with Gal{alpha}1-4GalNAc (NOR-di). The antibodies reacted also with Gal{alpha}1-4Gal and Gal{alpha}1-4Galß1-4GlcNAc, similarly as with NOR-di but not with other tested compounds. In turn, anti-Gal{alpha}1,3Gal antibodies reacted most strongly with Gal{alpha}1-3Gal and were very weakly inhibited by the NOR-related oligosaccharides (weaker than by galactose), and NOR-tri was less active than NOR-di. GSL-IB4 reacted with all tested {alpha}-galactosylated saccharides and conjugates, including the similarly active NOR-tri and NOR-di. These results showed that anti-NOR represent a new species of anti-{alpha}-galactosyl antibodies with high affinity for the Gal{alpha}1-4GalNAcß1-3Gal sequence present in rare NOR erythrocytes.

Key words: anti-{alpha}-galactosyl / anti-NOR / Griffonia simplicifolia IB4 lectin / oligosaccharides / polyagglutination


    Introduction
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
The polyagglutinable erythrocytes of NOR phenotype, identified so far in two families (Harris et al., 1982Go; Kusnierz-Alejska et al., 1999Go), contain two unusual neutral glycosphingolipids (designated NOR1 and NOR2) strongly reactive with Griffonia simplicifolia IB4 lectin (GSL-IB4; Kusnierz-Alejska et al., 1999Go). GSL-IB4 recognizes terminal {alpha}-galactose residues and reacts strongly with the Gal{alpha} 1-3Gal sequence (reviewed by Goldstein and Winter, 1999Go). The sequence Gal{alpha}1-3Galß1-4GlcNAc is abundant in mammalian glycoproteins and glycosphingolipids but is absent in humans. In turn, antibodies against this xenoglycotope (called anti-{alpha}Gal) are present in all human sera and are a major reason of acute rejection of xenotransplants (reviewed by Galili, 2001Go). Therefore, we initially suspected that the NOR phenotype may be a rare case of presence of the Gal{alpha}1-3Galß1-4GlcNAc sequence in humans and that the NOR erythrocytes were agglutinated by known anti-{alpha}Gal antibodies. However, determination of the structure of the GSL-IB4-reactive NOR1 glycolipid showed that it is a derivative of globoside that has galactose {alpha}1,4-linked to the terminal GalNAc residue (Duk et al., 2001Go).

Our recent (unpublished) studies indicated that the NOR2 glycolipid is the NOR1 elongated by Gal{alpha}1-4GalNAcß unit. We also found that affinity-purified anti-Gal{alpha}1,3Gal antibodies did not react with NOR erythrocytes or glycolipids, and human anti-NOR antibodies, which reacted with NOR1 and NOR2 glycolipids, did not react with rabbit glycolipid terminated with Gal{alpha}1-3Galß1-4GlcNAc- sequence (Duk et al., 2001Go). These results provide evidence that the identified NOR glycolipids are responsible for the polyagglutination and that the NOR-related glycotope and the xenoglycotope Gal{alpha}1-3Gal (both reactive with GSL-IB4) are recognized by different human antibodies.

In this article we present characterization of fine specificity of anti-NOR antibodies with the use of model oligosaccharides and oligosaccharide conjugates. We include in these studies the recently synthesized novel oligosaccharides corresponding to the terminal sequence of NOR1 and NOR2 glycolipids, Gal{alpha}1-4GalNAc (NOR-di) and Gal{alpha} 1-4GalNAcß1-3Gal (NOR-tri), which inhibited anti-NOR antibodies in a hemagglutination assay (Westerlind et al., 2002Go). Comparison of the reactivity of anti-NOR antibodies, anti-Gal{alpha}1,3Gal antibodies, and GSL-IB4 with the same panel of oligosaccharides and glycoconjugates showed that these three reagents have entirely different fine specificities.


