Patent Application: US-85235410-A

Abstract:
a method of generating and isolating a recombinant high affinity anti - sialic acid antibody molecule comprises the steps of immunising a host with an immunogen comprising a conjugate of sialic acid and a carrier protein to generate an anti - sialic acid polyclonal serum , isolating a sample of rna from the immunised avian host , and generating and screening of a library of recombinant antibody molecules from the rna sample , and isolating a recombinant high affinity anti - sialic acid antibody molecule . the antibody molecule is selected from the group consisting of : whole antibodies ; scfv fragments ; and fab fragments , and the host is gallus domesticus . a recombinant avian antibody fragment having high binding affinity to sialic acid and obtainable by the method of the invention , and an anti - sialic acid polyclonal serum obtainable by immunising an avian host with a conjugate of sialic acid and carrier protein , are also described .

Description:
the generation of anti - carbohydrate antibodies is a notoriously difficult task . carbohydrate antigens are self - antigens and thus have low antigenic potential . as a consequence , a poor immune response is generated from carbohydrate antigens and the antibody produced is typically a low - affinity immunoglobulin m ( igm ). 7 conventional hybridoma technologies have been shown to be ineffective at generating high - affinity monoclonal antibodies against a range of carbohydrate structures . in contrast , display technologies such as phage display offer greater potential , as this technology can sometimes generate antibodies against ‘ self - antigens ’. 8 moreover , the complementarity - determining regions ( cdrs ) of scfv fragments can be targeted and mutagenised to enhance their sensitivity and specificity for particular carbohydrate elements . 9 the vast majority of anti - carbohydrate antibodies generated have relatively low - affinities and are therefore not suitable for in vitro diagnostics . to overcome the problem of immunological tolerance , a carefully designed protocol was developed for the generation of a suitable immune response to sialic acid . for our study , gallus domesticus was selected as the animal model . several reports have suggested that the primary antigen on neu5gc , the hanganutziu - deicher ( hd ) antigen , is absent on avian cells . this observation permitted the selection of a novel neu5gc - containing immunogen for immunisations . sialic acid was seen as foreign by the avian immune system and thus generated a strong immune response . for t - cell recognition and the subsequent generation of an immune response , the neu5gc monosaccharide was conjugated to a suitable carrier protein . human serum albumin ( hsa ) was deemed to be appropriate as it facilitated the conjugation of multiple neu5gc residues . a second sialic acid protein conjugate was also designed . this conjugate , bovine serum albumin ( bsa ), was used for phage display screening , recombinant protein screening and the measurement of the avian polyclonal serum response . both the neu5gc - bsa and neu5gc - hsa conjugates were custom synthesised by carbohydrate synthesis , u . k . an overview of the synthesis scheme is given in fig1 . all procedures involving the use of animals were sanctioned by the local ethics committee at dublin city university ( dcu , dublin , ireland ). in addition , these experiments were approved and licensed by the irish department of health and children ( dublin , ireland ) and were performed with the highest standards of care . a white male leghorn chicken ( aged one month ) was injected with 250 μg / ml of the hsa - neu5gc conjugate ( 35 monosaccharide units of neu5gc per mole of hsa protein ) and an equal volume of freund &# 39 ; s complete adjuvant ( fca ). the chicken was injected subcutaneously ( 200 μl ) at four different sites . following the first injection , the second , third and fourth