2Division of Biochemical and Medical Microbiology, Borstel Research Center, Parkallee 22, D-23845 Borstel, Germany, 3Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada K1A 0R6, 4Department of Biochemistry, University of Ottawa, Ottawa, Ontario, Canada K1H 8M5, and 5Institute of Chemistry, University of Agriculture, A-1190 Vienna, Austria
Received on May 17, 1999; revised on August 16, 1999; accepted on August 17, 1999.
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Abstract |
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Key words: anti-carbohydrate antibodies/Chlamydia/lipopolysaccharide/scFv/surface plasmon resonance
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Introduction |
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All chlamydiae possess in their outer membrane a lipopolysaccharide (LPS), which contains a genus-specific epitope composed of a 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) trisaccharide of the sequence Kdo-(2
8)-
Kdo-(2
4)-
Kdo (Brade et al., 1997
). This structure is highly immunogenic, resulting in the formation of high-affinity antibodies after natural or experimental infection or after immunization, and may therefore be particularly well suited as a target for vaccine development. To this end, we have synthesized a panel of neoglycoconjugates containing the synthetic Kdo-trisaccharide conjugated to bovine serum albumin (BSA) via a cysteamine spacer (Fu et al., 1992
), and have used these antigens to generate and characterize several anti-Kdo monoclonal antibodies. Some of these antibodies are strictly Chlamydia-specific, with no observed cross-reactivity with any other bacterial LPS structures. These specific antibodies (types C and D in Figure 1) require either the complete trisaccharide or the
-2
8-linked disaccharide for binding. (Fu et al., 1992
). This specificity is based on the fact that the
2
8-linkage between two Kdo residues occurs only in chlamydial LPS. Antibodies binding to a single Kdo or to the
2
4-linked disaccharide portion of chlamydial LPS (types A and B in Figure 1) cross-react with LPS of various bacterial species, since these structural elements are widespread in LPS molecules.
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Results |
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Antibody specificities
The specificities of S252 and S2523 for different Kdo mono-, di- and trisaccharides were determined by SPR monitoring of IgG binding to BSA-sugar conjugates immobilized on sensor chip surfaces. The binding profiles obtained (results not shown) for the two antibodies at concentrations of 50 nM and similar surface densities (15001800 RU (resonance units)) showed that S252 IgG bound to all conjugates. Kinetic analyses of the sensorgrams indicated that the strongest binding occurred with the Kdo-(2
8)-
Kdo-(2
4)-
Kdo-trisaccharide conjugate. This was attributable to a much slower dissociation rate for this conjugate relative to the other six. Since similar conjugate surface densities were used in all experiments, the lack of correlation between the affinity of the interaction and the response levels may be due to varying degrees of substitution of BSA with ligand and/or differences in the accessibility of the conjugated ligands to antibody. The S2523 antibody showed a strong preference for the two trisaccharide antigens
Kdo-(2
8)-
Kdo-(2
4)-
Kdo and
-Kdo-(2
8)-
KdoC1red-(2
4)-
Kdo and exhibited little or no binding to the other glycoconjugates.
Antibody affinities
Binding constants were determined by SPR for the binding of Kdo-(2
8)-
Kdo-(2
4)-
Kdo by the IgG, Fab, and scFv forms of S252 and S2523 (Table I). When purified monovalent preparations were assayed, very similar affinity constants were obtained for the Fab and scFv forms of each antibody (Table I). Much higher functional affinities were observed for whole antibodies because of the avidity effect resulting from IgG bivalency. This manifested itself in slower dissociation rate constants and led to a 100-fold increase in functional affinity for S252 IgG relative to Fab and a somewhat smaller increase, approximately 40-fold, for S2523 on
-Kdo-(2
8)-
-Kdo-(2
4)-
-Kdo surfaces. Varying proportions of monovalent and bivalent binding can lead to these discrepancies.
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Scatchard analysis of the data for S252 binding to the -Kdo-(2
8)-
-Kdo-(2
4)-
-Kdo-trisaccharide (Figure 6) gave a KD value of 1.1 µM (Table II). This value is in reasonable agreement with the values of 590 nM and 770 nM derived from the rate constants obtained by local and global fitting, respectively. Due to low yields, the S2523 scFv was not available at the concentrations needed for accurate Scatchard analysis.
