Detection of free and protein-bound non-human gal-alpha(1-3)-gal epitope

Abstract

The present invention relates to the provision of antibody fragments capable of binding selectively to the Gal--(1.fwdarw.3)-Gal epitope. The invention further relates to assay systems comprising these antibody fragments for use in testing transplantation tissue for possible rejection complications. This epitope is often found on porcine tissue destined for human transplantation. The epitope is also found on biopharmaceuticals and on some infectious agents and accordingly the invention also provides assay systems for these applications.

Claims

1. An antibody capable of binding selectively to Gal--(1.fwdarw.3)-Gal epitope, the antibody comprising SEQ ID NOs 2, 3 or 4.

2. The antibody of claim 1 wherein the antibody is a monoclonal antibody.

3. The antibody of claim 2, wherein the antibody comprises SEQ ID No. 2.

4. An assay kit for the determination of the presence or the quantification of a Gal--(1.fwdarw.3)-Gal motif in tissues or cells or on proteins, comprising the antibody of claim 1.

5. The assay kit of claim 4 further comprising reagents and/or instructions for an ELISA assay, a competition/inhibition ELISA assay, a sandwich ELISA assay, a microarray based assay, a rapid assay platform which comprises quantum dots or fluorescent tags, an immunohistochemistry assay, or a flow cytometry assay.

6. A method of detecting the presence of, or quantifying the amount of a Gal--(1.fwdarw.3)-Gal motif in tissues or cells or on proteins, comprising detecting and determining the degree of binding of the antibody of claim 1 to the tissue, cell or protein.

7. A composition comprising the antibody of claim 1 together with a pharmaceutically acceptable carrier or excipient.

8. A method for the detection of a Gal--(1.fwdarw.3)-Gal epitope in a sample comprising contacting a sample comprising the epitope with the antibody of claim 1 and detecting the antibody bound to the sample.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1: (A) Chicken serum response to immunisation with Gal--(1.fwdarw.3)-Gal-BSA as determined by direct ELISA (Serum dilution 1:20,000). (B) Demonstration of the (i) insertion of the encoding scFv fragment and (ii) the diversity within this fragment across randomly selected clones examined by restriction digestion. (C) Binding to Gal--(1,3)-Gal-BSA of biopanning round 2 output phage particles

(2) FIG. 2: Evaluation of 96 individual scFv-phage particles in direct ELISA against Gal--(1,3)-Gal-BSA. Indicated in blue are 6 scFv selected for further analysis from this population

(3) FIG. 3: Schematic alignment of CDRs from the VL and VH regions from 3 scFv sequenced clones demonstrating amino acid variants. (A) a generic schematic of a fused scFv and (B) alignment of scFv CDR. (LCDR 1=SEQ ID NO. 5, LCDR 2=SEQ ID NO. 6, LCDR 3=SEQ ID NO. 7, HCDR 1=SEQ ID NO. 8, HCDR 2=SEQ ID NO. 9, HCDR 3=SEQ ID NO. 10, Linker sequence=SEQ ID NO. 11).

(4) FIG. 4: (A) IMAC purification of soluble scFv from bacterial culture media. Image of FPLC output using a gradient elution, showing absorbance at 280 nm mAU (in blue), flow rate (mL min-1, in black), and gradient slope (in green). (B) SDS-PAGE on a 4-12% Bis-Tris NuPage gel of affinity-purified scFv fractions with Coomassie staining.

(5) FIG. 5: Specificity profile of the three anti-Gal--(1.fwdarw.3)-Gal scFvs in direct ELISA. Wells were coated as follows: 1. Gal--(1.fwdarw.3)-Gal-BSA; 2. Gal--(1.fwdarw.3)-Gal--(1.fwdarw.4)-GlcNAc-HSA; 3. Gal--1-O-spacer-ITC-BSA; 4. Gal--1-O-spacer-ITC-BSA; 5. Gal--(1>2)-Gal-BSA; 6. Gal--(1.fwdarw.4)-Gal-BSA; 7. -xyl-4AP-BSA; 8. Fuc--4AP-BSA; 9. Fuc--4AP-BSA; 10. Neu5Ac-4AP-BSA; 11. GlcNAc-BSA; 12. Lac--4AP-BSA; 13. LacNAc-BSA; 14. HSA, 15. BSA and 16. BSA-4AP. The error bars indicate the standard error of the mean of triplicate determinations.

(6) FIG. 6: Standard curves for competitive ELISA given by scFv clones G12, A4, and A11. Plates were coated with immobilized Gal--(1.fwdarw.3)-Gal-BSA [10 g/ml] and incubated with 2.5 g/ml of the scFvs in the presence of an increasing concentrations of free Gal--(1.fwdarw.3)-Gal. The error bars indicate the standard error of the mean of triplicate determinations.

(7) FIG. 7: Composite standard curve given by scFv-A4 from 9 independent runs.

(8) FIG. 8: Comparison of the specificity of selected scFv fragments A4, A11 and G12 against a panel of related NGCs compared directly to the specificity binding pattern of the lectin GS-1-B4, considered as highly specific for -Gal residues.

(9) FIG. 9: Alignment of fused light chain and heavy chain amino acid sequences of the selected scFv fragments A4, A11 and G12 as expressed as soluble protein and presented for recognition of the epitope (SEQ ID NOS. 2-4).

(10) FIG. 10: Generic antibody sequence (SEQ ID NO. 1) showing complementarity determining regions (SEQ ID NOS. 5-10), and linker sequence (SEQ ID NO. 11).

(11) FIG. 11. Near UV CD spectra, expressed as molar ellipticity per residue recorded for scFvs G12 (A), A4 (B) and A11 (C) in the absence (black) and presence of 4 mM melibiose (red) and Gal(1,3)Gal1-OMe (green), in NaPi (10 mM)-NaCl (150 mM) pH 7.2 at 20 C. Samples were prepared by ultrafiltration.

(12) FIG. 12. Far UV CD spectra, expressed as molar ellipticity per residue, of scFvs in NaPi (10 mM)-NaCl (150 mM) pH 7.2 at 20 C.

(13) FIG. 13. Far UV CD spectra, expressed as molar ellipticity per residue of scFvs G12 (A), A4 (B) and A11 (C) in the absence and presence of 4 mM Gal(1,3)Galb1-OMe in NaPi (10 mM)-NaCl (150 mM) pH 7.2. Pure scFv at 20 C. (black), pure scFv at 90 C. (red), scFv with sugar at 20 C. (green), scFv with sugar at 90 C. (blue). Samples of G12 and A11 were prepared by ultrafiltration and samples of A4 by dialysis.