boosts were given at two , three , and two weekly intervals respectively . for boosting injections , an equal volume of freund &# 39 ; s incomplete adjuvant was used . a bleed was taken after the fourth boost and a serum - based polyclonal response was determined by elisa . for comparative analysis , a pre - bleed sample ( taken from the same host pre - immunisation ) was also selected . a direct elisa was used to determine the serum antibody titre from an immunised chicken ( fig2 a ). a maxisorp plate ( nunc a / s , denmark ) was coated overnight at 4 ° c . with 5 μg / ml of the bsa - neu5gc conjugate ( custom synthesised by carbohydrate synthesis , u . k .). the plate was blocked with 3 % ( w / v ) bsa in phosphate - buffered saline ( pbs ( ph 7 . 2 ); nacl 5 . 84 g / l , na 2 hpo 4 4 . 72 g / l and nah 2 po 4 2 . 64 g / l ) for 1 hour at 37 ° c . the plate was washed three times with pbst , ph 7 . 2 ( pbs containing 0 . 5 % ( v / v ) tween ) followed by three times with 1 × pbs , ph 7 . 2 . a series of dilutions ranging from neat to 1 in 1 , 000 , 000 of the chicken serum , diluted in 1 % ( v / v ) bsa 1 × pbst ( ph 7 . 2 ), were added to the elisa plate in triplicate and incubated for 1 hour at 37 ° c . the plate was washed three times with 1 × pbst ( ph 7 . 2 ) followed by three times with 1 × pbs ( ph 7 . 2 ). 100 μl of rabbit anti - chicken igy , conjugated with horseradish peroxidase ( hrp ) ( sigma , u . k ., 1 : 2000 1 % ( v / v ) bsa pbst ), was added to the plate and then incubated for 1 hour at 37 ° c . the plate was washed 3 times with 1 × pbst ( ph 7 . 2 ) and 1 × pbs ( ph 7 . 2 ) and 100 μl of tmb substrate ( sigma - aldrich , u . k .) solution ( 2 mg / ml tmb in citrate 0 . 05m phosphate - citrate buffer , ph 5 . 0 , sigma - aldrich , u . k .) was added to each well . the plate was incubated at room temperature to allow chromophore development , after which the reaction was stopped by the addition of 100 μl of 10 % ( v / v ) hcl . the optical density ( o . d .) was determined at 450 nm with a tecan safire plate reader ( tecan , u . k .). to ensure that the avian polyclonal response was directed towards the neu5gc component of the conjugate and not the synthetic linker or protein element , the polyclonal serum was also tested against a synthetic carbohydrate that consisted of a multivalent biotinylated polyacrylamide ( paa ) polymer that contained 0 . 2 moles of neu5gc per mole of paa ( glycotech , usa ) ( fig2 b ). the serum igy response against the neu5gc antigen was measured by direct elisa . a 96 well maxisorp plate was coated overnight at 4 ° c . with 5 μg / ml of neutravidin ( pierce , u . k .) prepared in coating buffer ( 1 × pbs , ph 7 . 2 ). the plate was washed three times with 1 × pbst ( ph 7 . 2 ) and three times with 1 × pbs ( ph 7 . 2 ). 100 μl of biotinylated - paa - neu5gc ( 25 μg / ml ) was added to the plate and incubated for one hour at 37 ° c . the plate was then blocked with 3 % ( w / v ) bsa solution in 1 × pbs ( ph 7 . 2 ) for 1 hour at 37 ° c . after washing three times with 1 × pbst ( ph 7 . 2 ) and 1 × pbs ( ph 7 . 2 ), 100 μl of serially - diluted serum ( in 1 % ( v / v ) bsa 1 × pbst ( ph 7 . 2 ) blocking buffer ) was added to the relevant wells . after 1 hour at 37 ° c ., the plates were washed as before and 100 μl of rabbit anti - chicken igy conjugated with hrp was added and the plate was then incubated for a further 1 hour . the plate was washed 3 times with 1 × pbs ( ph 7 . 2 ) and 1 × pbst ( ph 7 . 2 ). subsequently , 100 μl of tmb substrate ( sigma - aldrich , u . k .) solution ( 2 mg / ml tmb in citrate 0 . 05m phosphate - citrate buffer , ph 5 . 0 , sigma - aldrich , u . k .) was added to each well . the plate was incubated at room temperature to allow chromophore development , after which the reaction was stopped by the addition of 100 μl of 10 % ( v / v ) hcl . the optical density ( o . d .) was determined at 450 nm with a tecan safire plate reader ( tecan , u . k .). a maxisorp nunc plate was coated overnight at 4 ° c . with 100 μl of 5 μg / ml of the bsa - neu5gc conjugate . the plate was blocked with 3 % ( w / v ) bsa prepared in 1 × pbs ( ph 7 . 