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Discussion |
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Several precautions were taken to ensure the reliability of the biosensor data. Binding constants for the interactions of the two antibodies with different ligands were derived from data for the binding of high quality monovalent scFv to immobilized ligand, thus avoiding the pitfalls arising from avidity effects (MacKenzie et al., 1996; Myszka, 1997
). The consistency between KD values obtained from kinetic and equilibrium binding data attested to the quality of the samples and validity of the experimental design. The data withstood the demands of global analysis, which showed good fitting to a simple one-to-one reaction model.
The gene usage by the two Kdo-specific antibodies is similar to that of other anti-carbohydrate antibodies and does not appear to contribute to their high affinities relative to other carbohydrate-specific antibodies. The molecular analysis of anti-carbohydrate antibodies has indicated that the antibody response to some carbohydrate antigens involves a restricted set of germline genes (Crews et al., 1981; Lutz and Davie, 1988
; Scott et al., 1989a
,b) whereas the antibody response to LPS from Pseudomonas aeruginosa was encoded by diverse VH and V
genes (Emara et al., 1995
). The VH of S252 showed a high sequence homology to Yst9.1 (Bundle et al., 1989
), which is specific for the A antigen of Brucella, suggesting usage of a gene that is preferentially used by anti-carbohydrate antibodies. Similarly, the VL of S2523 showed a high sequence homology with BR96 (Hellström et al., 1990
), which binds the Ley oligosaccharide and may be further indication that certain germline families are preferentially selected in the immune response to carbohydrates. However, the S252 VL showed sequence homology to the HyHEL-5 VL, an antibody against lysozyme (Padlan, 1994
). The usage of the same gene family in response to lysozyme and carbohydrates shows that the restriction is not exclusive. In contrast to the antibody response against the cell-wall polysaccharide of group A Streptococcus, which elicits an antibody response with highly restricted VH-gene usage (Harris et al., 1997
) and is presumed to recognize very similar or even identical epitopes the primary structures differed to a great extent in both Chlamydia-specific mAbs. A common feature, however, is a high number of basic amino acids, which are located at the center of the combining sites, creating a highly positively charged surface. Ionic interactions may, therefore, play a key role in recognition of the anionic antigens by these antibodies. In a previous study (Brade et al., 1997
), we investigated the importance of negative charges on the ligand in antigen binding by S252 and S2523. Binding to ligands in which the carboxyl groups were selectively reduced was abolished for antibody S2523. An exception was the binding to the Kdo-trisaccharide ligand in which the negative charge on the internal Kdo-residue was eliminated by reduction. In this case the affinity was considerably reduced, as assessed by ELISA and SPR analyses. NMR analyses showed that the removal of a negative charge on the trisaccharide was accompanied by a conformational change in the ligand. The conformation of the Kdo-trisaccharide in which the internal Kdo residue had been reduced showed the closest structural similarity to the natural ligand (Brade et al., 1997
), suggesting that steric hindrance may explain the decreased binding. The low affinities of most anti-carbohydrate antibodies appear to be related to rapid dissociation rates (MacKenzie et al., 1996
). A tight interaction involving opposite charges may explain the slower dissociation rates observed with S252 and S2523. Relatively fast association rates also contribute to the high affinities of these antibodies and could arise from the conformational properties of the antigens. Transfer NOE measurements of the antigen in complex with mAb S252 in combination with conformational analysis performed on the free
2
8-linked Kdo-disaccharide provided evidence that the conformation of this ligand does not change upon binding (Sokolowski et al., 1998
). The NMR-determined conformation was similar to the conformation of the crystallized ligand (Mikol et al., 1994
).
The involvement of ionic interactions in antigen binding would represent a different type of recognition than has been observed in the two crystal structures of antibodies in complex with carbohydrate antigens that have been determined at high-resolution (Cygler et al., 1991; Jeffrey et al., 1995
). In these complexes, stacking interactions and hydrogen bonds formed between antigen and antibody were predominantly responsible for the interactions resulting in comparatively low affinity binding. These examples showed that the binding of carbohydrate ligands by antibodies is different from other carbohydrate-binding proteins such as transport proteins and lectins, in which the interactions involve mainly carboxy and amide side chains of aspartic acid, asparagine, glutamic acid, and glutamine, which form hydrogen bonds and aromatic groups stack with hexose rings (Vyas, 1991
; Bundle and Young, 1992
).