(14) FIG. 14. Denaturation profiles for scFvs G12 (A), A4 (B) and A11 (C). The continuous line corresponds to the fit of a sigmoidal function to the experimental data, using the equation mentioned in the text. Pure scFv (black), scFv+Gal(1,3)Galb1-OMe (red) and scFv+melibiose (green).

(15) FIG. 15 CL-Elisa data for scFvs and neoglycoproteins showing relative luninescence.

(16) FIG. 16. Raw data for FIG. 15.

DETAILED DESCRIPTION OF THE DRAWINGS

Materials and Methods

(17) Chicken Immunization.

(18) A pooled glycoconjugate preparation composing of human -acid glycoprotein, porcine lactoferrin, honey bee phospholipase A2, Gal--(1.fwdarw.3)-Gal-BSA and soluble glycoprotein fraction taken from CHO cell media were combined at an equal ratio. Adult male Leghorn chickens were immunized subcutaneously, at 21-day intervals, with 50 g of the glycoconjugate preparation in a total volume of 400 l. The first dose was prepared with complete Freund's adjuvant, and three subsequent doses were administered with incomplete Freund's adjuvant. Serum anti-Gal--(1.fwdarw.3)-Gal response was evaluated, seven days after the third inoculation, via direct enzyme-linked immunosorbent assay (ELISA) analysis, FIG. 1a. Briefly, 96-well immunoassay plates (Maxisorp; Nunc) were coated overnight at 4 C. with 100 l/well Gal--(1.fwdarw.3)-Gal-GlcNAc-HSA at 10 g/ml. Wells were then blocked for 1 hour at 37 C. with 0.5% BSA-PBS (PBS-B). Serum samples were serially diluted in PBS-B containing 0.1% Tween 20 (PBS-BT) and added to the plates 100 l/well. Plates were then incubated at 37 C. for 1 hour, washed 10 times with PBS-0.1% Tween 20 (PBS-T), and probed for 1 hour at 37 C. with horseradish peroxidase (HRP)-conjugated anti-chicken IgG antibody (Pierce) in PBS-BT. After washing with PBS-T, the wells were developed with 100 l/well HRP substrate, tetramethylbenzidine (DAKO), and reactions were stopped with 100 l/well 1 M H.sub.2SO.sub.4. All analyses were performed in triplicate.

(19) Chicken scFv Library Generation.

(20) Library generation and phage display was performed using the protocols as described by Andris-Widhopf et al. (Andris-Widhopf et al., 2000). Briefly, total RNA was isolated from the spleen and bone marrow of one femur from each chicken (TRIzol Reagent, Invitrogen) and first-strand cDNA synthesised (Superscript II, Invitrogen Inc) both as per manufacturers protocol. The scFv library was generated using a two step approach with initial amplification of heavy and light chains, followed by overlap extension PCR, with the scFv cDNA products introduced into the pComb3 phagmid system via ligation (Andris-Widhopf et al., 2000) and subsequently transformed into electrocompetent E. coli XL1-Blue cells [F proAB lacIqZH15 Tn10 (Tetr)] (Stratagene). All amplification oligonucleotides were as described by Andris-Widhopf et al. Total library size were estimated by antibiotic resistance plate counting on Luria-Bertani (LB) agar containing 100 g/ml carbenicillin (Sigma). scFv insertion was validated by colony PCR and library sequence diversity assessed using endonuclease BstNI digestion of PCR products (Promega) and visualised by agarose gel electrophoresis, FIG. 1b. The completed library preparations were propagated in XL1-Blue cells, and held as glycerol stocks.

(21) Rescue of scFv-Displaying Phage.

(22) To rescue scFv-displaying phage, 50 ml super broth (SB) supplemented with 2% glucose, 50 g/ml ampicillin and 10 g/ml tetracycline was inoculated with approximately 210.sup.9 cells from the glycerol stock library. The culture was then incubated with rotation at 37 C. until an OD.sub.600 of 0.5, was reached. At this point the culture is co-infected with 10.sup.12 colony-forming unit of VCSM13 helper phage (Stratagene) and increased to a volume of 200 ml with SB. After 90 minutes incubation, 50 g/ml kanamycin was added and incubation 30 C. overnight carried out. Phage particles were purified and concentrated from the liquid medium by PEG/NaCl precipitation (Andris-Widhopf et al., 2000), and resuspended in 1% OVA-PBS (PBS-O).

(23) Selection by Biopanning of scFv Phage Libraries Against Gal--(1.fwdarw.3)-Gal-BSA.

(24) Biopanning against Gal--(1.fwdarw.3)-Gal-BSA was performed using Maxisorp immunotubes (Nunc). Briefly, tubes were coated overnight at 4 C. with 1 ml of 10 g/ml Gal--(1.fwdarw.3)-Gal-BSA (Dextra, UK) and then blocked with PBS-B. 110.sup.11-10.sup.12 scFv-displaying phage suspended in PBS-0 were then added and mixed by rotation for 1 hour at room temperature. Follow by repeated washing with PBST, and then with PBS only. Bound phage were eluted with the addition of 1 ml 100 mM glycine-HCl, pH 2.2 for 10 min. Eluted phage were neutralized using 50011 of 1M Tris-HCl, pH 8.8. Propagation of eluted phage and further manipulations were then carried out, as previously described (Andris-Widhopf et al., 2000). Three rounds of panning were performed, with increasing selection stringency mediated by a progressive increase in washing steps from 10 in pan 1 to 15 in pan 2 and 3.

(25) ELISA Analysis of the scFv-Phage Population Pool after Each Round of Biopanning.

(26) Eluted scFv-displaying phage suspensions from each round of panning were assessed for Gal--(1.fwdarw.3)-Gal binding by direct ELISA analysis. Immunoassay plates (Maxisorp, Nunc) were coated and blocked as described above, and threefold dilution of phage-scFv preparations (in PBS-BT) added to the wells. BSA and HSA were included as negative references. After 1 hour of incubation at 37 C., plates were washed with three times with PBS-T and probed with HRP-conjugated anti-M13 antibody (GE Healthcare) in PBS-BT for 1 hour at 37 C. After extensive washing with distilled water, the wells were developed as described above.

(27) Single scFv-Phage Clones and Direct ELISA Analysis.