    Results
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Isolation of human antibodies
Anti-NOR antibodies were isolated from human sera by adsorption on glutaraldehyde-fixed NOR erythrocytes, followed by elution with galactose. Anti-Gal{alpha}1,3Gal antibodies were purified by affinity chromatography on immobilized porcine erythrocyte glycophorin carrying multiple O-glycans terminated with Gal{alpha}1-3Gal sequence (reviewed by Krotkiewski, 1988Go). Both antibodies tested by Ouchterlony double diffusion showed the presence of IgG accompanied by small amount of IgM.

Interaction of human antibodies and GSL-IB4 with oligosaccharide conjugates
The NOR-tri–human serum albumin (HSA) conjugate and seven other oligosacharide-polyacrylamide (PAA) conjugates (listed in Table I) were used for coating the enzyme-linked immunosorbent assay (ELISA) plates and determination of binding of the antibodies and GSL-IB4 (Figure 1, Table I). Neither antibodies nor GSL-IB4 bound to GalNAc{alpha}1-4GlcNAc1ß-PAA, showing a strict requirement for terminal {alpha}-galactose residue in recognized oligosaccharide chains. Both antibodies and the lectin showed different patterns of binding to seven remaining conjugates terminated with {alpha}-galactose. Anti-NOR antibodies bound strongly to NOR-tri-HSA and moderately to Gal{alpha}1-4Galß1-4GlcNAcß-PAA (P1-tri-PAA), corresponding to the terminal sequence of blood group P1 antigen (Marcus et al., 1981Go). They did not bind or bound negligibly to PAA conjugates with Gal{alpha}1-3Galß, Gal{alpha}1-4GlcNAcß, Gal{alpha}1-3GalNAcß, Gal{alpha}1-6Glcß, and Gal{alpha}1-2Galß structures. The anti-Gal{alpha}1,3Gal antibodies bound most strongly to Gal{alpha}1-3Galß-PAA, less strongly to Gal{alpha}1-4GlcNAcß-PAA and Gal{alpha}1-6Glcß-PAA, and did not bind to the remaining conjugates. The cross-reactivity of anti-Gal{alpha}1, 3Gal antibodies with Gal{alpha}1-6Glc is known (Galili et al., 1984Go), but their binding to Gal{alpha}1-4GlcNAcß-PAA was not expected. GSL-IB4 bound to all {alpha}-galactosylated conjugates, but the binding to P1-tri-PAA and Gal{alpha}1-3GalNAcß- PAA was weaker than to the remaining conjugates.


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Table I. Summary of binding (%) of anti-NOR antibodies, anti-Gal{alpha}1,3Gal antibodies, and GSL-IB4 to oligosaccharide conjugates

 


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Fig. 1. Binding of anti-NOR antibodies, anti-Gal{alpha}1-3Gal antibodies, and GSL-IB4 to oligosaccharide conjugates. The ELISA plate was coated with the following conjugates: NOR-tri-HSA ({bullet}), P1-tri-PAA ({diamondsuit}), Gal{alpha}1-6Glcß-PAA ({square}), Gal{alpha}1-2Galß-PAA ({triangleup}), Gal{alpha}1-4GlcNAcß-PAA ({diamond}), Gal{alpha}1-3GalNAcß-PAA (+), Gal{alpha}1-3Galß-PAA ({circ}), and GalNAc{alpha}1-4GlcNAcß-PAA (x). Binding of serially diluted antibodies and GSL-IB4 was detected as described in Materials and methods.

 
Inhibition of human antibodies and GSL-IB4 by mono- and oligosaccharides
Specificity of anti-NOR antibodies was tested by inhibition of their binding to NOR-tri-HSA- and P1-tri-PAA-coated ELISA plates and by inhibition of agglutination of papain-treated NOR erythrocytes (Figure 2 and Table II). Inhibition of high-affinity binding of anti-NOR antibodies to NOR-tri-HSA required high concentrations of oligosaccharides. To make the inhibition assay more sensitive, the time of incubation of the antibody-oligosaccharide mixture (and antibody alone) on the NOR-tri-HSA-coated plate was shortened to 40 min, versus 2 h on the P1-tri-PAA-coated plate.



