2 Department of Applied Biological Chemistry, Shizuoka University, Ohya 836, Shizuoka 422-8529, Japan
3 Department of Biochemistry, University of Shizuoka, School of Pharmaceutical Science, 52-1 Yada, Shizuoka 422-8526, Japan
4 Department of Molecular Design and Engineering, Graduate School of Engineering, Nagoya University, Chikusa-ku, Nagoya 464-8603, Japan
5 Department of Applied Molecular Biology, Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Oiwake-cho, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan
Received on June 10, 2002; revised on November 18, 2002; accepted on November 23, 2002
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Abstract |
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Key words: glycopolymers / influenza virus / inhibition / poly(L-glutamic acid) / sialyloligosaccharides
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Introduction |
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Various synthetic glycopolymers carrying multivalent Sia residues that target viral HAs have been prepared as influenza virus inhibitors using polyacrylamide (PA) (Gamian et al., 1991; Spaltenstein and Whitesides, 1991
; Lees et al., 1994
; Itoh et al., 1995
; Sigal et al., 1996
; Furuike et al., 2000
; Wu et al., 2000
), poly(acrylic acid) (PAA) (Mammen et al., 1995
; Choi et al., 1997
), and polystyrene (Tsuchida et al., 1998
) as polymer backbones. These conventional glycopolymers inhibit the binding of influenza viruses to host cell receptors with high affinity (Ki < 10-5 M of Sia residue in solution). Monomeric Sia derivatives have shown inhibition with relatively lower affinity (Ki = 10-2
10-5 M) (Sauter et al., 1989
; Toogood et al., 1991
). Generally, synthetic glycopolymers could have several problems for in vivo use, such as low solubility, significant cytotoxicity (Reuter et al., 1999
), and immunogenecity (Iurovskii et al., 1986
; Tanaka et al., 2002
). On the other hand, sialoglycoproteins (e.g., mucins) containing a number of Sia residues have shown inhibitory activities toward influenza virus infection comparable to those of conventional glycopolymers (Boat et al., 1978
; Burness and Pardoe, 1983
; Rogers et al., 1983
; Hanaoka et al., 1989
; Pritchett and Paulson, 1989
; Ryan-Poirier and Kawaoka, 1991
; Suzuki et al., 1994
).
2,6-Sialyl-N-acetyllactosamine, a typical carbohydrate determinant, is expressed on these natural sialoglycoproteins. We planned to synthesize glycopolymers that mimic these sialoglycoproteins. In these glycopolymers, the sialyl sugar units were clustered in a pendant manner on a simple polypeptide backbone poly(L-glutamic acid) (PGA).
In the present study, enzymatically synthesized p-nitrophenyl ( pNP) disaccharides were introduced to various lengths of PGAs and subsequently sialylated to highly water-soluble glycopolymers carrying clustered identical sialyldisaccharide segments. Using these glycopolymers, the inhibition of infection by human influenza viruses was investigated by measurement of the degree of the cytopathic effect in virus-infected Madine-Darby canine kidney (MDCK) cells. The physiological merits of PGA as a backbone of glycopolymers in terms of cytotoxicity and immunogenicity are discussed in this article.
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Results |
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Discussion |
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Among the glycopolymers tested for viral neutralization assays, 3c carrying multivalent Neu5Ac2-6Galß1-4GlcNAcß- units most significantly inhibited (IC50 3 µg/ml) the infection by A/Memphis/1/71. This glycopolymer also inhibited (IC50 30 µg/ml) infection by B/Lee/40. Therefore, the
2,6-sialoglycopolymer with a high-molecular-weight PGA backbone is possibly an effective inhibitor of infection by both influenza A and B viruses. To date, infections by influenza viruses have been successfully inhibited by Sia-conjugated PA (Gamian et al., 1991
; Spaltenstein and Whitesides, 1991
; Sparks et al., 1993
; Lees et al., 1994
; Itoh et al., 1995
; Sigal et al., 1996
; Wu et al., 2000
), PAA (Mammen et al., 1995
; Choi et al., 1997
), dendrimers (Reuter et al., 1999
), sialylphosphatidylethanolamine derivatives (Guo et al., 1998
), sialyl lactose-conjugated polystyrene (Tsuchida et al., 1998
), lyso-GM3-conjugated PGA (Kamitakahara et al., 1998
), GM3 lactone (Sato et al., 1999
), and sialyl LacNAc-conjugated PA (Furuike et al., 2000
). Among these inhibitors, IC50 of glycopolymer 3c used in the present study is almost equal to that of polymers synthesized by Tuchida et al. (1998) or Furuike et al. (2000)
(Table III).