It has been shown in many instances (Smith-Gill et al., 1986; Radic et al., 1991
; Kang et al., 1991
; Collet et al., 1992
; Cooper et al., 1993
; Jespers et al., 1994
; Burton and Barbas, 1994
) that the VH-domain was the main determinant for the specificity of an antibody, and that the affinity was modulated by VL. Interchange of light chains between antibodies has been performed successfully in some instances, particularly between DNA-binding antibodies, without abolishing the reactivity. However, other examples have shown that the interchange of VL abolishes binding (Dinh et al., 1996
). Interchanging the domains of scFv 2523 with scFv 252 abolished the activity although both bind the same antigen with high affinity. We hypothesize that this may be due to a conformational change that results in the loss of contact sites on VL, an alteration in the conformation of VH induced by the new VL or from obstruction of contact sites on VH. Elucidation of this mechanism is beyond the scope of the present study. It has become evident that carbohydrate epitopes are quite small, comprising only a few functional groups on the antigen. Therefore, even a trisaccharide may contain a number of different epitopes. Although S252 and S2523 can both bind the same antigen, the interaction with the terminal disaccharide may be different. This hypothesis is supported by the different kinetics of binding observed for the two antibodies. Whereas kon is shorter for the bivalent antibody S252 than for S2523, the slower dissociation rate of S2523 leads to an overall higher affinity of S2523. The same holds true for monovalent Fab fragments and scFv.
The molecular analysis of antibodies that are directed against chlamydial LPS has provided insight into the molecular mechanisms that are involved in the high affinity interaction of these antibodies with carbohydrate antigens. The exceptional affinities of the anti-Kdo antibodies described here imply that there may be a biological reason for the low affinities of anti-carbohydrate antibodies, in some instances at least. If the acidic nature of the Kdo antigens is responsible for these high affinities, it would be expected that other anti-carbohydrate antibodies with similar affinities could arise in an immune response since acidic residues, notably sialic acid, are commonly found in carbohydrates in many mammalian glycolipids and glycoproteins. For example, specific antibodies reactive against tumor cells in pathological conditions may cross-react with common epitopes on normal cells with reasonable affinity and are therefore eliminated by antibody-mediated cytotoxicity. A well-characterized mAb, R24, which is specific for the disialoganglioside GD3, is known to have low intrinsic affinity for its acidic glycolipid antigen, but relies on an avidity gained from homophilic binding for efficient binding to GD3 bearing membranes (Kaminski et al., 1998). The R24 antibody recognizes tumor cells that overexpress GD3. It has been proposed that the homophilic binding provides a mechanism whereby the antibody binds effectively only above a threshold level of GD3. This mechanism would prevent binding to normal cells that express lower levels of GD3 (Kaminski et al., 1998
). The immune system may therefore use different strategies to discriminate between normal and pathological conditions in bacterial infections vs. self-antigens. Since Kdo is not found in mammalian cells, the immune system does not require any precautionary treatment during immune surveillance.
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Materials and methods |
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PCR amplifications and construction of scFvs
All DNA manipulations were carried out essentially as described by Sambrook et al., (Sambrook et al., 1989). Plasmid isolation was performed using kits from Qiagen. DNA sequencing was carried out using a Taq fluorescent dideoxy termination cycle sequencing kit (Applied Biosystems Inc.) and a 373 automated DNA sequencer (Applied Biosystems Inc.).