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Fig. 2. Inhibition of anti-NOR antibodies with mono- and oligosaccharides. Anti-NOR (without or with inhibitor) were bound for 40 min to ELISA plates coated with NOR-tri-HSA (A) or for 2 h to P1-tri-PAA (B). The following compounds were tested: Gal{alpha}1-3Gal ({bullet}), Gal{alpha}1-4GalNAc ({diamondsuit}), Gal{alpha}1-4Gal ({blacktriangleup}), Gal{alpha}1-4GalNAcß1-3Gal ({blacksquare}), Gal{alpha}1-4Galß1-4GlcNAc-(CH2)3-NH2 ({square}), and galactose ({circ}).

 

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Table II. Comparison of inhibition (mM) of anti-NOR antibodies, anti-Gal{alpha}1,3Gal antibodies, and GSL-IB4 with mono- and oligosaccharides

 


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Fig. 3. Inhibition of anti-Gal{alpha}1,3Gal antibodies (A) and GSL-IB4 (B) with mono- and oligosaccharides. Antibodies and GSL-IB4 were bound to porcine asialoglycophorin-coated plates. The following compounds were tested: Gal{alpha}1-3Gal ({bullet}), Gal{alpha}1-4GalNAc ({diamondsuit}), Gal{alpha}1-4Gal ({blacktriangleup}),Gal{alpha}1-4GalNAcß1-3Gal ({blacksquare}), galactose ({circ}), andN-acetygalactosamine ({triangleup}).

 
In all assays anti-NOR antibodies were inhibited most strongly by NOR-tri (Gal{alpha}1-4GalNAcß1-3Gal), which was 100–500 times stronger as an inhibitor (depending on the assay) than NOR-di (Gal{alpha}1-4GalNAc). These results indicated that a binding site of anti-NOR antibodies is extended to accommodate at least a trisaccharide structure. Anti-NOR antibodies were also inhibited by the Gal{alpha}1-4Gal and P1-tri, but this inhibition was much weaker than that by NOR-tri and comparable to the inhibition by NOR-di. Anti-NOR antibodies were weakly inhibited by galactose (five to eight times less strongly than by NOR-di) and were not inhibited by Gal{alpha}1-3Gal. Similar pattern of inhibition of antibodies binding to NOR-tri-HSA and P1-tri-PAA indicated that the binding to P1-tri-PAA occurred due to relatively weak cross-reactivity of anti-NOR antibodies with Gal{alpha}1-4Gal and not due to the presence of other anti-Gal{alpha}1,4Gal antibodies in the preparation studied.

Anti-Gal{alpha}1,3Gal antibodies were tested with the same panel of saccharides by inhibition of antibodies binding to porcine asialoglycophorin-coated ELISA plates and inhibition of agglutination of rabbit erythrocytes (Figure 3, Table II). As expected, in ELISA the anti-Gal{alpha}1,3Gal antibodies were inhibited most strongly by Gal{alpha}1-3Gal, and their inhibition by Gal{alpha}1-4Gal was over 100 times weaker and close to the inhibition by galactose. The NOR-related oligosaccharides were even weaker inhibitors than galactose; particularly, the NOR-tri was very weakly active. In the case of anti-NOR antibodies, the results of the ELISA and the hemagglutination inhibition were generally similar, but agglutination of rabbit erythrocytes by anti-Gal{alpha}1,3Gal antibodies was very weakly inhibited, as compared to the ELISA results. The agglutination was inhibited only by Gal{alpha}1-3Gal, at four times higher concentration by galactose, and was not inhibited by other oligosaccharides (Table II).

In contrast to antibodies, binding of GSL-IB4 to porcine asialoglycophorin and agglutination of human blood group B erythrocytes were inhibited by all oligosaccharides used and only slightly less strongly by galactose (Figure 3, Table II). The difference in activity of Gal{alpha}1-3Gal (the strongest inhibitor) and galactose was lower than 10-fold (Table II). GSL-IB4 binding was also weakly inhibited by N-acetylgalactosamine, at concentrations over 50 times higher than those required for inhibition by galactose.