(28) Ninety six random clones were picked from output plates from rounds 2 and 3 of panning and cultured in deep-well plates in super broth (SB) in the presence of 100 g/ml carbenicillin for 6 hours at 37 C., 300 rpm. Cultures were then induced with 2 mM IPTG and grown overnight. Cells were pelleted by centrifugation at 2,500 g for 15 min at 4 C., and supernatants removed and stored at 4 C. Periplasmic scFv was liberated by resuspending the cell pellets in 100 l of PBS and two rounds of freeze-thawing at 80 C. and 37 C. Plates were then centrifuged again as above, and supernatants were removed and added to their respective culture supernatant. Direct ELISA analysis of binding of single scFv-phage to Gal--(1.fwdarw.3)-Gal-BSA was then performed as described previously using HRP-conjugated anti-M13 antibody (GE Healthcare) in PBS-BT for 1 hour at 37 C. After extensive washing with distilled water, the wells were incubated with 100 l/well HRP substrate (DAKO), and reactions were stopped with 1M H.sub.2SO.sub.4 as detailed previously.

(29) Establishment of Individual scFv Clones for Stable Expression.

(30) Colonies selected based upon observed binding to Gal--(1.fwdarw.3)-Gal-BSA were used to perform phagemid purifications (NucleoBond, Macherey-Nagel). The resulting plasmid preparations were transformed by electroporation into E. coli strain TOP 10F (Invitrogen). An aliquot of each transformation was plated on LB carbenicillin agar to provide single colonies, permitting the testing of single-clone scFv properties.

(31) Expression and Purification of Soluble scFv.

(32) Colonies were cultured overnight in 5 ml SB containing 50 g/ml carbenicillin (37 C., 250 rpm shaking). 50 l of each clone culture was then added to 50 ml SB (50 g/ml carbenicillin and 20 mM MgCl.sub.2), and scFv production induced with the addition of 2 mM IPTG and cultured overnight. To isolate soluble antibody, the bacterial cells were pelleted by centrifugation and resuspended in equilibration buffer (50 mM NaH.sub.2PO.sub.4, 300 mM NaCl, 10 mM C.sub.3H.sub.4N.sub.2, pH 8.0) containing 20 mM phenylmethylsulfonyl fluoride. Periplasmic scFv was then liberated by sonication and cell debris removed by centrifugation, the supernatant was filtered through a 0.22 m syringe filter. Soluble scFv antibodies were purified by nickel chelation chromatography (Ni-NTA Superflow, Qiagen). For large scale production, culture volumes were increased to liter scale, with purification performed using an AKTA purifier FPLC (GE Healthcare) under nave conditions using HisTrap HP 1-ml (GE Healthcare) using ten column washes and a linear gradient up to 250 mM C.sub.3H.sub.4N.sub.2 for all elutions. Purified samples were dialysed against PBS and concentrated by membrane filtration (5 kDa filtration columns; Vivascience). Protein concentration on final products was estimated by BCA assay (Pierce). Purified scFv were stored at a concentration of 1 mg/ml at 4 C.

(33) DNA Sequencing of Single scFv Clones.

(34) Plasmid DNA was isolated from the bacterial pellets as above for expression and purification of soluble scFv. Multiple read sequencing reactions were performed for both strands of the complete scFv inserts (Eurofins MWG Operon). Multiple sequence alignment, translation and recognition of CDRs was performed using BLAST (ncbi.nlm.nih.gov/BLAST), ClustalW2 from EBI (ebi.ac.uk/Tools/msa/) and the CDR repository held at bioinf.org.uk/abs.

(35) Evaluation of Purified scFv by ELISA.

(36) Prior to analysis a sub-saturating concentration of scFv was determined by testing a gradient of scFv concentrations against 10 ug/ml Gal--(1.fwdarw.3)-Gal-BSA. A concentration of 4 ug/ml was determined to be optimal and was applied throughout further testing. Direct ELISA analysis of the specificity of purified scFv was assessed using a panel of 13 NGCs comprising Gal--(1.fwdarw.3)-Gal-BSA, Gal--(1.fwdarw.3)-Gal--(1.fwdarw.4)-GlcNAc-HSA, Gal--1-O-spacer-ITC, Gal--1-O-spacer-ITC, Gal--(1>2)-Gal-BSA, Gal--(1.fwdarw.4)-Gal-BSA, -Xyl-BSA, Fuc--4AP-BSA, Fuc--4AP-BSA, Neu5Ac-4AP-BSA, GlcNAc-BSA, Lac--4AP-BSA, LacNAc-BSA and two negative controls; HSA and BSA. ELISA performed as described with the exception of HRP-conjugated anti-HA antibody (Roche) in PBS-BT for 1 hour at 37 C. as the secondary antibody. After extensive washing with distilled water, the wells were incubated with 100 l/well HRP substrate (DAKO), and reactions were stopped with 1M H.sub.2SO.sub.4. Biotinylated GS-I-B4 lectin (Ey Laboratories Inc, San Mateo, Calif., USA) and -Gal Epitope (Gal1-3Gal1-4GlcNAc-R), mAb (M86) (Enzo Life Sciences, Inc.) were used as control references for assay assessment. Secondary antibodies used were streptavidin-conjugated HRP (Thermo Fisher Scientific) and a polyclonal rabbit anti-mouse Ig HRP (P 0260, Dako).

(37) Development of scFv-Based Competitive ELISAs for Gal--(1,3)-Gal.

(38) Each scFv was evaluated in a competitive ELISA format using free Gal--(1,3)-Gal as standard. Microtitre plate wells were coated and blocked as previously described. Standard solutions were prepared in PBS (pH 7.2), ranging in concentration from 2 g/ml (5.84 M) to 20 mg/ml (58.4 mM). For the assay, 50 l of each standard and buffer blank were added to designated wells in triplicate, followed by 50 l of a sub-saturating concentration (1 g/ml) of the appropriate scFv, giving a final standard concentration in the assay of 1 g/ml (2.92 M) to 10 mg/ml (29.2 mM). The plate was incubated for 1 hour at 37 C. with shaking After washing, bound scFv was detected as previously described. The ratios of the OD for each standard versus the OD given by the blank (B/Bo) were plotted versus standard concentration and standard curve plotted using SigmaPlot (v11, Systat Software Inc). The detection limits were determined as the concentration corresponding to the mean response for zero standard minus three times the standard deviation (SD). Inhibition of scFv by a panel of free sugars. scFv binding to Gal--(1.fwdarw.3)-Gal-BSA was determined against a panel of 21 free form sugars; D-Cellibiose, Melibiose, D-Raffinose, Lactulose, Palatinose, -Gentibiose, D+ Trehalose, D+ Turanose, L Fructose, D+ Glucose, Sucrose, Galacto-N-biose, 4-Galactobiose, gal-(1-3)-gal--(1-4)-gal, gal-(1-3)-gal--(1-4)-glc, gal-(1-3)-gal--(1-4)-gal-all-3)-gal, Laminarbiose, 1-4-D-Xylobiose, N-Acetyl-D-glucoseamine (GlcNAc), Galactose, N-Acetyl-D-lactosamine (LacNAc), (sourced from Sigma Aldrich and Dextra, UK). Inhibitions were performed by serial dilutions of free sugars and a sub-saturating concentration scFv added to each dilution and pre-incubated for 60 minutes at 37 C., prior to performing ELISA against Gal--(1.fwdarw.3)-Gal-BSA as used throughout. Inhibition concentration range used is provided in Table 1. The percent binding of the scFv was plotted against the free Gal--(1.fwdarw.3)-Gal-BSA concentration, and the 50% effective dose determined (ED.sub.50; dose of free sugar causing 50% displacement of the scFv).