The following primers were used for isolation of the antibody variable genes of S252 and S2523: S252 VL reverse, 5'-GGGGAATTCGA(C,T)AT(ACT)GTNATGTCNCA(AG)TCNCC-3'; S252 VL forward, 5'-GGGAAGCTTCAAGAAGCACACGACTGAGGCAC-3'; S252 VH reverse, 5'-GGGGAATTCGAGGTGAAGCTGGTNGA(AG)TCNGGNGGNG-G-3'; S252 VH forward, 5'-GGGAAGCTTGTCACCATGGAGTTAGTTTGGGC-3'; S2523 VL reverse, 5'-GGGGAATTCGA(CT)GTNCTNATGACNCA(AG)ACNCC-3'; S2523 VL forward, 5'-GGGAAGCTTCAAGAAGCACACGACTGAGGCAC-3'; S2523 VH reverse, 5'-GGGGAATTC(CG)AGGTN(AC)A(AG)CTN(CG)(AT)G(CG)AGTC-3'; S2523 VH forward, 5'-GGGAAGCTTGTCACCATGGAGTTAGTTTGGGC-3'. Reverse primers were based on the N-terminal amino acid sequence of VL and VH domains of S252 and the VL domain of S2523. A degenerate reverse primer was used for the S2523 VH sequence since an amino acid sequence could not be determined because of N-terminal blockage. Total mRNA was isolated from 4.3 x 107 hybridoma cells using the RNeasy kit from Qiagen (Mississauga, ON) and antibody genes were reverse transcribed into first strand cDNA using the forward primers listed above and a cDNA-synthesis kit (Pharmacia Biotech, Baie dUrfé, QC). For PCR amplification, the above primers and Taq polymerase were used in 50 µl reactions under the following conditions: 94°C/3 min, then 30 cycles of 94°C/30 s, 50°/60 s, 70°/30 s. The annealing temperature was reduced to 40°C for reverse transcription of S2523 VH due to the higher degeneracy. The obtained PCR products were purified, digested with EcoRI/HindIII, ligated into pUVC-9, and used for transformation of E. coli TG1 by electroporation (Gene Pulser electroporator, Bio-Rad Laboratories Ltd., Mississauga, Ontario, Canada) according to the manufacturers instructions. Clones harboring each VH and VL gene were sequenced and compared to known mouse antibody sequences (Martin, 1998). Based on the obtained sequences, the following primers were used to allow for cloning into the expression vector pSJF8 by introduction of EcoRI, BspEI, and BglII sites: S252 VL reverse, 5'-GGGGAATTCATGAAAAAAACCGCTATCGCGATCGCAGTTGCACTGGCTGGTTTCGCTACCGTT-GCGCAGGCCGATATTGTGATGTCACAGTCTCCA-3'; S252 VL forward, 5'-GGGTCCGGAACCGCCACCGCCGGAACCGCCACCGCCAGCCCGTTTGATTTCCAGCTT-3'; S252 VH reverse, 5'-GGGTCCGGAGCGGTGGCTCCGGCGGTGGCGGTGAGGTGAAGCTGGTCGAATCG-3'; S252 VH forward, 5'-GGGAGATCTTGCAGAGACAGTGACCAG-3'; S2523 VL reverse, 5'-GGGGAATTCATGAAAAAAACCGCTATCGCGATCGCAGTTGCACTGGCTGGTTTCGCTACCGTTGCGCAGGCCGACGTCCTAATGACACAGACTCCA-3'; S2523 VL forward, 5'-GGGTCCGGAACCGCCACCGCCGGAACCGCCACCGCCAGCCCGTTTTATTTCCAGCTTGGT-3'; S2523 VH reverse, 5'-GGGTCCGGA-GGCGGTGGCTCCGGCGGTGGCGGTGAGGTACAACTGCAGGAGTCA-3'; S2523 VH forward, 5'-GGGGGATCCGTTCAAATCTTCCTCACTGATTAGCTTCTGTTCAGATCTTGCAGAGACAGTGACCAGAGTCCC-3. Single-chain Fv assembly was achieved by separate cloning of VL and VH genes using EcoRI and Kpn2 I sites for VL and Kpn2 I and BglII sites for VH. The above primers add extensions to the VL sequences and to the VH sequences which encode the ompA-leader sequence (5' of VL), half of the linker (3' of VL, and 5' of VH), c-myc and 5xHis tags (3' of VH). The linker sequence was GGGG(SGGGG)3. Appropriately cut VL and VH PCR-products were ligated into pSJF8 and used for transformation of E.coli TG1. Screening by DNA sequencing and SDS-PAGE/Western-blotting revealed clones harboring scFv genes. Cultures grown for 16 h in 25 ml TB/Amp were subjected to periplasmic extraction by an osmotic shock procedure and the obtained fractions were analyzed for the presence of soluble scFv by SDS-PAGE/Western blotting as described previously (Anand et al., 1991).