    Discussion
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Human sera contain many "natural" anticarbohydrate antibodies that are most likely formed due to continous immunization by the gastrointestinal microbial flora. Among them, a distinct group are naturally occurring anti-{alpha}-galactosyl antibodies that display a variety of specificities. Most abundant and widely studied are anti-Gal{alpha}1,3Gal antibodies, which are also heterogeneous in terms of subspecificities. They cross-react with Gal{alpha}1-6Glc (mellibiose), which was initially used for their affinity purification (Galili et al., 1984Go). Later it was found that besides the cross-reacting antibodies, human sera also contain antibodies specific for one of these sequences only, Gal{alpha}1-3Gal or Gal{alpha}1-6Glc (Parker et al., 1999Go). Moreover, natural antibodies against Gal{alpha}1-2Gal and Gal{alpha}1-4Gal, not reactive with Gal{alpha}1-3Gal, were identified in human sera (Wieslander et al., 1990Go). This list is now extended by anti-NOR antibodies described in this article.

A distinct difference between specificities of anti-NOR and anti-Gal{alpha}1,3Gal antibodies was shown. The latter antibodies recognize the Gal{alpha}1-3Gal unit with high affinity, where the second Gal residue cannot be replaced by GalNAc, as indicated by the lack of binding to Gal{alpha}1-3GalNAcß-PAA (Figure 1). Inhibition of these antibodies by Gal{alpha}1-4Gal was comparable to inhibition by galactose and required more than 100 or 200 times higher concentration of these compounds, respectively, as compared to Gal{alpha}1-3Gal. The NOR-related oligosaccharides, especially NOR-tri, were even weaker inhibitors than galactose. In contrast, anti-NOR antibodies recognized only those glycotopes in which terminal galactose is {alpha}1,4-linked to N-acetylgalactosamine or galactose. Moreover, the combining site of anti-NOR antibodies must accomodate at least a trisaccharide epitope because the NOR-tri was over two orders more active than NOR-di. The third residue of NOR-tri (C3-substituted galactose) must play a specific role in the epitope because Gal{alpha}1-4Gal and NOR-di were similarly active, but addition of reducing GlcNAc residue to Gal{alpha}1-4Gal (to form P1-tri) did not improve its activity (Table II). Similarly, a trisaccharide epitope is probably recognized by anti-Gal{alpha}1,3Gal antibodies, but in this case the role of the third monosaccharide residue is less pronounced because these antibodies react only 5–10 times more strongly with Gal{alpha}1-3Galß1-4GlcNAc than with Gal{alpha}1-3Gal (Nethling et al., 1996Go; Parker et al., 1996Go).

In contrast to the antibodies, GSL-IB4 reacts with galactose and various saccharides terminated with an {alpha}-galactosyl residue (including the blood group B epitope) showing relatively low quantitative differences in reactivity and, in some cases, weaker binding of a trisaccharide than the respective disaccharide (Wood et al., 1979Go; Wu et al., 1995Go; Kirkeby and Moe, 2001Go). Our results showed that GSL-IB4 recognized the NOR-related oligosaccharides with high sensitivity and, in contrast to anti-NOR antibodies, reacted similarly with NOR-tri and NOR-di.

Recent crystallographic and molecular dynamics simulation studies on the complex of this lectin with Gal{alpha}1-3Gal showed that binding cavity of GSL-IB4 accomodates only terminal {alpha}-galactosyl residue and recognizes the lowest energy conformation of Gal{alpha}1-3Gal (Tempel et al., 2002Go). Therefore, the quantitative differences in binding various {alpha}-galactosyl oligosaccharides by GSL-IB4 most likely reflect the differences in availability of terminal {alpha}-galactose residue in these compounds and not a preference of the lectin for particular larger oligosaccharide structures.