The relationship between viral inhibitory activities and core sugar chain structure in glycopolymers is not clear. In our study, the contribution of asialo-portions in sugar chains was examined. Infection by A/PR/8/34 was inhibited equally by 2,3-sialoPGAs 2c, 5f, and 7 (IC50
20 µg/ml), suggesting that there is no contribution by the asialo-portion to inhibitory activity of the glycopolymers. Infection by A/Memphis/1/71 was inhibited by 2c carrying Neu5Ac
2-3Galß1-4GlcNAcß- but not by 5f with Neu5Ac
2-3Galß1-3GalNAc
- or 7 with Neu5Ac
2-3Galß1-3GalNAcß- units (Figure 3D and E), indicating that the asialo-portion (LacNAc) in sugar chain is important for the binding of A/Memphis/1/71 (H3N2) to terminal Neu5Ac residues.
- or ß-GalNAc residues in 5f and 7 may present unfavorable steric interactions to the virus HAs to decrease their affinity to the terminal Neu5Ac residues. Paulson and colleagues have demonstrated that the binding of influenza virus to Sia residues is influenced by the asialo-portion of the carbohydrate structure based on the inhibition of adsorption of A/Memphis/102/72 (H3) to erythrocytes using natural and synthetic monovalent sialosides (Rogers and Paulson, 1983
; Pritchett et al., 1987
; Matrosovich et al., 1993
). B/Gifu/2/73 is known to have binding characteristics that are different to those of other influenza B viruses (Xu et al., 1994
). The fact that the inhibitory activities of 5f and 7 were less than that of 2c indicates that the asialo-portion in sugar chains also affected the binding of B/Gifu/2/73 (Figure 4C).
PA-based glycopolymers (homopolymers) gave somewhat different results compared to those obtained using PGA-based glycopolymers. Glycopolymer 9 with a PA backbone showed no inhibitory activity toward A/Memphis/1/71 infection (Figure 3F), whereas 2c with a PGA backbone inhibited the infection by this strain, as previously mentioned. These findings suggest that the backbone of a glycopolymer (PA or PGA) affects virus inhibitory activity. Roy and colleagues found that the type of carrier molecules of Sia in neoglycoconjugates also affected the inhibitory activity toward an influenza virus (Gamian et al., 1991). A similar effect by carriers of sugar chains was reported by Kojima et al. (2002)
in adhesion of Helicobactor pylori to neoglycoconjugates carrying Leb saccharides. Our data on virus inhibition by 2c with a PGA backbone are in accordance with the results using a copolymer with a PA backbone (Furuike et al., 2000
). The density of sugar chains in glycopolymers, which is higher in homopolymers and lower in copolymers, may affect the inhibitory activities of glycopolymers toward viruses.
Whitesides and colleagues reported that the crucial factors for influenza virus inhibition by conventional Sia-conjugated glycopolymers are the cluster effect of Sia, molecular weights, charges, bulky and hydrophobic groups, and steric stabilization of the glycopolymer (Spaltenstein and Whitesides, 1991; Sparks et al., 1993
; Lees et al., 1994
; Mammen et al., 1995
; Sigal et al., 1996
). The effect of molecular weight (length) of backbone was also found to be critical in our glycopolymers. Glycopolymers 2c and 3c were roughly 10-fold more effective than were 2b and 3b in viral inhibition (Figure 3). As shown in Figure 5, the inhibitory activity toward virus infections increased with increases in molecular weights (or DPs) of PGAs and Sia contents (%) in glycopolymers. This indicates that much stronger inhibition can be expected if a longer PGA is used in a sufficient Sia content.