Expression and isolation of soluble scFv
The expression of soluble scFv and its isolation from the periplasm was achieved as described previously (Anand et al., 1991). Periplasmic extracts were dialyzed against 10 mM HEPES pH 7.0 and the scFv isolated by Interaction Metal Affinity Chromatography (IMAC) using HiTrap columns (Pharmacia Biotech). Bound protein was eluted using a gradient of 50 mM to 500 mM imidazole in HEPES buffer pH 7.0. The eluent was collected in 1 ml fractions and fractions were further analyzed by SDS-PAGE (12.5%) and staining with Coomassie brilliant blue.
Preparation of monoclonal antibodies and Fab fragments
S252 was purified from ascites by Protein G affinity chromatography. Protein G Sepharose (Pharmacia Biotech) was equilibrated with 20 bed volumes of binding buffer (100 mM sodium citrate, 300 mM NaCl, pH 5.3). The ascite sample was then diluted with equal parts of binding buffer and incubated with the Protein G Sepharose for 30 min with continuous mixing. The mixture was centrifuged at 200 x g for 5 min, and the unbound fraction was decanted. The Protein G resin was resuspended in binding buffer, loaded into a column, and washed with binding buffer until the A280 of the flowthrough reached ~0.02. The S252 IgG was then eluted with 100 mM sodium citrate pH 3.0 and collected in 1.5 ml fractions. Fractions containing IgG, as determined by A280, were pooled and dialyzed against 20 mM sodium acetate, 100 mM NaCl, pH 5.4. S2523 IgG was prepared as described previously (Fu et al., 1992).
Fab fragments of S252 and S2523 mAbs were prepared by digestion with mercuripapain (Sigma, St. Louis, MO) activated by ß-mercaptoethanol at an IgG:papain ratio of 100:1 (w/w) (Yamaguchi et al., 1994). Digestion of S252 was carried out in the presence of 1 mM DTT, 2 mM EDTA, and 15 mM Tris pH 8.5; the reaction mixture was brought to a final volume using 20 mM sodium acetate pH 5.4. Time trials established the optimal reaction time at 4 h after which time the reaction was quenched with iodoacetamide at a final concentration of 4 mM. The digest mixture was dialyzed against 20 mM sodium acetate pH 5.4. Fab fragments were separated by ion exchange HPLC using a Shodex CM-825 column (Phenomenex) in a mobile phase of 20 mM sodium acetate against a 01 M sodium chloride gradient. Fab fragments of the S2523 IgG were prepared with mercuripapain (Sigma, #P-9886) activated by ßmercaptoethanol (Yamaguchi et al., 1994
). IgG and papain were added in a 200:1 (w/w) ratio in the presence of 0.5 mM DTT, 2 mM EDTA; the mixture was brought to a final volume with 50 mM Tris, 150 mM NaCl pH 8.0. After 4 h, the reaction was quenched with 23 mM iodoacetamide and dialyzed against 20 mM HEPES pH 7.5. The Fab fragments were separated by ion exchange HPLC using a CM-5PW column (Toshaas) in a mobile phase of 20 mM sodium acetate pH 3.8 against a 0500 mM sodium chloride gradient. Both Fab fragments eluted as separate peaks which were collected, pooled and concentrated using Centricon 10 microconcentrators to final concentrations of ~3.5 mg/ml.
Surface plasmon resonance
The phenomenon of SPR originally observed by Otto (1968) and Kretschmann and Raether (1968)
was used as a method for studying interactions of different forms of antibodies recognizing chlamydial lipopolysaccharide. Analyses were carried out using an automated BIACORE 1000 biosensor instrument (Biacore, Inc., Piscataway, NJ (Jönsson et al., 1991
)). Neoglycoconjugates consisting of bovine serum albumin (BSA) linked, via a cysteamine spacer, to
Kdo-monosaccharide,
-Kdo-(2
4)-
-Kdo-disaccharide,
-Kdo-(2
8)-
Kdo-disaccharide,
-Kdo-(2
8)-
-Kdo-(2
4)-
-Kdo-trisaccharide,
-KdoC1red-(2
8)-
-Kdo-disaccharide,
Kdo-(2
8)-
KdoC1red-disaccharide, and
-Kdo-(2
8)-
-KdoC1red-(2
4)-
Kdo-trisaccharide were immobilized on research grade CM5 sensor chips in 10 mM sodium acetate, pH 4.5, using the amine coupling kit supplied by the manufacturer. Unreacted moieties were blocked with ethanolamine. Control BSA surfaces were prepared in the same manner. All measurements were performed in 10 mM HEPES, pH 7.4, containing 150 mM NaCl, 3.4 mM EDTA and 0.005% Surfactant P-20 (Biacore, Inc.) at a flow rate of 10 or 50 µl/min. Surfaces were regenerated by normal dissociation or with 5 µl of 10 mM HCl and a contact time of 6 s. The amount of immobilized ligand on BSA was determined from the amount of Kdo in 1 mg/ml solutions of neoglycoconjugates. Kdo was determined by the thiobarbituric acid(TBA)-assay as described previously (Kaca et al., 1988
). The concentration of BSA was determined using the Bradford-assay (Bio-Rad). Immediately prior to SPR-analysis, Fab and scFv preparations were subjected to size exclusion chromatography on Superdex 75 FPLC-column (Pharmacia Biotech) (Anand et al., 1991
) to remove aggregated material and scFv dimers and higher oligomers. Concentrations of IgG, Fab, and scFv were assayed by adsorption at 280 nm on the basis of 1 mg/ml giving A280 = 1.35.