2 ) and incubated for 1 hour at 37 ° c . the plate was washed three times with 1 × pbst ( ph 7 . 2 ) and 1 × pbs ( ph 7 . 2 ). the neu5gc - bsa conjugate was added at varying concentrations to a 1 : 50 , 000 dilution of avian serum in 1 % ( v / v ) bsa in 1 × pbst ( ph 7 . 2 ). samples containing no conjugate ( a 0 ) were diluted in 1 × pbst ( ph 7 . 2 ) to ensure the same serum concentration . sample dilutions were incubated for 2 hours at 37 ° c . and 100 μl of sample was added to the relevant wells . after a 1 hour incubation at 37 ° c ., the plates were washed three times with 1 × pbst ( ph 7 . 2 ) and 1 × pbs ( ph 7 . 2 ) and 100 μl of rabbit anti - chicken igy conjugated with hrp was added and the plate was then incubated for 1 hour . the plate was washed three times with 1 × pbst ( ph 7 . 2 ) and 1 × pbs ( ph 7 . 2 ). subsequently , 100 μl of tmb substrate ( sigma - aldrich , u . k .) solution ( 2 mg / ml tmb in citrate 0 . 05m phosphate - citrate buffer , ph 5 . 0 , sigma - aldrich , u . k .) was added to each well . the plate was incubated at room temperature to allow chromophore development , after which the reaction was stopped by the addition of 100 μl of 10 % ( v / v ) hcl . the optical density ( o . d .) was determined at 450 nm with a tecan safire plate reader ( tecan , u . k .). results are shown in fig2 c . novel sialic acid binding clones were identified from an avian immune library using the phage display technique . phage display is a well - established and powerful technique for the discovery and characterisation of antibody fragments ( scfv or fab ) that bind to a panel of specific ligands ( proteins , carbohydrates or haptens ). in this method , antibody fragments are displayed on the outer surface of filamentous phage by inserting short gene fragments in - frame , most commonly into gene iii of the phage . this technique couples a polypeptide or peptide of interest to the dna that encodes it , thus making it possible to select these two characteristics together . the gene iii minor coat protein is important for proper phage assembly and for infection by attachment to the pili of escherichia coli . the gene iii fusions are translated into chimeric proteins and phage that display proteins with high binding affinities for the target ligand are readily selected . affinity can be enriched ( matured ) through multiple rounds of biopanning , a process that involves binding to reducing concentrations of the immobilised ligand . phage that are weakly bound , or have low affinity for the antigen are removed by stringent washing steps during biopanning . the high - affinity bound phage are removed from the surface of an immunotube / maxisorb plate by acid or trypsin elution , and amplified through infection of mid - logarithmic growth - phase e . coli cells . typically , 4 to 6 rounds of panning and amplification are sufficient to select for phage displaying high - affinity antibody fragments . 10 many publications exist that describe the display of antibody fragments on the surface of the bacteriophage . 11 however , the use of this technique to generate anti - carbohydrate antibody fragments is a much less developed area , although some examples of this have been described . 12 however , no publications currently exist that have used an avian host to generate a recombinant antibody fragment ( scfv ) that can recognise both major forms of sialic acid ( neu5gc / neu5ac ) by phage display . 2 . 1 . isolation and quantification of total cellular rna from the spleen and bone marrow of an immunised leghorn chicken . the immunised leghorn chicken was sacrificed . both the spleen and the femurs were immediately harvested and processed in a laminar flow hood ( gelaire bsb 4 ) that was thoroughly cleaned with 70 % ( v / v ) industrial methylated spirits ( ims , lennox ) and rnasezap © ( invitrogen , usa ). the bone marrow from the chicken femurs was washed out with 10 mls of chilled trizol ® reagent ( invitrogen , usa ) using a 25 gauge needle and 5 ml syringe . 