In conclusion, the NOR glycolipids are recognized by GSL-IB4 due to its rather broad specificity for terminal {alpha}-galactose residues and by anti-NOR antibodies due to their high specificity for Gal{alpha}1-4GalNAcß1-3Gal, the terminal segment of the NOR glycolipids. These data are in perfect agreement with the recently elucidated structure of the NOR1 glycolipid (Duk et al., 2001Go). Oligosaccharides terminated with Gal{alpha}1-4GalNAc have not been identified so far in glycoconjugates of humans, except the rare NOR phenotype, but the Gal{alpha}1-4Gal sequence is present in blood group P system-related antigens, including P1 (Marcus et al., 1981Go). Inhibition of the NOR polyagglutination by avian P1 glycoprotein was the most characteristic feature of this phenotype (Harris et al., 1982Go) and helped us identify a new case of polyagglutination as NOR (Kusnierz-Alejska et al., 1999Go).

A cross-reactivity of anti-NOR antibodies with Gal{alpha}1-4Gal and P1-tri, described herein, confirmed an antigenic relationship between the NOR phenotype and the blood group P system. Anti-P1 isoantibodies are found in sera of P2 individuals lacking the P1 antigen and comprising only 21% of Caucasians (Reid and Lomas-Francis, 1997Go). NOR erythrocytes are agglutinated by ~70% of blood group compatible sera (most of them from P1 individuals), and anti-NOR antibodies are likely to be even more common because we isolated them also from the sera that did not agglutinate NOR erythrocytes, probably due to a lower level of anti-NOR antibodies. The results of the present article show that anti-NOR antibodies represent a distinct species of natural human anti-{alpha}-galactosyl antibodies, different from anti-Gal{alpha}1,3Gal antibodies. However, their relation to anti-Gal{alpha}1,4Gal antibodies, described by Wieslander et al. (1990)Go, and to anti-P1 antibodies remains to be established.


    Materials and methods
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 References
 
Oligosaccharides and oligosaccharide-HSA and -PAA conjugates
Two NOR-related oligosaccharides, NOR-di and NOR-tri, were obtained by chemical syntheses, as described by Westerlind et al. (2002)Go. The N-acryloyl derivative of NOR-tri was obtained (Kallin, 1994Go; Kallin et al., 1989Go) and used for copolymerization with HSA. For preparation of the conjugate, Michael addition conditions were used as described elsewhere (Romanowska et al., 1994Go). Analysis by matrix-assisted laser desorption ionization time-of-flight mass spectroscopy revealed an average 80,000 Da molecular mass of the conjugate, corresponding to an average substitution degree of 23 trisaccharides per HSA molecule. The P1 trisaccharide with spacer, P1-tri, and the oligosaccharide-PAA conjugates (of Mr around 30,000 Da and containing ~20% sugars) were synthesized by the previously described methods (Bovin, 1998Go). The disaccharides Gal{alpha}1-3Gal and Gal{alpha}1-4Gal were purchased from Glycorex (Lund, Sweden).

Porcine erythrocyte glycophorin
Glycophorin was obtained by the phenol extraction of porcine erythrocyte membranes, as described for isolation of human glycophorin; desialylation of porcine glycophorin was performed by mild acid hydrolysis (Lisowska et al., 1987Go).

Antibodies and lectin
Anti-NOR and anti-Gal{alpha}1,3Gal antibodies were isolated from normal human sera by adsorption on glutaraldehyde-fixed NOR erythrocytes or porcine erythrocyte glycophorin linked to Sepharose 4B, respectively, and by elution of adsorbed antibodies with 0.5 M galactose, as described by Duk et al. (2001)Go. The fixed NOR erythrocytes could be reused many times. Although the untreated porcine glycophorin binds the anti-Gal{alpha}1,3Gal antibodies less strongly than asialoglycophorin, it was used for antibody purification to avoid binding of serum anti-T antibodies to the asialoglycophorin. GSL-IB4 was purchased from Sigma (St. Louis, MO) and was biotinylated with biotinamidocaproate-N-hydroxysuccinimide ester (Sigma), as described previously (Duk et al., 1994Go).