Generally, conventional glycopolymers have a problem of toxicity caused by a backbone such as PA (Ikeda et al., 1994; Reuter et al., 1999
). In our study, acrylamide monomer, which is known to be a potent neurotoxin (Spencer and Schaumburg, 1975
), and PAA-Na were cytotoxic to MDCK cells in the conditions used for the tests (Figure 6). These are materials for the synthesis of conventional glycopolymers (Lees et al., 1994
; Choi et al., 1997
), and this would limit its utility in vivo. It has also been shown that sugarbovine serum albumin(BSA) or PA conjugates act as antigens or immunogens. Sharp elevations (e.g., by 10,000-fold) of the titers in sera were observed after immunization of mice with a synthetic Galß1-3GalNAc
-BSA (Rittenhouse-Diakun et al., 1998
), PA-carrying Lea saccharides (Iurovskii et al., 1986
), or PA-carrying sulfated saccharides (Tanaka et al., 2002
). We tested this point with our PGA-based glycopolymer. When BALB/c mice were immunized with a PGA-carrying Galß1-3GalNAc
-(4b), no visible elevation of the titer of the antibody in sera reacting to the glycopolymer was observed (Figure 7). This might be because PGA has a relatively low immunogenicity (Hoes et al., 1993
). It should be emphasized that our PGA-based glycopolymers have extremely high solubility in water, more than 10% (w/v), compared to that of PA-based glycopolymers (less than 1%) and that they are remarkably heat-stable with no aggregation even in boiling water. Therefore, PGA-based glycopolymers have ideal characteristics as in vivo polymeric inhibitors.
Recently, noncytotoxic Sia-conjugated dendritic polymers have been successfully synthesized (Reuter et al., 1999). However, there have only been a few studies on in vivo inhibition of influenza virus infection by synthetic glycopolymers (Ikeda et al., 1994
; Gambaryan et al., 2002
). PGA has been extensively studied as a drug protectant in a drug delivery system (Jackman et al., 1993
; Lescure et al., 1995
; Akamatsu et al., 1997
, 1999
; Li et al., 1998
; Zou et al., 2001
) or as a biocompatible medical material (Honde et al., 1994
; Lescure et al., 1995
; Otani et al., 1996
). In vivo application of PGA-based glycopolymers is expected in the near future.
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Materials and methods |
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Analytical methods
High-performance liquid chromatography was done using a Jasco Gulliver system (Jasco Corp., Tokyo, Japan). 1H- and 13C-NMR spectra were recorded on a JEOL JNM-LA 500 spectrometer or a JEOL JNM-EX 270 spectrometer. Chemical shifts were expressed in relative to sodium 3- (tri-methylsilyl)-propionate as an external standard.
Synthesis of glycopolymers with PGA backbones
Glycopolymers 1a1c [Poly(Galß1-4GlcNAcß-pAP/Gln- co-Glu)], 4af [Poly(Galß1-3GalNAc-pAP/Gln-co-Glu)], and 6 [Poly(Galß1-3GalNAcß-pAP/Gln-co-Glu)] were prepared by our previously reported methods (Zeng et al., 1998
, 2000
). The compositions of pAP disaccarides, PGAs, and reagents used for the coupling reactions are summarized in Table I. The DS in the mole fraction of substituted residues in glycopolymers as a percentage was calculated from the relative intensities of 1H-NMR signal areas of phenolic protons, H-1 proton, peptide
- and ß-methylene protons, and acetyl protons. Glycopolymers 5af [Poly(Neu5Ac
2-3Galß1-3GalNAc
-pNP/Gln-co-Glu)] and 7 [Poly(Neu5Ac
2-3Galß1-3GalNAcß-pNP/Gln-co-Glu)] were prepared by
2,3-(O)-sialyltransferase from 4af and 6, respectively, as previously described (Zeng et al., 2000
).