10 mls of chilled trizol ® reagent was added to the avian spleen and all samples were fully homogenised using a sterile ( autoclaved and baked overnight at 180 ° c .) homogeniser ( ultra - turrax model tp 18 / 10 , ika ® werke gmbh & amp ; co . kg , germany ). the tubes were incubated at room temperature for 5 minutes and centrifuged ( eppendorf centrifuge 5810r ) at 3500 rpm for 10 minutes at 4 ° c . the supernatants were carefully removed and transferred to fresh ‘ rnase - free ’ 50 ml oakridge tubes ( thermo fisher scientific , usa ). for each sample , 3 mls of ‘ rnase - free ’ chloroform ( sigma - aldrich ) were added and tubes were shaken vigorously for 15 seconds , stored at room temperature for 15 minutes and subsequently centrifuged at 17 , 500 rpm at 4 ° c . for an additional 15 minutes . following centrifugation , the mixture separated into a lower phenol - chloroform phase , an interphase , and a colourless upper aqueous phase . the upper aqueous phase , containing the rna , was carefully removed and transferred to a fresh ‘ rnase - free ’ 50 ml oakridge tube . for each sample , 15 mls of propan - 2 - ol ( sigma - aldrich ) was added and tubes were shaken vigorously for 15 seconds , stored at room temperature for 10 minutes and centrifuged at 17 , 500 rpm at 4 ° c . for 30 minutes . rna precipitated as a white gel - like pellet on the bottom and side of the tube . the supernatant was removed and the pellet was washed with 30 mls of 75 % ( v / v ) ethanol ( sigma - aldrich ) and centrifuged at 17 , 500 rpm at 4 ° c . for 10 minutes . this step was repeated and after removal of the supernatant , the rna pellet was allowed to air dry for 5 minutes . the pellet was then resuspended in 250 μl of ‘ rnase - free ’ water ( sigma - aldrich ). the rna concentrations were determined by spectrophotometric measurement at 260 nm with a nanodrop ™ spectrophotometer nd - 1000 ( thermo fisher scientific , usa ). the purity of the rna preparation was assessed by measuring the ratio of absorbance at 260 nm and 280 nm . furthermore , sample purity was assessed by analysis on a 1 % ( w / v ) agarose gel . an aliquot of freshly isolated rna was used for cdna synthesis . the remaining rna solution was precipitated at − 20 ° c . with 1 / 10 the volume of ‘ rnase - free ’ sodium acetate ph 5 . 2 ( sigma - aldrich ) and 2 times the total sample volume of 100 % ( v / v ) ethanol . to enhance rna precipitation , nuclease - free glycogen ( fermentas ) was added at a final concentration of 1 μg / ul . the superscript ™ iii first - strand synthesis system for rt - pcr , ( invitrogen , usa ) was used to generate first strand - cdna from 5 μg of total rna using oligo dt 20 priming . all reactions were kept on ice at all times . two 20 × master mixes , hereafter referred to as 1 and 2 , were made using the recipe below . 25 μls of reaction mix 1 were added to 8 tubes that were subsequently incubated at 65 ° c . for 5 minutes ( biometra tgradient pcr machine ) and placed on ice for 1 minute . 25 μls of reaction mix 2 were added to the same 8 tubes , the tubes were further incubated at 50 ° c . for 50 minutes and the cdna reaction was terminated by incubation at 85 ° c . for 5 minutes . the samples were spun briefly and 1 μa of rnase h was added to each tube . the tubes were then incubated at 37 ° c . for 20 minutes , after which the cdna was pooled , aliquoted and stored at − 20 ° c . to assess cdna quality samples were run on a 1 % ( w / v ) agarose gel . 2 . 