Hemagglutination and hemagglutination inhibition
The assay was performed in U-shaped microtiter plates at room temperature. The anti-NOR antibodies, anti-Gal{alpha}1,3Gal antibodies, and GSL-IB4 were tested using papain-treated NOR erythrocytes, untreated rabbit erythrocytes, and human blood group B erythrocytes, respectively. The diluting solution for antibodies and their inhibitors was 0.01 M phosphate buffered saline (PBS) of pH 7.4, containing 0.15 M NaCl and 0.5% bovine serum albumin (BSA). For tests with GSL-IB4, PBS/BSA of pH 6.9 containing 1 mM Mg2+ and 1 mM Mn2+ was used. Serially diluted 30-µl samples of the antibodies or GSL-IB4 were mixed in wells with 30 µl of 2% erythrocyte suspension in 0.15 M NaCl, and agglutination was read after 30 min.

For inhibition assay, antibody or lectin solutions were diluted to agglutination titer 4–8, and 15-µl samples of the diluted solutions were mixed with 15-µl samples of serially diluted inhibitors. After 30 min, the agglutination assay was performed.

Antibody and lectin binding assays (ELISA)
All reagents were used in a volume of 50 µl per well; if not stated otherwise, incubations were performed at room temperature. Each binding was measured at least in duplicate; the differences did not exceed 5%. In all experiments the binding to wells coated with the buffer only served as a control for unspecific binding which was deducted from the values obtained. The conditions of the assays were experimentally selected to ensure an optimal specific binding and minimal unspecific binding of the reagents. For this reason some details of the assays were different for antibodies and the lectin.

Coating the plates
The plates (Nunc, MaxiSorp, Reskilde, Denmark) were coated with oligosaccharide conjugates in 0.05 M carbonate buffer of pH 9.6 for 2 h at 37°C and overnight at 4°C at concentration of 1 µg/well for binding the antibodies and 0.2 µg/well for binding the GSL-IB4. Coating with porcine asialoglycophorin was done at 1 µg/well, overnight at 4°C.

Binding of anti-NOR antibodies to saccharide-PAA and -HSA conjugates
The coated plates were washed several times with 0.01 M Tris–HCl buffer of pH 7.4/0.15 M NaCl (Tris-buffered saline, TBS), containing 0.05% Tween 20 and 1% HSA. The antibody was serially diluted with the same buffer. Dilutions of other reagents and washing the plates between the incubations were done with the buffer containing 0.1% HSA. The coated plates were consecutively incubated with (1) serially diluted anti-NOR antibody for 2 h; (2) biotinylated goat antibodies against human Ig (Sigma; diluted 1:1200) for 1 h; (3) ExtrAvidin-alkaline phosphatase conjugate (Sigma; diluted 1:10,000) for 1 h; (4) substrate, p-nitrophenyl phosphate (Signa 104 Phosphatase Substrate Tablets, 5 mg, dissolved in 5 ml of 0.05 M carbonate buffer of pH 9.8) until color developed, usually 30–60 min. The absorbance was read at 405 nm in an ELISA reader.

Binding of anti-Gal{alpha}1,3Gal antibodies
The buffer used for diluting the reagents and washing the plates was TBS of pH 7.4 containing 0.5% casein (Sigma) and 0.02% thimerosal (Kenna et al., 1985Go). Plates, coated with saccharide-PAA or -HSA conjugates or porcine asialoglycophorin, were incubated with (1) serially diluted anti-Gal{alpha}1,3Gal antibody for 1 h; (2) conjugate of goat anti-human Ig antibodies with alkaline phosphatase (Sigma; diluted 1:4000) for 1 h; (3) a phophatase substrate. Other details were the same as described for anti-NOR antibodies.

Binding of GSL-IB4
The TBS of pH 6.9, containing 1 mM Mg2+, 1 mM Mn2+, 0.02% Tween 20, and 0.1% BSA, was used for diluting the reagents and washing the plates. The binding procedure was based on the described method of Duk et al. (1994)Go. Briefly, the plates coated with saccharide conjugates or porcine asialoglycophorin were incubated with serially diluted biotinylated GSL-IB4 for 1 h, followed by incubations with ExtrAvidin-alkaline phosphatase conjugate (Sigma; diluted 1:10,000) for 1 h and with the phosphatase substrate.