Glycopolymers 2a2c [Poly(Neu5Ac2-3Galß1-4Glc-NAcß-pAP/Gln-co-Glu)] were enzymatically synthesized from 1a1c as follows. A mixture containing 5 mg of 1a, 1b, or 1c, 7.5 mg CMP-ß-Neu5Ac, 15 mU of rat recombinant
2,3-(N )-sialyltransferase, 2.5 mM MnCl2, 0.1% BSA, and 10 U calf intestine alkaline phosphatase (Boehringer-Mannheim, Mannheim, Germany) in 50 mM sodium cacodylate buffer ( pH 6.0) was incubated at 37°C for 48 h in a total volume of 0.5 ml. After heating at 100°C and centrifugation, the supernatant from the reaction mixture was charged onto a Sephadex G-25M PD-10 column Amersham Biosciences Corp. (NJ, USA) in the presence of 0.15 M NaCl. The high-molecular-weight fraction collected was dialyzed against distilled water for 3 days and lyophilized to afford the respective glycopolymers 2a, 2b, and 2c (6.3, 6.7, and 3.3 mg). 1H-NMR data of 2a (500 MHz, D2O, 30°C):
7.26 (o-Ph), 6.97 (m-Ph), 5.04 (H-1), 4.57 (H-1'), 4.3 (
-methine, Gln-co-Glu), 3.54.0 (m, 19H, from sugar), 2.75 (d, 1H, J33 8.0 Hz, H-3''eq), 2.22.5 (
-methylene, Gln-co-Glu), 2.03 (ß-methylene, Gln-co-Glu), 2.01 (CH3CO, GlcNAc, Neu5Ac), and 1.81 (dd, 1H, J33,34 11.6 Hz, H-3''ax). 13C-NMR data of 2a (500 MHz, D2O, 30°C):
177.9 (CH3CO, Neu5Ac), 177.7 (COOH, Gln-co-Glu), 176.5 (CH3CO, GlcNAc), 173.5 (C''-1), 155.9 (c-Ph), 135.6 ( p-Ph), 127.8 (m-Ph), 120.2 (o-Ph), 106.3 (C-1'), 104.5 (C-2''), 103.2 (C-1), 82.6 (C-4), 78.5 (C-5'), 78.1 (C-3'), 76.6 (C-5), 75.9 (C-6''), 75.4 (C-3), 74.9 (C-2'), 74.7 (C-8''), 71.3 (C-4'), 71.2 (C-4''), 71.1 (C-7''), 65.5 (C-9''), 63.9 (C-6'), 62.9 (C-6), 57.8 (C-2), 56.4 (
-methine, Gln-co-Glu), 54.9 (C-5''), 43.1 (C-3''), 36.3 (
-methylene, Gln-co-Glu), 30.9 (ß-methylene, Gln-co-Glu), 25.2 (CH3CO, Neu5Ac), and 25.0 (CH3CO, GlcNAc). Glycopolymers 2b and 2c gave similar NMR data.
Glycopolymers 3a3b [Poly(Neu5Ac2-6Galß1-4Glc-NAcß-pAP/Gln-co-Glu)] were also synthesized from 1a1c using rat recombinant
2,6-(N )-sialyltransferase (15 mU) in a similar manner to afford the respective glycopolymers 3a, 3b, and 3c (5.0, 6.1, and 5.7 mg). 1H-NMR data of 3a (500 MHz, D2O, 30°C):
7.29 (o-Ph), 7.01 (m-Ph), 5.08 (H-1), 4.45 (H-1'), 4.28 (
-methine, Gln-co-Glu), 3.54.0 (m, 19H, from sugar), 2.68 (dd, 1H, J33 12.5 and J34 4.6 Hz, H-3''eq), 2.22.5 (
-methylene, Gln-co-Glu), 2.04 (ß-methylene, Gln-co-Glu), 2.01 (CH3CO, GlcNAc, Neu5Ac), and 1.71 (dd, 1H, J33,34 12.2 Hz, H-3''ax). 13C-NMR data of 3a (500 MHz, D2O, 30°C):
184.2 (COOH, Gln-co-Glu), 177.7 (CH3CO, Neu5Ac), 176.3 (CH3CO, GlcNAc), 174.1 (C''-1), 155.9 (c-Ph), 134.9 (p-Ph), 126.6 (m-Ph), 120.0 (o-Ph), 106.3 (C-1'), 103.0 (C-2''), 102.4 (C-1), 82.9 (C-4), 77.5 (C-5'), 76.4 (C-5), 75.4 (C-6''), 75.2 (C-3'), 75.1 (C-3), 74.5 (C-8''), 73.6 (C-2'), 71.2 (C-4'), 77.1 (C-4''), 71.0 (C-7''), 66.1 (C-6'), 65.5 (C-9''), 62.9 (C-6), 57.6 (C-2), 56.5 (
-methine, Gln-co-Glu), 54.3 (C-5''), 42.9 (C-3''), 36.4 (
-methylene, Gln-co-Glu), 30.7 (ß-methylene, Gln-co-Glu), 25.1 (CH3CO, Neu5Ac), and 24.9 (CH3CO, GlcNAc). Glycopolymers 3b and 3c gave similar NMR data.