(39) Results

(40) Immune Response and Library Construction.

(41) Analysis of the serum antibody response following immunization with Gal--(1.fwdarw.3)-Gal-BSA indicated a strong response to the target glycan motif in both immunized chickens (FIG. 1a). scFv libraries displayed on filamentous phage were generated from the combined RNA extracted from spleen and bone marrow of each chicken (Andris-Widhopf et al., 2000). Colony PCR revealed that all individual clones tested contained full size inserts and sequence diversity was confirmed by BstN1 restriction mapping of amplified products (FIG. 1b). The initial size of each library was estimated at approximately 510.sup.7 transformants. Libraries were combined and panned as a single scFv-phage library.

(42) Isolation of Anti-Gal--(1.fwdarw.3)-Gal scFv-Phage Particles.

(43) Three rounds of biopanning were performed at room temperature against Gal--(1.fwdarw.3)-Gal-BSA under identical conditions and bound phage were eluted at low pH. Following the second round of panning, the presence of an enriched population of phage-displayed scFvs demonstrating binding to Gal--(1.fwdarw.3)-Gal-BSA was confirmed by direct ELISA (FIG. 1c). No further enrichment was observed after the third round. Minimal cross reactivity was observed against the carrier proteins, BSA or HSA. From the output phage of the second and third panning rounds, 96 individual phage clones were selected at random, cultured individually and analysed for specific binding activity. Of the 96 clones tested, 74 showed binding greater than 3 times background to the immobilised NGC (FIG. 2). Six of the higher binding clones (A4, A11, D9, F1, G12 and H3) were chosen for sequencing, and their encoding phagemids were transformed into TOP10 cells for inducible expression and generation of soluble scFv for downstream affinity purification.

(44) Sequence Analysis of Isolated Clones.

(45) Analysis of the nucleotide sequences of the V.sub.H and V.sub.L regions of the six scFvs demonstrated a high degree of consensus with germline chicken V.sub.H and V.sub.L regions. All clones exhibited considerable divergence in the V.sub.L complementarity determining region 1 (CDR1), CDR2 and CDR3 regions, with less variability in the V.sub.H region. Indeed the V.sub.H CDR 1 was conserved across all clones (FIG. 3). The sequence data suggests that the scFvs isolated were generated in an antigen-driven response, with particular reference to the V.sub.L regions, rather than being naive antibody sequences. Four unique sequences, representing clones A11/D9, F1/G12, H3 and A4, were identified (FIG. 3). Within the V.sub.L CDRs, single transitions occurred in a number of positions; (i) V.sub.L CDR1 where four codons were identified alternating from tyrosine (Y) to histidine (H) to asparagine (N) to lysine (K); (ii) in V.sub.L CDR2 where asparagine (N) to glutamine (Q) to lysine (K) occurred and (iii) V.sub.L CDR3 where threonine (T) to serine (S) to glycine (G) to alanine (A) was observed. In contrast, fewer polymorphic sites were observed across the V.sub.H regions, with A11/D9, H3 and A4 showing consensus across the CDR1, CDR2 and CDR3. F1/G12 differed from the other clones within the CDR3 region. Across the linker region two substitutions from the germline sequence were observed (serine (S) to glutamine (Q) and a glycine (G) to serine (S)) along with 2 serine (S) deletions shortening the linker region of A11/D9. Such observations of linker region alterations have been reported within similar libraries previously with no effect on library efficiency (Finlay et al., 2006).

(46) Expression and Purification of Soluble scFv.

(47) The four unique scFv clone sequences represented by A4, A11, G12 and H3, were carried forward for expression and purification of soluble scFv. Three were successfully expressed in bacterial culture (A4, A11 and G12), purified using Ni-NTA affinity chromatography, and purity confirmed by gel electrophoresis (FIG. 4). Yields of purified scFv were approximately 6.5 mg/L of bacterial culture medium. All purified scFv demonstrated stability at 4 C. for periods of up to 6 months in PBS. One clone, H3, failed to purify under both native and denatured conditions.

(48) ScFv Specificity Evaluation by Direct ELISA.

(49) The specificities of the purified scFv clones, A4, A11 and G12, were tested in a direct ELISA format against a range of Gal--R related neoglycoconjugates (NGCs). All three scFvs were specific for Gal--(1.fwdarw.3)-Gal-BSA, binding with slightly higher intensity to the Gal--(1.fwdarw.3)-Gal--(1.fwdarw.4)-GlcNAc-HSA compared to Gal--(1.fwdarw.3)-Gal-BSA (FIG. 5). This difference was likely due in part to the different molar substitution ratios of the glycan motif per molecule of albumin (16 and 23 for the disaccharide and trisaccharide NGCs, respectively), although the difference was not as great as expected suggesting that the scFvs having slightly greater affinity for the Gal--(1.fwdarw.3)-Gal disaccharide compared to the trisaccharide. The binding to the disaccharide and trisaccharide epitopes and not to the monosaccharide (Gal--1-O-spacer-ITC) or the Gal--(1>2)-Gal-BSA and Gal--(1.fwdarw.4)-Gal-BSA structures demonstrates that the -(1.fwdarw.3) linkage is a requirement for binding. Similar binding specificity for Gal--(1.fwdarw.3)-Gal epitope was shown by all three scFvs tested, regardless of sequence diversity in the CDR regions (FIG. 3). No binding was observed to the other structures tested in the direct ELISA (Gal--1-O-spacer-ITC-BSA, Gal--1-O-spacer-ITC-BSA, Gal--(1>2)-Gal-BSA, Gal--(1.fwdarw.4)-Gal-BSA, D-xylose--BSA, Fuc--4AP-BSA, Fuc--4AP-BSA, Neu5Ac-4AP-BSA, GlcNAc-BSA, Lac--4AP-BSA, LacNAc-BSA) or to the carrier proteins, HSA, BSA and BSA-4AP. By comparison, the lectin GS-1-B4 showed significantly higher binding to the trisaccharide NGC compared to the disaccharide NGC and also bound to all Gal--1-R-containing NGCs (FIG. 5s), in consensus with previous reports. Therefore, the scFvs generated demonstrate a higher specificity than the GS-1-B4 lectin, for Gal--(1.fwdarw.3)-Gal. In parallel with the lectin GS-1-B4 and the monoclonal M86, all scFvs were able to detect the target motif on the natural glycoprotein, murine laminin, in direct ELISA (data not shown). Murine laminin is known to display the Gal--(1.fwdarw.3)-Gal motif.