2 . pcr primers and conditions used for the construction of the avian library the following sets of oligonucleotides were used to generate a chicken scfv library with a short linker from both the bone marrow and spleen . all primers were high purity , salt free and were purchased from eurofins mwg operon ( ebersberg , germany ). for v h and v l , gene amplification , a 100 μl pcr reaction contained the following : 1 μl of cdna , 60 pmole of cscho - f and cscg - b , 5 × pcr buffer ( promega , usa ), 1 . 5 mm mgcl 2 ( promega , usa ), 200 μm dntps ( promega , usa ), and 0 . 5 μl gotaq ® dna polymerase ( promega , usa ). for v l , gene amplification the pcr reaction components were the same except that 60 pmole of cscvk and ckjo - b were used in place of the v h primers . the hybaid thermal cycler ( thermo px2 , thermo fisher scientific , usa ) was used for all pcr reactions . touchdown pcr was performed with the following cycling conditions : 4 minutes at 94 ° c . ( initial denaturation ), followed by 30 cycles of 15 sec at 94 ° c . ( denaturation ), 30 sec at 60 ° c . ( annealing )— the annealing temperature of each cycle was decreased by 0 . 1 ° c ., 45 sec at 72 ° c . ( extension ) and the reaction was terminated after 5 minutes at 72 ° c . ( final extension ). the resulting pcr products were run analysed a 1 % ( w / v ) agarose gel and purified with the wizard ® sv gel and pcr clean - up system ( promega , usa ) according to the manufacturer &# 39 ; s instructions . the v h and v l , purified fragments were joined with a glycine - serine linker ( gly 4 ser ) 3 using splice overlapping extension ( soe ) pcr . the resulting pcr product was an amplicon approximately 750 bp in length . for soe - pcr , a 100 μl pcr reaction contained the following : 100 ng of the v l , and v h purified products , 60 pmolar of csc - f and csc - b , 10 × pcr buffer ( invitrogen , usa ), 1 . 5 mm mgso 4 ( invitrogen , usa ), 200 μm dntps and 1 μl platinum ® taq dna polymerase ( invitrogen , usa ). pcr was performed with the following cycling conditions : 5 minutes at 94 ° c . ( initial denaturation ), followed by 30 cycles of 30 sec at 94 ° c . ( denaturation ), 30 sec at 57 ° c . ( annealing ), 1 minutes at 72 ° c . ( extension ) and the reaction was terminated after 10 minutes at 72 ° c . ( final extension ). the resulting pcr products were run on a 1 % ( w / v ) agarose gel and purified with the wizard ® sv gel and pcr clean - up system according to the manufacturer &# 39 ; s instructions . 2 . 2 . 1 . soe - pcr restriction digestion and ligation into pcomb3xss vector for phage display . the scfv fragment and the cloning vector pcomb3xss were digestion with the sfi 1 restriction enzyme . prior to digestion , the vector and scfv dna concentrations were determined by absorbance measurement at 260 nm with the nanodrop ™ nd1000 spectrophotometer . for scfv digestion , a 100 μl reaction contained the following : 12 μg of gel - purified short linker scfv , 200 units of sfi 1 ( new england biolabs , usa ), 10 × nebuffer 2 ( new england biolabs , usa ) and 10 × bsa ( new england biolabs , usa ). the pcomb3xss 100 μl digestion reaction contained the following : 40 μg of gel - purified vector , 240 units of sfi1 , 10 × nebuffer 2 and 10 × bsa . the digestion of purified insert ( scfv ) and vector ( pcomb3xss ) was performed for 5 hours at 50 ° c . following digestion , the cut pcomb3xss vector and the scfv fragment were purified from a 1 % ( w / v ) agarose gel using the wizard ® sv gel and pcr clean - up system and dna quantification was determined at 260 nm using the nanodrop ™ nd1000 spectrophotometer . the ligation of the scfv fragment with the pcomb3xss vector ( ratio of vector to insert 2 : 1 ) was performed using t4 dna ligase ( new england biolabs , usa ) overnight at room temperature . the 200 μl ligation mixture contained the following : 1 . 4 μg of gel - purified and stuffer free pcomb3xss vector , 700 ng of gel - purified scfv , 5 × ligase buffer , and 200 units of t4 dna ligase . after ligation , the solution was precipitated at − 20 ° c . with 1 / 10 the volume of ‘ rnase - free ’ sodium acetate ( ph 5 . 