Inhibition of binding
A binding of anti-NOR antibodies to NOR-tri-HSA and P1-tri-PAA, and of anti-Gal{alpha}1,3Gal antibodies and GSL-IB4 to porcine asialoglycophorin were used for inhibition assays. Antibody or lectin samples at constant dilution were mixed with an equal volume of serially diluted inhibitor (or respective buffer) and incubated for 1 h. These samples were applied on the coated plates; further steps were as in the binding assays. An exception was the inhibition of anti-NOR antibodies binding to NOR-tri-HSA, for which a 40-min incubation of antibody/inhibitor samples on the NOR-tri-HSA-coated plates was used. Dilutions of antibodies and lectin were selected from the binding curves to give absorbances of control samples (diluted twice with buffer) at the range of 0.5–1.0. The results are presented as percent of inhibition of the control binding.


    Acknowledgements
 
This work was supported by the grant 4PO5A 124 18 (to M.D. and E.L.) of the State Committee for Scientific Research (KBN), Warsaw.

1 To whom correspondence should be addressed; e-mail: lisowska{at}iitd.pan.wroc.pl Back


    Abbreviations
 
BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; GSL-IB4, Griffonia simplicifolia IB4 isolectin; HSA, human serum albumin; PAA, polyacrylamide; PBS, phosphate buffered saline; TBS, Tris-buffered saline.


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Bovin, N.V. (1998) Polyacrylamide-based glycoconjugates as tools in glycobiology. Glycoconj. J., 15, 431–446.[CrossRef][ISI][Medline]

Duk, M., Lisowska, E., Wu, J.H., and Wu, A.M. (1994) The biotin/ avidin-mediated microtiter plate lectin assay with the use of chemically modified glycoprotein ligand. Anal. Biochem., 221, 266–272.[CrossRef][ISI][Medline]

Duk, M., Reinhold, B.B., Reinhold, V.N., Kusnierz-Alejska, G., and Lisowska, E. (2001) Structure of a neutral glycosphingolipid recognized by human antibodies in polyagglutinable erythrocytes of the rare NOR phenotype. J. Biol. Chem., 276, 40574–40582.[Abstract/Free Full Text]

Galili, U. (2001) The {alpha}-gal epitope (Gal{alpha}1-3Galß1-4GlcNAc-R) in xenotransplantation. Biochimie, 83, 557–563.[CrossRef][ISI][Medline]

Galili, U., Rachmilewitz, E.A., Peleg, A., and Flechner, I. (1984) A unique natural human IgG antibody with anti-{alpha}-galactosyl specificity. J. Exp. Med., 160, 1519–1531.[Abstract]

Goldstein, I.J. and Winter, H.G. (1999) The Griffonia simplicifolia I-B4 isolectin. A probe for {alpha}-D-galactosyl end groups. Subcell. Biochem., 32, 127–140.[Medline]

Harris, P.A., Roman, G.K., Moulds, J.J., Bird, G.W.G., and Shah, N.G. (1982) An inherited RBC characteristic, NOR, resulting in erythrocyte polyagglutination. Vox Sang., 42, 134–140.[ISI][Medline]

Kallin, E. (1994) Use of glycosylamines in preparation of oligosaccharide polyacrylamide copolymers. Methods Enzymol., 242(A), 221–226.[ISI][Medline]

Kallin, E., Lönn, H., Norberg, T., and Elofsson, M. (1989) Derivatization procedures for reducing oligosaccharides. 3. Preparation of oligosaccharide glycosylamines and their conversion into oligosaccharide-acrylamide copolymers. J. Carbohydr. Chem., 8, 597–611.[ISI]

Kenna, J.G., Major, G.N., and Williams, R.S. (1985) Methods for reducing non-specific antibody binding in enzyme-linked immunosorbent assays. J. Immunol. Methods, 85, 409–419.[CrossRef][ISI][Medline]

Kirkeby, S. and Moe, D. (2001) Binding of Griffonia simplicifolia 1 isolectin B4 to {alpha}-galactose antigens. Immunol. Cell Biol., 79, 121–127.[CrossRef][ISI][Medline]