(50) The spectacular specificity of the scFvs has also been determined by analysis on an NGC microarray containing over 50 different structures. As shown in FIG. 8 the antibody fragments of the present invention, A4, G12 and A11, have a much higher specificity for the Gal--(1.fwdarw.3)-Gal epitope than does the prior art lectin, GS-1-B4. ScFv-based competitive ELISA. Competitive ELISAs were established with each of the three scFvs examined using as standard the free Gal--(1.fwdarw.3)-Gal disaccharide within the concentration range 0.001 to 10000 g/ml (2.93 nM to 29.3 mM). The curves given by scFvs G12 and A4 were very similar with ED.sub.50 values of 158 g/ml and 311 g/ml (0.46 and 0.91 mM), respectively (FIG. 6). ScFv A11 gave a less sensitive standard curve (ED.sub.50=1225 g/ml, 3.58 mM; FIG. 6). No standard curve could be generated with either the lectin GS-1-B4 or monoclonal M86 within the same concentration (data not shown). A panel of 21 related sugars were tested in the scFv-based competitive assays (Table 1). Of the sugars tested, only those bearing the Gal--(1,3)-Gal epitope demonstrated inhibition at detectable levels. All three scFvs were more reactive to the disaccharide than Gal--(1,3)-Gal-containing trisaccharides, as shown by lower inhibitory activity of the trisaccharides and supporting the results obtained in the direct ELISA. Also, scFvs A4 and A11 showed slightly higher inhibition (3 to 10%) when presented with the free tetrasaccharide (Gal--(1,3)-Gal--(1,4)-Gal--(1,3)-Gal), compared to the trisaccharides (Gal--(1,3)-Gal--(1,4)-Gal or Gal--(1,3)-Gal--(1,4)-Glc). No inhibition was observed in the presence of any monosaccharide within the ranges indicated, supporting the conclusion from the direct assay that monosaccharide structures are not capable of inhibiting binding to any of the scFvs to the Gal--(1,3)-Gal structure (Table 1).

(51) ScFv A4 gave the most consistent readings and was selected for further in evaluation. FIG. 7 shows the A4 composite standard curve, giving an ED.sub.50 of 327 g/ml [0.95 mM] and detection limit of 3.9 ng/ml [10 nM]. The assay was able to detect the Gal--(1.fwdarw.3)-Gal motif when protein bound in the form of NGCs and on the murine glycoprotein, laminin. The disaccharide NGC interacted with the scFvs in a similar manner to the free sugar, as indicated by the parallelism of the response obtained over the concentration range 0.25 to 1 mg/ml. Assay results indicated that 1 mg/ml of the Gal--(1.fwdarw.3)-Gal containing NGC contained 42 g/ml of the disaccharide. This corresponded to a disaccharide:protein molar ratio of 9.4:1. The range as determined by MALDI trace is 10 to 25 residues, with an average of 16:1 per albumin. Similarly, the Gal--(1.fwdarw.3)-Gal--(1,4)-Gal containing NGC with glycan:protein molar ratio range of 15 to 31, with an average of 23:1 by MALDI was determined to have a molar incorporation ratio of 19.7:1 by competitive ELISA. Native laminin was too viscous to be analysed in the competitive ELISA. However, heat treatment reduced the viscosity and assay results indicated that there were 39 moles of the Gal--(1.fwdarw.3)-Gal residue per mole of murine laminin.

(52) Affinity Values

(53) Dissociation constants (Kd) for the scFvs were calculated from the measured association (kon) and dissociation (koff) rate constants by using surface plasmon resonance (SPR) BIAcore instrumentation and software (GE). Determined Kd's for the scFv are:

(54) A4: 5.7310.sup.8 M

(55) G12: 1.8010.sup.8 M

(56) A11: 8.0410.sup.9M

(57) Under the same conditions, no binding was observed with the prior art lectin, GS-1-B4 or prior art monoclonal antibody, M86. In general, it is accepted that lectin-carbohydrate interactions are weaker than antigen-antibody interactions, with lectins having lower affinity constants in the range, K.sub.d=10.sup.3 to 10.sup.6 M for glycans. Thus the scFvs have greater affinity for their target glycan than existing binders.

(58) With reported anti carbohydrate antibodies against a number of glycan epitopes having affinity constants in the range of K.sub.d=10.sup.5 to 10.sup.8 M (determined at steady state equilibrium in SPR) indicating that those scFv identified here in are amongst the highest affinity antibody fragments reported against glycan epitopes as defined by SPR.