Because of low product yields by clones expressing scFv 2523, purified preparations of the scFv were too dilute for spectrophotometric determination of concentration. In these instances, concentrations were determined by SPR monitoring of scFv binding to anti-c-myc surfaces under mass transport limiting conditions. Monomeric scFv of known concentration was used to prepare a standard curve using immobilized anti-c-myc antibody surfaces. Sensorgram data were analyzed using the BIAevaluation 3.0 software (Biacore, Inc.). Association and dissociation rate constants were derived by both separate and simultaneous fitting of the association and dissociation phases of individual sensorgrams. Sensorgrams were also fitted globally using data collected at a series of concentrations.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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Brade,H., Brabetz,W., Brade,L., Holst,O., Löbau,S., Lucakova,M., Mamat,U., Rozalski,A., Zych,K. and Kosma,P. (1997) Chlamydial lipopolysaccharide. J. Endotoxin Res., 4, 6784.[ISI]
Brade,L., Zych,K., Rozalski,A., Kosma,P., Bock,K. and Brade,H. (1997) Structural requirements of synthetic oligosaccharides to bind monoclonal antibodies against Chlamydia lipopolysaccharide. Glycobiology, 7, 819827.[Abstract]
Bundle,D.R., Cherwonogrodzki,J.W., Gidney,M.A., Meikle,P.J., Perry,M.B. and Peters,T. (1989) Definition of Brucella A and M epitopes by monoclonal typing reagents and synthetic oligosaccharides. Infect. Immun., 57, 28292836.[ISI][Medline]
Bundle,D.R. and Young,N.M. (1992) Carbohydrate-protein interactions in antibodies and lectins. Curr. Opin. Struct. Biol., 2, 666673.
Burton,D.R. and Barbas III,C.F. (1994) Human antibodies from combinatorial libraries. Adv. Immunol., 57, 191280.[ISI][Medline]
Caldwell,H.D. and Hitchcock,P.J. (1984) Monoclonal antibodies against a genus specific antigen of Chlamydia species: location of the epitope on chlamydial lipopolysaccharide. Infect. Immun., 44, 306314.[ISI][Medline]
Collet,T.A., Roben,P., OKennedy,R., Barbas III,C.F., Burton,D.R. and Lerner,R.A. (1992) A binary plasmid system for shuffling combinatorial antibody libraries. Proc. Natl Acad. Sci. USA, 89, 1002610030.[Abstract]
Cooper,L.J., Shikhman,A.R., Glass,D.D., Kangisser,D., Cunningham,M.W. and Greenspan,N.S. (1993) Role of heavy chain constant domains in antibody-antigen interaction. J. Immunol., 150, 22312242.