2 ), 2 times the volume of 100 % ( v / v ) ethanol and 41 of pellet paint ® nf co - precipitant ( merck , u . k .) after overnight precipitation , the sample was centrifuged at 14000 rpm for 20 minutes at 4 ° c . and the pellet was washed with 70 % ( v / v ) ice - cold ethanol . the mixture was centrifuged at 14000 rpm for 10 minutes at 4 ° c . and the pellet was resuspended in 5 μl of molecular grade water ( sigma - aldrich , usa ). 2 . 3 . electro - transformation of xl - 1 blue e . coli cells with scfv - containing plasmid . commercially available electrocompetent xl - 1 blue e . coli cells ( stratagene , usa ) were transformed with the ligated scfv vector construct . this was achieved using a gene pulser xcell electroporation system ( bio - rad laboratories , usa ) with the controls set at 25 μf , 1 . 25 kv and the pulse controller at 200ω . the e . coli cells ( 50 μl ) were thawed on ice . the ligated product ( 2 μl ) was added to the cells , mixed , left to incubate for 30 seconds and immediately transferred to an ice - cold 0 . 2 cm electroporation cuvette ( bio - rad laboratories , usa ). the cuvette was tapped so that the suspension was at the base and was placed in the shockpod and pulsed once . the cuvette was quickly removed from the chamber and 1 ml of soc medium ( sob medium containing 20 mm glucose ; sob medium contains : tryptone 20 g / l , yeast extract 5 g / l , nacl 0 . 5 g / 1 , 186 mg / l kcl , 10 mm mgcl 2 and 10 mm mgso 4 ( sigma - aldrich , usa )) was added immediately to the cuvette . the cells were quickly but gently resuspended with a sterile pasteur pipette . the 1 ml suspension was transferred to a 20 ml sterile universal container containing 2 mls of soc media . to facilitate recovery of the cells , the universal container was shaken for 1 hour at 250 rpm at 37 ° c . the pcomb3xss transformants were plated on tye plates ( tryptone 10 g / l , yeast extract 5 g / l , nacl 8 g / l and bacto agar 15 g / l ( sigma - aldrich , usa )), supplemented with 100 μg / ml carbenicillin ( sigma - aldrich , usa ) and 1 % ( v / v ) glucose ( sigma - aldrich , usa ). untransformed xl - 1 blue e . coli cells ( negative control ) were plated out in parallel on agar plates with 100 μg / ml carbenicillin and 1 % ( v / v ) glucose . the plates were incubated overnight at 37 ° c . the pcomb3xss transformant colonies were scraped off the plates and used as library stocks . these cells were suspended in 20 % ( v / v ) glycerol , snap frozen in liquid nitrogen and stored at − 80 ° c . the anti - sialic acid spleen and bone marrow libraries were propagated using 2 × 600 μl inoculums of cells ( from the frozen glycerol stocks ) into 2 × 600 mls cultures of 2 × ty ( tryptone 12 g / l ; yeast extract 10 g / l ; nacl 5 g / l ; final ( ph 7 . 2 ) containing 100 μg / ml carbenicillin and 2 % ( w / v ) glucose . these libraries were propagated at 200 rpm and 37 ° c . until mid - exponential phase of growth ( o . d . ˜ 0 . 600 @ 600 nm ). the cultures were spun down at 4000 rpm at 4 ° c . for 10 minutes . the pellets were resuspended in fresh 2 × ty media ( 600 mls ) containing 100 μg / ml carbenicillin and 1 × 10 11 plaque - forming units ( pfu )/ ml of m13ko7 helper phage ( new england biolabs , usa ). the cultures were incubated at 37 ° c . for 30 minutes without agitation after which time they were propagated at 200 rpm and 37 ° c . for 2 hours . subsequently , carbenicillin ( 100 μg / ml ) and kanamycin ( 50 μg / ml , sigma - aldrich , usa ) were added and the cultures were grown overnight ( 200 rpm , 30 ° c .). the cultures were centrifuged at 4000 rpm for 15 minutes at 4 ° c . and the supernatants transferred to clean sterile 250 ml sorval centrifuge tubes ( thermo fisher scientific , usa ). the phage particles were precipitated by the addition of polyethyleneglycol 8000 ( to 4 % ( w / v )) and nacl ( to 3 % ( w / v )) ( sigma - aldrich , usa ). the peg - nacl solution was dissolved by shaking at 200 rpm for 10 minutes at 37 ° c . the 250 ml centrifuge tube was placed on ice for 1 hour at 4 ° c . and centrifuged at 8000 rpm for 25 minutes at 4 ° c . the phage / bacterial pellet was resuspended in 2 ml tris - edta ( sigma - aldrich , usa ) buffer in 2 % ( w / v ) bsa ( sigma - aldrich , usa ) solution . after the phage pellet was transferred to 1 . 