(59) TABLE-US-00003 SequenceInformation SeqIdNo.1:Genericsequenceofthefragments oftheinvention: (SEQIDNO:1) QAALTQPSSVS[T/A]NPGGTVKITCSGG[N/G/-][S/-][Y/-][G/-] [G/-][S/-][G/Y][N/H/Y]YGWYQQKSPGSAPVTVIYSN[N/D][K/ Q/N]RPS[D/G]IPSRFSGS[T/K]S[G/D]ST[A/G/S]TLTITGVQ[V/ A]DDEAVY[F/Y]CG[A/S/T]YD[N/S][S/-][N/-][T/S]Y[V/A]G [V/I]FGAGT[T/A]LTVLGQSSRSS[S/-]GGGSSGGGGSAVTLDESGGG LQTPGG[G/A]LSLVCKASGFTFSSYSMQWVRQ[T/A]PGKGLEFVAGIG [Y/N]SD[S/R]YTYFGPAVKGRATISRDNGQ[N/S]T[V/L]RLQLNNLR AEDTATY[Y/F]CARS[A/G]D[T/S][I/G][Y/N]GCTHPWCSADNI [D/N]AWGHGTEVIVSSTSGQAGQ Uniqueindividualsequencesforeachclone SeqIdNo.2:scFv-A4 QAALTQPSSVSTNPGGTVKITCSGGNGNYGWYQQKSPGSAPVTVIYSNN KRPSDIPSRFSGSKSGSTATLTITGVQVDDEAVYFCGAYDNTYVGVFGA GTTLTVLGQSSRSSSGGGSSGGGGSAVTLDESGGGLQTPGGGLSLVCKA SGFTFSSYSMQWVRQTPGKGLEFVAGIGYSDSYTYFGPAVKGRATISRD NGQNTVRLQLNNLRAEDTATYYCARSADTIYGCTHPWCSADNIDAWGHG TEVIVSSTSGQAGQ SeqIdNo.3:scFv-G12 QAALTQPSSVSANPGETVKITCSGGSYHYGWYQQKSPGSAPVTVIYSNN QRPSGIPSRFSGSTSDSTGTLTITGVQADDEAVYFCGSYDSSNTYAGIF GAGTTLTVLGQSSRSSSGGGSSGGGGSAVTLDESGGGFQTPGGALSLVC KASGFTFSSYSMQWVRQAPGKGLEFVAGIGNSDRYTYFGPAVKGRATIS RDNGQSTLRLQLNNLRAEDTATYFCARSGDSGNGCTHPWCSADNINAWG HGTEVIVSSTSGQAGQ SeqIdNo.4:scFv-A11 QAALTQPSSVSANPGETVKITCSGGGSYGGSYYYGWYQQKSPGSAPVTV IYSNDNRPSDIPSRFSGSTSGSTSTLTITGVQVDDEAVYYCGTYDSSYV GIFGAGTALTVLGQSSRSSGGGSSGGGGSAVTLDESGGGLQTPGGGLSL VCKASGFTFSSYSMQWVRQTPGKGLEFVAGIGYSDSYTYFGPAVKGRAT ISRDNGQNTVRLQLNNLRAEDTATYYCARSADTIYGCTHPWCSADNIDA WGHGTEVIVSSTSGQAGQ [ ] indicating a transition; - indicating deletion
scFvs: Interaction Studies by Circular Dichroism

(60) The scFvs A4, A11 and G12 were used to check their interaction with Gal(1,3)Gal1-OMe (Carbosynth) and with melibiose, the latter as negative control.

(61) Two types of experiments were performed: firstly, the near UV circular dichroism (CD) spectra of the scFvs in the absence and presence of 4 mM of both disaccharides have been compared. Secondly, the effect of the disaccharides on the thermal stability of the scFvs has been studied.

(62) Experimental Procedure:

(63) Samples were diluted in NaPi (10 mM)-NaCl (150 mM) pH 7.2 and either concentrated by ultrafiltration twice (for small amounts) or exhaustively dialyzed. CD spectra were acquired at 20 C. in a J-810 spectropolarimeter, equipped with a Peltier temperature control system, using a bandwidth of 1 nm, a scan rate of 20 nm/min and a response time of 4 s, collecting data every 0.2 nm and 3 accumulations for each spectrum. Near-UV spectra were registered at around 1.0 mg ml.sup.1 protein concentration in 1-cm path-length cells while far-UV spectra were recorded in 0.1 cm path-length quartz cells at a protein concentration of around 0.2 mg ml.sup.1. For all CD spectra, the corresponding buffer baseline was subtracted. Thermal stability was evaluated by measuring ellipticity changes at 208 nm while increasing temperature from 20 to 90 C., with a constant rate of 40 C./h. Ellipticity changes were collected every 0.2 C. and spectra were collected every 10 C. The thermal denaturation profiles were fitted to a sigmoidal function using the equation:

(64) $\left(T\right)={}_N+\underset{i=1}{\overset{n}{.Math.}}{}_i\left\{\exp \left[-{\mathrm{HD}}_i\left(T_{\mathrm{mi}}-T\right)/R.Math.T_{\mathrm{mi}}.Math.T\right]\right\}/\left\{1+\exp \left[-{\mathrm{HD}}_i\left(T_{\mathrm{mi}}-T\right)/R.Math.T_{\mathrm{mi}}.Math.T\right]\right\}$

(65) where (T) represents the ellipticity at temperature T (Kelvin), .sub.N the ellipticity of the native protein, n the number of transitions, .sub.1 the ellipticity increment of transition i, R the gas constant and T.sub.mi and HD.sub.i the temperature at the transition midpoint (melting temperature) and the parameter which describes the cooperativity (slope) of the corresponding transition, respectively.

(66) Results:

(67) In general, molar ellipticity values per residue in the near CD were between 80 and 40 grad.Math.cm.sup.2.Math.dmol.sup.1 for the three scFvs, which tend to be low in terms of usual values in proteins. Starting with G12, the spectrum (in black, FIG. 1A) showed negative ellipticity between 250-280 nm (region dominated by Phe and Tyr electronic transitions) and a small positive band at 292 nm (region dominated by Trp electronic transitions). While melibiose did not cause any significant changes in the shape/intensity of the spectrum (spectrum in red, FIG. 1A), a clear change was detected in the presence of Gal(1,3)Gal1-OMe: the band at 292 nm tended to disappear while higher negative values were observed in the 250-290 nm region (spectrum in green, FIG. 1A). Thus, the spectrum in the presence of Gal(1,3)Gal1-OMe seems to indicate a change in the environment of aromatic residues contributing to the spectrum.

(68) In the case of A4, the CD spectrum (in black, FIG. 1B) is also negative between 250 and 290 nm and there is a band (less intense than for G12) at 291 nm too. In addition, there is a clear negative band at 300 nm (assigned to Trp). As for G12, the effect of melibiose remained unchanged (spectrum in red, FIG. 1B). However, the molar ellipticity per residue in general decreased in the presence of Gal(1,3)Gal1-OMe, especially between 260-280 nm, giving rise to lower negative values (spectrum in green, FIG. 1B). The band at 300 nm also showed a decrease in intensity, while the band at 291 nm hardly changed.

(69) Regarding A11, the CD spectrum (in black, FIG. 1C) was similar to those of scFvs G12 and A4, with negative values of the molar ellipticity per residue between 250-290 nm and two bands, one positive at 292 nm and the second one negative at 302 nm. No significant changes were observed in the presence of either melibiose (spectrum in red, FIG. 1C) or Gal(1,3)Gal1-OMe (spectrum in green, FIG. 1C). This indicates that, if there is any interaction with the sugar, the environment of the aromatic groups of the protein is not significantly affected.