Krotkiewski, H. (1988) The structure of glycophorins of animal erythrocytes. Glycoconj. J., 5, 35–48.[ISI]

Kusnierz-Alejska, G., Duk, M., Storry, J.R., Reid, M.E., Wiecek, B., Seyfried, H., and Lisowska, E. (1999) NOR polyagglutination and Sta glycophorin in one family. Relation of NOR polyagglutination to terminal {alpha}-galactose residues and abnormal glycolipids. Transfusion, 39, 32–38.[CrossRef][ISI][Medline]

Lisowska, E., Messeter, L., Duk, M., Czerwinski, M., and Lundblad, A. (1987) A monoclonal anti-glycophorin A antibody recognizing the blood group M determinant: studies on the subspecificity. Mol. Immunol., 24, 605–613.[CrossRef][ISI][Medline]

Marcus, D.M., Kundu, S.K., and Suzuki, A. (1981) The P blood group system: recent progress in immunochemistry and genetics. Semin. Hematol., 18, 63–71.[ISI][Medline]

Nethling, F.A., Joziasse, D., Bovin, N., Cooper, D.K., and Oriol, R. (1996) The reducing end of {alpha}-Gal oligosaccharides contributes to their efficiency in blocking natural antibodies of human and baboon sera. Transpl. Int., 9, 98–101.[ISI][Medline]

Parker, W., Lateef, J., Everett, M.L., and Platt, J.L. (1996) Specificity of xenoreactive anti-Gal{alpha}1-3Gal IgM for {alpha}-galacosyl ligands. Glycobiology, 6, 499–506.[Abstract]

Parker, W., Lin, S.S., Yu, P.B., Sood, A., Nakamura Y.C., Song, A., Everett, M.L., and Platt, J.L. (1999) Naturally occurring anti-{alpha}-glactosyl antibodies: relationship to xenoreactive anti-{alpha}-galactosyl antibodies. Glycobiology, 9, 865–873.[Abstract/Free Full Text]

Reid, M.E. and Lomas-Francis, C. (1997) The blood group antigens. Facts book. San Diego: Academic Press.

Romanowska, A., Meunier, S.J., Tropper, F.D., Lafferriere, C.A., and Roy, R. (1994) Michael additions for syntheses of neoglycoproteins. Methods Enzymol., 242(A), 90–101.[ISI][Medline]

Tempel, W., Tschampel, S., and Wood, R.J. (2002) The xenograft antigen bound to Griffonia simplicifolia lectin 1-B4. X-ray crystal structure of the complex and molecular dynamics characterization of the binding site. J. Biol. Chem., 277, 6615–6621.[Abstract/Free Full Text]

Westerlind, U., Hagback, P., Duk, M., and Norberg, T. (2002) Synthesis and inhibitory activity of a di- and a trisaccharide corresponding to an erythrocyte glycolipid responsible for the NOR polyagglutination. Carbohydr. Res., 337, 1517–1522.[CrossRef][ISI][Medline]

Wieslander, J., Mansson, O., Kallin, E., Gabrielli, A., Nowack, H., and Timpl, R. (1990) Specificity of human antibodies against Gal{alpha}1-3Gal carbohydrate epitope and distinction from natural antibodies reacting with Gal{alpha}1-2Gal or Gal{alpha}1-4Gal. Glycoconj. J., 7, 85–100.[ISI]

Wood, C., Kabat, E.A., Murphy, L.A., and Goldstein, I.J. (1979) Immunochemical studies of the combining site of the two isolectins, A4 and B4, isolated from Bandeiraea simplicifolia. Arch. Biochem. Biophys., 198, 1–11.[ISI][Medline]

Wu, A.M., Song, S.-C., Wu, J.H., and Kabat, E.A. (1995) Affinity of Bandeiraea (Griffonia) simplicifolia lectin-I, isolectin B4 for Gal{alpha}1-4Gal ligand. Biochem. Biophys. Res. Commun., 216, 814–820.[CrossRef][ISI][Medline]