Synthesis of glycopolymers with PA backbones
PA-based asialoglycopolymer 8 [poly(pAP ß-N-acetyllactosaminide-carrying acrylamide): PAP(LacNAcß-pAP)] was prepared by coupling LacNAcß-pAP to an acrylate monomer and its homopolymerization according to our previously reported method (Kobayashi et al., 1994). Glycopolymer 9 [PAP(Neu5Ac
2-3Galß1-4GlcNAcß-pAP), 5.7 mg] was enzymatically synthesized from 5 mg of 8 and 10 mg of CMP-ß-Neu5Ac using
2,3-(N )-sialyltransferase (20 mU) in the same manner as that for the syntheses of 2a2c. Glycopolymer 10 [PAP(Neu5Ac
2-6Galß1-4GlcNAcß-pAP), 5.6 mg] was synthesized using
2,6-(N )-sialyltransferase (20 mU) in a similar manner. The structures of 9 and 10 were confirmed with reference to NMR data of PA-based glycopolymers reported previously (Kojima et al., 2002
).
Synthesis of pNP oligosaccharides
Neu5Ac2-3Galß1-4GlcNAcß-pNP was enzymatically synthesized from LacNAcß-pNP prepared by our previously reported method (Usui et al., 1993
) as follows: a mixture containing 12.5 mg LacNAcß-pNP, 16 mg CMP-ß-Neu5Ac, 30 mU rat recombinant
2,3-(N)-sialyltransferase, 2.5 mM MnCl2, 0.1% BSA, and 30 U alkaline phosphatase in 50 mM sodium cacodylate buffer (pH 6.0) was incubated at 37°C for 48 h in a total volume of 1.0 ml. After terminating the reaction, the supernatant from the reaction mixture was charged onto a Toyopearl HW-40 s column (
2.5x85 cm) (Tosoh Corp., Tokyo, Japan) and developed with 25% MeOH monitoring at 210 nm (carbonyl group), 300 nm ( pNP group), and 485 nm (sugar content, phenolsulfuric acid method). The flow rate was 1.0 ml/min, and the fraction size was 6 ml/tube. The fractions (tubes 3137) in which profiles of three absorbances agreed were lyophilized to afford Neu5Ac
2-3Galß1-4GlcNAcß-pNP (18.1 mg). Neu5Ac
2-6Galß1-4GlcNAcß-pNP (18.7 mg) was synthesized using
2,6-(N )-sialyltransferase (30 mU) in a similar manner. The structures of these compounds were confirmed with reference to NMR data reported previously (Sabesan and Paulson, 1986
).
Binding of influenza A viruses to glycopolymers
Glycopolymers 1b, 2b, and 3b were covalently immobilized on a microtiter plate (Corning-Costar, Labcoat 2504, Cambridge, MA). The wells were treated with a glycopolymer solution (10 µg/ml) in 10 mM sodium acetate buffer (pH 4.0) at 25°C for 1 h and irradiated under UV light at 254 nm for 1 min. The wells were blocked with 100 µl phosphate buffered saline (PBS) containing 2% BSA at 25°C for 1 h and then washed with PBS five times. Influenza viruses (A/PR/8/34 and A/Memphis/1/71) were twofold serially diluted with PBS to 2027 HAU, added to the glycopolymer-immobilized wells, and then incubated at 4°C for 12 h. The virions that had bound to fetuin on the wells were reacted with rabbit anti-influenza virus antiserum (anti-P-50) and detected with horseradish peroxidase (HRP)conjugated protein A (Organon Teknika N. V. Cappel Products, Turnhout, Belgium) as described previously (Suzuki et al., 1992). The absorbance was measured at 492 nm with 630 nm as a reference wavelength. All assays were carried out in duplicate (Figure 1A and B).