Crews,S., Griffin,J., Huang,H., Calame,K. and Hood,L. (1981) A single VH gene segment encodes the immune response to phosphorylcholine: somatic mutation is correlated with the class of antibody. Cell, 25, 5966.[ISI][Medline]
Cygler,M., Rose,D.R. and Bundle,D.R. (1991) Recognition of a cell surface oligosaccharide of pathogenic Salmonella by antibody Fab fragment. Science, 253, 442445.[ISI][Medline]
Dinh,Q., Weng,N.P., Kiso,M., Ishida,H., Hasegawa,A. and Marcus,D.M. (1996) High-affinity antibodies against Lex and sialyl Lex from a phage display library. J. Immunol., 157, 732738.[Abstract]
Emara,M.G., Tout,N., Kaushik,A. and Lam,J.S. (1995) Diverse VH and Vk genes encode antibodies to Pseudomonas aeruginosa LPS. J. Immunol., 155, 39123921.[Abstract]
Fu,Y., Baumann,M., Kosma,P., Brade,L. and Brade,H. (1992) A synthetic glycoconjugate representing the genus-specific epitope of chlamydial lipopolysaccharide exhibits the same specificity as its natural counterpart. Infect. Immun., 60, 13141321.[Abstract]
Harris,S.L., Craig,L., Mehroke,J.S., Rashed,M., Zwick,M.B., Kenar,K., Toone,E.J., Greenspan,N., Auzanneau,F.I., Marino-Albernas,J.R., Pinto,B.M. and Scott,J.K. (1997) Exploring the basis of peptide-carbohydrate crossreactivity: evidence for discrimination by peptides between closely related anti-carbohydrate antibodies. Proc. Natl. Acad. Sci. USA, 94, 24542459.
Hellström,I., Garrigues,H.J., Garrigues,U. and Hellström,K.E. (1990) Highly tumour-reactive, internalizing, mouse monoclonal antibodies to Le (y)-related cell surface antigens. Cancer Res., 50, 21832190.[Abstract]
Jeffrey,P.D., Bajorath,J., Chang,C.Y., Yelton,D., Hellström,I., Hellström,K.E. and Sheriff,S. (1995) The X-ray struture of an anti-tumour antibody in complex with antigen. Nat. Struct. Biol., 2 (6), 466471.
Jespers,C.S., Roberts,A., Mahler,S.M., Winter,G. and Hoogenboom,H.R. (1994) Guiding the selection of human antibodies from phage display repertoires to a single epitope of an antigen. BioTechnology, 12, 899903.[ISI][Medline]
Jönsson,U., Fägestam,L., Ivarsson,B., Johnsson,B., Karlsson,R., Lundh,K., Löfås,S., Persson,B., Roos,H., Rönnberg,I., Sjölander,S., Stenberg,E., Ståhlberg,R., Urbaniczky,S., Östlin,H. and Malmqvist,M. (1991) Real-time biospecific interaction analysis using surface plasmon resonance and a sensor chip technology. Biotechniques, 11, 620627.[ISI][Medline]
Kabat,E.A., Wu,T.T., Reid-Miller,M., Perry,H.M. and Gottesmann,U.S. (1987) Sequences of Immunological Interest. National Institutes of Health, Bethesda, MD.
Kaca,W., de Jongh-Leuvenink,J., Zähringer,U., Rietschel,E.Th., Brade,H., Verhoef,J. and Sinnwell,V. (1988) Iolation and chemical analysis of 7-O- (2-amino-2-deoxy-- D-glucopyranosyl-L-glycero- D-manno-heptose as a constituent of the lipopolysaccharides of the UDP-galactose epimerase less mutant J-5 of Escherichia coli and Vibrio cholerae. Carbohydr. Res., 179, 289299.[ISI][Medline]
Kaminski,M.J., MacKenzie,C.R., Moolibroek,M.J., Dahms,T.E.S., Hirama,T., Houghton,A.N., Chapman,P.B. and Evans,S.V. (1998) The role of homophilic binding in anti-tumor antibody R24 recognition of molecular surfaces. Demonstration of an intermolecular-sheet interaction between VH domains. J. Biol. Chem., 274, 55975604.
Kang,A.S., Jones,T.M. and Burton,D.R. (1991) Antibody redesign by chain shuffling from random combinatorial immunoglobulin libraries. Proc. Natl. Acad. Sci. USA, 88, 1112011123.[Abstract]
Kretschmann,E. and Raether,H. (1968) Radiative decay of non radiative surface plasmons excited by light. Z. Naturforsch., 23, 21352136.
Lutz,C.T. and Davie,J.M. (1988) Genetics and primary structure of Vk gene segments encoding antibody to streptococcal group A carbohydrate. J. Immunol., 140, 641645.
MacKenzie,C.R., Hirama,T., Deng,S., Bundle,D.R., Narang,S.A. and Young,N.M. (1996) Analysis by surface plasmon resonance of the influence of valence on the ligand binding affinity and kinetics of an anti-carbohydrate antibody. J. Biol. Chem., 271, 15271533.