(70) In the second set of experiments, the thermal stability of the scFvs in the absence and presence of the disaccharides was investigated. With this type of experiment, we were able to get: 1) the far UV CD spectra at increasing temperatures, which gave information on changes in the secondary structure of the proteins upon heating and 2) the profile of the change in ellipticity at a certain wavelength, also with increasing temperatures, that allowed the calculation of the temperature of transition (Tm). Direct comparison of the Tm in the absence and presence of sugar provided information about the effect of the sugar on the scFvs, a thermal stabilisation (i.e. an increase in the Tm in the presence of the sugar) being indicative of interaction.

(71) The far UV CD spectra of all scFvs at 20 C. showed positive values of molar ellipticity at short wavelengths (below 208 nm) and a negative band between 208-240 nm, features that are typical of secondary structure (FIG. 2 and spectra in black in FIG. 3). FIG. 2 shows the good superimposition of the spectra for the three scFvs, which indicates a similar secondary structure for the three of them. No changes were observed in the spectra in the presence of either Gal(1,3)Gal1-OMe (FIG. 3, spectra in green) or melibiose (not shown). After heating at 90 C., the positive band disappeared and the spectrum tended to higher negative values of ellipticity in almost the whole range of wavelengths (FIG. 3, spectra in red), indicating a loss of their native secondary structure. The global change in the spectrum from 20 C. to 90 C. for G12 (FIG. 3A) and A4 (FIG. 3B) was similar in the absence or presence of the sugars; however, the final species after heating A11 are different in the absence and presence of Gal(1,3)Gal1-OMe (FIG. 3, spectra in blue). This difference was not observed in the presence of melibiose.

(72) Comparison of the profiles of the change in ellipticity at a given wavelength with increasing temperature (FIG. 4) clearly showed that Gal(1,3)Gal1-OMe induced an important stabilisation (between 7.7 and 10.2 C.) of the three scFvs (Table 1). On the contrary, the presence of melibiose did not result in a significant stabilisation (0.2 C.) of either G12 or A11 (not checked for A4).

(73) Denaturation was monitored at two different wavelengths for scFv G12 (Table 1). Comparative analysis of the effect of sugars on thermal stability of G12, by measuring changes in ellipticity at 218 nm in their absence and presence, shows that the addition of 4 mM melibiose does not alter either the T.sub.m or the HD (describing cooperativity) of the unfolding process, whereas the presence of 4 mM Gal(1,3)Gal1-OMe increased both parameters. Data obtained at 208 nm also revealed an increment of 8.8 C. in the T.sub.m and a slightly more cooperative unfolding (+6.8 Kcal/mol) in the presence of Gal(1,3)Gal1-OMe. For clarity, only denaturation profiles collected at 208 nm, in the absence and presence of Gal(1,3)Gal1-OMe, are shown in FIG. 4A.

(74) TABLE-US-00004 TABLE 1 Transition temperature (T.sub.m) and cooperativity parameter (HD) of the thermal denaturation of scFvs in the absence and presence of 4 mM melibiose or Gal(1,3)Gal1-OMe. scFv G12 scFv A4 scFv A11 HD HD HD T.sub.m ( C.) (Kcal/mol) T.sub.m ( C.) (Kcal/mol) T.sub.m ( C.) (Kcal/mol) Pure 218 66.4 0.4* 116 21 n.d. n.d. n.d. n.d. 208 64.7 0.2** 64 4 64.6 0.2** 57 3 61.3 0.1* 134 7 +melibiose 218 66.5 0.3* 116 16 n.d. n.d. n.d. n.d. 208 n.d. n.d. n.d. n.d. 61.1 0.1* 141 7 +Gal(1,3)Gal 218 74.3 0.4* 137 24 n.d. n.d. n.d. n.d. 1-OMe 208 73.5 0.3** 71 5 74.8 0.3** 52 3 69.0 0.2* 95 6 N.d.: not determined. *Samples prepared by ultrafiltration. **Samples prepared by dialysis.

(75) As the change of molar ellipticity at 208 nm was significantly bigger than at 218 nm for G12 (see FIG. 3), monitoring at 208 nm was chosen for the rest of scFvs. In the case of A4, while the cooperativity of the process (shown in Table 1 and evidenced by the slope of the denaturation curve in FIG. 4B) did not change significantly (4.7 Kcal/mol), the T.sub.m increased around 10 C. in the presence of 4 mM Gal(1,3)Gal1-OMe. Finally, for A11, we observed a difference not only in the value of the T.sub.m, but also in the cooperativity of the process, which is clearly higher than for G12 and A4 (Table 1), and in the molar ellipticity value at the end of the denaturation (Table 1 and FIG. 4C). While there is no significant difference between the curves obtained with or without melibiose, there is an increase of 7.7 C. in the T.sub.m of the antibody when Gal(1,3)Gal1-OMe is present and interestingly, a large decrease in the cooperativity when comparing to the same scFv in the absence of this disaccharide (38.5 Kcal/mol). When comparing to the cooperativity of the process for G12 and A4 in the presence of Gal(1,3)Gal1-OMe, the value for the three proteins gets closer than in the absence of this disaccharide.

(76) In summary, the results obtained clearly prove the existence of interaction between the three scFvs, A4, A11 and G12 and Gal(1,3)Gal1-OMe. Moreover, the lack of interaction with melibiose evidences the specificity in the recognition. G12 showed a significant change in the near UV CD spectra, indicating that the binding of the disaccharide significantly affects the environment of aromatic residues. Furthermore, thermal denaturation experiments unequivocally demonstrated a stabilizing effect of Gal(1,3)Gal1-OMe on the three scFvs, with a stabilisation of 10.2 C. for A4, 8.8 C. for G12 and 7.7 C. for A11.

DISCUSSION

(77) Although known for over 30 years, there is still considerable interest in the Gal--(1,3)-Gal motif, commonly found as a terminal motif on glycoproteins and glycolipids in a range of species, with the exception of humans and chickens which lack the enzyme involved in the synthesis of the disaccharide. This glycan structure still remains relevant to xenotransplantation efforts and the potential to exploit the relatively large quantities of natural anti-Gal--(1,3)-Gal antibodies present in human serum to increase immunogenicity in cancer immunotherapy has been widely explored. Modification of cancer cells or cancer-associated molecules, such as mucins, with the Gal--(1,3)-Gal epitope has been shown to increase uptake by antigen-presenting cells and enhance the immune response. The ability to measure the loading of the motif on cells is important for this work, but the recently reported occurrence of this immunogenic motif on glycoprotein biopharmaceuticals, with resulting adverse reactions in patients, has particularly highlighted the need for specific binding agents and a convenient analytical method for Gal--(1,3)-Gal.