Inhibition of influenza virus binding to fetuin by glycopolymers
Fetuin was adsorbed on a polystyrene-surface microtiter plate (Nunc-Immuno Plate, MaxiSorp, Nalge Nunc International, Roskilde, Denmark). The wells were treated at 37°C for 1 h with fetuin (10 µg/ml) in 50 µl PBS. The wells were washed with PBS three times and blocked at 25°C for 2 h with 100 µl PBS containing 1% BSA. The wells were then washed with PBS five times. Influenza A viruses (A/PR/8/34 and A/Memphis/1/71) were diluted with 50 µl PBS to 2728 HAU and bound on the fetuin-coated surface at 4°C for 12 h in the presence or absence of glycopolymers 1a, 2a, 3a, or 5b (0.5500 µg/ml) or fetuin. As negative controls, diluted viruses were added to wells that had not been coated with fetuin. The virions that had bound to fetuin on the wells were detected as described previously (Suzuki et al., 1992). All assays were carried out in triplicate.
Neutralization of infection of MDCK cells by influenza virus
The TCID50 (50% tissue-culture infectious dose) of each virus to MDCK cells was determined as described previously (Suzuki et al., 1996). One hundred microliters of TCID50 of each virus, which corresponds to 2-221 HAU, was preincubated at 4°C for 1 h in the presence or absence of glycopolymers (0.011000 µg/ml) in PBS. The preincubated mixtures were inoculated at 34.5°C for 5 h on MDCK cell monolayers in 96-well microtiter plates (Nunclon Delta Surface, Nalge Nunc International Corp., Roskilde, Denmark). After removal of the inoculums, the cells were washed three times with EMEM and incubated at 34.5°C for 18 h in 100 µl EMEM supplemented with 5% FBS. Viral-induced cytopathic effect was monitored by light microscopy. The activity of LDH released from MDCK cells was determined using a slightly modified calorimetric assay as previously described (Watanabe et al., 1995
; Suzuki et al., 1996
; Tsuchida et al., 1998
). The culture medium (40 µl) was mixed with fivefold concentrated PBS (10 µl). The plates were preincubated at 37°C for 5 min. LDH reagent (50 µl) was added, and the mixture was incubated 37°C for 10 min. The reaction was stopped by the addition of 100 µl of 0.5 M HCl. The absorbance was measured at 550 nm with 630 nm as a reference wavelength. Each experiment was carried out in duplicate.
MDCK cellular toxicity assay
MDCK cell monolayers in 96-well microtiter plates (Delta Surface) were washed with 200 µl of serum-free EMEM three times and incubated with 100 µl (110,000 µg/ml) of 4d, 8, PA, PAA-Na, or acrylamide monomer at 37°C for 24 h. The cell viability was quantified using the MTT spectrophotometric assay (Watanabe et al., 1994; Reuter et al., 1999
).
Immunization of mice with a glycopolymer
Glycopolymer 4b (2 mg/ml in saline) was mixed with an equal volume of Freund's complete adjuvant (Difco Laboratories, Detroit, MI) and ID injected into female BALB/c mice (4b, 200 µg/animal, N = 3). Three and five weeks later, the mice were boosted by intraperitoneal injection of 0.2 ml of the Freund's-antigen mixture (Rittenhouse-Diakun et al., 1998).
ELISA for antisera
Sera from the immune and control mice (N = 3) were diluted with PBS, pH 7.4, 0.05% Tween 20 containing 1% BSA, and 50 µl were added to each well of a 4b-immobilized microtiter plate (Labcoat 2504, Corning International K.K., Tokyo, Japan). The samples were analyzed in duplicate. The plates were incubated for 18 h at 4°C, and bound antibodies were detected by 1:1000-diluted HRP-conjugated anti-mouse immunoglobulins (IgG, IgA, and IgM) (Cappel, ICN Pharmaceuticals, Aurora, OH). The absorbance was measured at 492 nm as described earlier. To confirm the immobilization of 4b on the plate, HRP-conjugated PNA (from Arachis hypogaea, Honen, Honen Corp., Tokyo, Japan) was serially diluted with PBS, pH 7.4, 0.05% Tween 20 and shown to bind to the plate as described earlier. PNA is known to bind to 4b strongly (Zeng et al., 2000).
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Acknowledgements |
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1 To whom correspondence should be addressed; e-mail: actusui{at}agr.shizuoka.ac.jp
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Abbreviations |
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References |
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