Martin,A., http://www.biochem.ucl.ac.uk/~martin/abs/index.html
Mikol,V., Kosma,P. and Brade,H. (1994) Crystal and molecular structure of allyl-O (sodium-3-deoxy--D-manno-2-octulopyranosylonate)- (28)-3-deoxy-
- D-manno-2-octulopyranosidonate)-monohydrate. Carbohydr. Res., 263, 3542.
Moulder,J.W. (1991) Interaction of Chlamydiae and host cells in vitro. Microbiol. Rev., 55, 143190.[ISI]
Myszka,D.G. (1997) Kinetic analysis of macromolecular interactions using surface plasmon resonance biosensors. Curr. Opin. Biotechnol., 8, 5057.[ISI][Medline]
Nurminen,M., Leinonen,M., Saikku,P. and Mäkelä,P.H. (1983) The genus specific antigen of Chlamydia: resemblance to the lipopolysaccharide of enteric bacteria. Science, 220, 12791281.[ISI][Medline]
Otto,A. (1968) Excitation of nonradiative surface plasma waves in silver by the method of frustrated total reflection. Z. Phys., 216, 398410.[ISI]
Padlan,E.A. (1994) Anatomy of the antibody molecule. Mol. Immunol., 31, 169217.[ISI][Medline]
Patenaude,S.I., Vijay,S.M., Yang,Q.L., Jennings,H.J. and Evans,S.V. (1998) Crystallization and preliminary x-ray diffraction analysis of antigen-binding fragments which are specific for antigenic conformations of sialic acid homopolymers. Acta Crystallogr. D Biol. Crystallogr., 54, 10051007.
Radic,M.Z., Masceli,M.A., Ericson,J., Shan,H. and Weigert,M. (1991) IgH and L chain contribution to autoimmune specificities. J. Immunol., 146, 176182.
Sambrook,J., Fritsch,E.F. and Maniatis,T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Sato,C., Kitajima,K., Inoue,S., Seki,T., Troy,F.A., Inoue,Y. (1995) Characterization of the antigenic specificity of four different anti- ( 2
8-linked polysialicacid) antibodies using lipid-conjugated oligo/polysialic acids. J. Biol. Chem., 270, 1892318928
Scott,M.G., Crimmins,D.L., McCourt,D.W., Zocher,I., Thiebe,R., Zachau,H.G. and Nahm,M.H. (1989a) Clonal characterization of the human IgG antibody repertoire to Haemophilus influenzae type b polysaccharide. III. A single VkII gene and one of several Jk genes are joined by an invariant arginine to form the most common L chain V region. J. Immunol., 143, 41104116.
Scott,M.G., Tarrand,J.J., Crimmins,D.L., McCourt,D.W., Siegel,N.R., Smith,C.E. and Nahm,M.H. (1989b) Clonal characterization of the human IgG antibody repertoire to Haemophilus influenzae type b polysaccharide. II. IgG antibodies contain VH genes from a single VH family and VL genes from at least four VL families. J. Immunol., 293298.
Smith-Gill,S.J., Hamel,P.A., Klein,M.H., Rudikoff,S. and Dorrington,K.J. (1986) Contribution of the Vk4 light chain to antibody specificity for lysozyme and ß- (16)-D-galactan. Mol. Immunol., 23, 919926.[ISI][Medline]
Sokolowski,T., Haselhorst,T., Scheffler,K., Weisemann,R., Kosma,P., Brade,H., Brade,L. and Peters,T. (1998) Conformational analysis of a Chlamydia-specific disaccharide -Kdo- (28)-
-Kdo- (2-O)-allyl in aqueous solution and bound to a monoclonal antibodyobservation of intermolecular transfer NOEs. J. Biomol. NMR,
Vyas,N.K. (1991) Atomic features of protein carbohydrate interactions. Curr. Opin. Struct. Biol., 1, 732740.
Yamaguchi,Y., Kim,H., Kato,K., Masuda,K., Shimada,I. and Arata,Y. (1994) Proteolytic fragmentation with high specificity of mouse immunoglobulin G. Mapping of proteolytic cleavage sites in the hinge region. J. Immunol. Methods, 181, 259267.[ISI]