(78) The most commonly utilised biomolecule for the detection and profiling of the Gal--(1.fwdarw.3)-Gal epitope is the lectin GS-1-B4, being highly specific for -Gal residues. The affinity of GS-1-B4 to different -Gal carbohydrates is a drawback in using this lectin as a reliable marker for Gal--(1.fwdarw.3)-Gal. The lectin from mushroom Marasmius oreades (MOA) has been reported to be more specific for Gal--(1,3)-Gal and Gal--(1,3)-Gal--(1,4)-GlcNAc epitopes, but yet binds strongly to the B blood group antigen, which contains an L-Fuc -(1,2) linked to the Gal at the reducing end. Naturally occurring serum human antibodies against this and other non-human glycan epitopes have not been reliably used as an analytical tools. Traditional immunisation approaches are not very reliable for generation of high affinity antibodies against specific carbohydrate moieties and do not often yield antibodies with characteristics suitable for assay development. The use of phage display technology with recombinant antibody fragment libraries offers more promise, giving access to a larger repertoire of antibodies including anti-self antibodies that would not normally be available due to tolerisation. However, only a few successful examples have been published to date. The generation of antibodies toward carbohydrate moieties has to date been difficult due to their low affinity with only few successful examples published. The selection of initial library source and potential immunization regimes need careful thought and design, permitting for glycan presentation to mimic that was encountered under natural conditions. The selection here to utilize immunized chicken libraries allowed for single primers targeting the conserved regions flanking the unique functional V.sub.H and V.sub.L genes in to be amplified and fused as scFv. This enables the complete spectrum of rearranged variable fragments with subsequent cloning of highly diverse chicken immunoglobulin repertoires, as opposed to more complicated systems.

(79) Chicken recombinant antibodies, like mouse or human derived antibodies, can be expressed in various forms (Fab or scFv), with a number of groups reporting the construction of chicken recombinant antibodies against a variety of protein targets. The optimization of panning conditions is keys to successful phage display against carbohydrate targets, taking into account the lower affinity of carbohydrate-protein interactions in comparison with protein-protein interactions.

(80) Competitive binding assays are widely used for determination of low molecular weight compounds in complex samples, with often no viable alternative. They represent a versatile, robust assay configuration, being adaptable for high throughput or single-use, point-of-care formats. Competitive assays are, however, demanding in terms of the binding agent, with assay sensitivity being directly dependent on affinity of the binder for the analyte. We have demonstrated that all three scFvs that were purified could be used in competitive assays for Gal--(1,3)-Gal, allowing detection of the disaccharide at low ng/ml levels (detection limit estimated at 3.9 ng/ml [10 nM]), which exceeds reported detection limits of most lectins, typically at the g/ml level, when tested in similar plate assays. The exceptional specificity of the scFvs was also retained in this format. We have shown that the assay can detect the motif in the context of a glycoprotein and can be used to determine the level of incorporation of the motif on the protein. Thus, the assay provides the option of either direct analysis of the motif on a glycoprotein of interest or measurement of the disaccharide following release by, for example, the enzyme endo-beta-galactosidase C from Clostridium perfringens.

(81) The observed differences in the CDR sequences between the three scFvs best characterised (scFv-G12, scFv-A4 and scFv-A11) may have resulted from a diverse in vivo population of Gal--(1.fwdarw.3)-Gal specific B cells in each individual animal or possibly from the pooled cDNA of the two animals used to produce the library. Several amino acid changes (S.fwdarw.Q; G.fwdarw.S) and deletions, typically serine, occurred in the linker sequences of each of the sequenced fragments. The modifications within the linker region did not appear to affect the stability or function of the linker significantly. Single scFv clones, induced in bacterial culture, produced soluble scFv that was readily purified by Ni-NTA chromatography. Without any optimization, the soluble yield was satisfactory and reproducible. scFv stability and presentation can be influenced by concentration, pH and temperature along with other factors. It is possible that individual preparation of scFvs may contain an unknown proportion of dimeric antibody, which is functionally bivalent and can lead to changes in observed kinetics.

(82) The purified and tested scFv antibodies (A4, A11 and G12) demonstrated high specificity to the Gal--(1.fwdarw.3)-Gal epitope, which may indicate that they have future use as fine specificity reagents against this epitope.

(83) TABLE-US-00005 TABLE 1 Specificity study of competitive ELISAs based on the three anti-gal-alpha1-3 gal scFvs Highest concen- Inhibition % Maximum inhibition tration observed at Max Conc Free Sugars (mg/mL) (Y/N) A11 A4 G12 gal-(1,3)-gal- at 1 mg/ml Y 51.06 36.4 33.93 BSA (NGC) gal--(1,3)-gal- 1 Y 76.83 68.18 64.43 -(1,4)-gal gal-(1,3)-gal- 1 Y 73.88 66.20 83.03 -(1,4)-glc gal-(1,3)-gal- 1 Y 78.41 73.48 73.54 -(1,4)-gal-- (1,3)-gal Galcato-N-biose 1 N / / / 4 galactobiose 1 N / / / Laminarbiose 1 N / / / 1-4-D-Xylobiose 1 N / / / N-Acetyl-D- 1 N / / / lactosamine . . . (LacNAc) D-cellibiose 10 N / / / Melibiose 10 N / / / D-Raffinose 10 N / / / Lactulose 10 N / / / Palatinose 10 N / / / -Gentibiose 10 N / / / D+ Trehalose 10 N / / / D+ Turanose 10 N / / / L- Frucose 10 N / / / D+ Glucose 10 N / / / Sucrose 10 N / / / N-Acetyl-D- 10 N / / / glucoseamine . . . (GlcNAc) Galactose 10 N / / /

(84) The words comprises/comprising and the words having/including when used herein with reference to the present invention are used to specify the presence of stated features, integers, steps or components but does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

(85) It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination.

REFERENCES

(86) Andris-Widhopf J, Rader C, Steinberger P, Fuller R, Barbas C F (2000) Methods for the generation of chicken monoclonal antibody fragments by phage display. J. Immunological Methods 242:159-81 Available at: http://www.ncbi.nlm.nih.gov/pubmed/10986398. Bosques C J, Collins B E, Meador J W, Sarvaiya H, Murphy J L, Dellorusso G, Bulik D a, Hsu I-H, Washburn N, Sipsey S F, Myette J R, Raman R, Shriver Z, Sasisekharan R, Venkataraman G (2010) Chinese hamster ovary cells can produce galactose--1,3-galactose antigens on proteins. Nature biotechnology 28:1153-6 Available at: http://www.ncbi.nlm.nih.gov/pubmed/21057479 [Accessed Aug. 12, 2011].