5 ml sterile centrifuge tubes , it was centrifuged at 14000 rpm for 5 minutes at 4 ° c . the supernatant containing the phage scfv was placed on ice and stored at 4 ° c . 2 . 4 . 1 selection of sialic acid binding phage scfv fragment by panning with immobilised neu5gc - bsa . maxisorp immuno - tubes ™ ( thermo fisher scientific , usa ) were coated overnight at 4 ° c . with 500 μl of 100 μg / ml neu5gc - bsa conjugate . the tubes were blocked with 4 mls of 3 % ( w / v ) bsa in 1 × pbs ( ph 7 . 2 ) for 2 hours at room temperature . the blocking solution was removed and 500 μl of rescued phage was added and incubated on a tube roller - mixer srt1 ( bibby scientific , u . k .) for 2 hours at room temperature . the solution was removed and non - binding phage were discarded by washing three times with 1 × pbst ( ph 7 . 2 ) and 1 × pbs ( ph 7 . 2 ). excess pbs was discarded and bound phage particles were eluted with 500 μl of 10 mg / ml type ii porcine trypsin ( sigma - aldrich ) in 1 × pbs ( ph 7 . 2 ) solution . the immuno - tubes ™ were then incubated at 37 ° c . for 30 minutes half the eluted phage particles ( 250 μl ) were stored at 4 ° c . the other 250 μl of phage were infected into 2 mls of mid - exponential phase xl - 1 blue e . coli cells . after a static 30 minute incubation at 37 ° c ., 20 μl of culture was removed and serial diluted ( 10 − 1 - 10 − 12 ) in 2 × ty media . serial dilutions ( 10 − 8 - 10 − 4 ) were spread on 2 × ty agar plates containing 100 μg / ml carbenicillin and incubated overnight at 37 ° c . the remaining culture was propagated at 200 rpm for 1 hour at 37 ° c . cells were harvested by centrifugation at 4000 rpm for 10 minutes at 4 ° c . library plates were prepared by resuspending the cell pellet in 600 μl of fresh 2 × ty media and by spread - plating on tye plates containing 1 % ( w / v ) glucose and 100 μg / ml carbenicillin . plates were incubated overnight at 37 ° c . input titres were performed by infecting mid - exponential growth phase xl - 1 blue e . coli cells ( 180 μl ) with 20 μl of precipitated phage ( stored at 4 ° c .) for 15 minutes at 37 ° c . and serial dilutions were performed ( 10 − 1 - 10 − 12 ) and spread on 2 × ty agar plates containing 100 μg / ml carbenicillin and incubated overnight at 37 ° c . in the subsequent rounds of biopanning ( 2 , 3 , 4 , and 5 ) the bone marrow and spleen libraries , only 100 mls of 2 × ty medium was used for cell propagation . in addition , two different phage elution strategies were followed namely ( a ) competitive elution and ( b ) standard tyrpsin elution . for the standard trypsin elution method , the neu5gc coating concentrations of the immuno - tubes ™ were reduced in rounds 3 , 4 , and 5 of biopanning to a concentration of 30 μg / ml , 20 μg / ml , and 10 μg / ml , respectively . in addition , the washing of the immuno - tubes ™ was increased as follows : round three , 6 times 1 × pbst ( ph 7 . 2 ) and 1 × pbs ( ph 7 . 2 ), round four , 9 times 1 × pbst ( ph 7 . 2 ) and 1 × pbs ( ph 7 . 2 ) and round five , 12 times 1 × pb st ( ph 7 . 2 ) and 1 × pbs ( ph 7 . 2 ). for competitive elution the neu5gc - bsa and neu5gc - paa conjugates were added to the immuno - tubes ™ and incubated overnight at 4 ° c . the next day the eluted phage were infected into 2 mls of mid - exponential phase xl - 1 blue e . coli cells . after a static 30 minute incubation at 37 ° c ., 20 μl of culture was removed and serial diluted ( 10 − 1 - 10 − 12 ) in 2 × ty media . serial dilutions ( 10 − 8 - 10 − 4 ) were spread on 2 × ty agar plates containing 100 μg / ml carbenicillin and incubated overnight at 37 ° c . the remaining culture was propagated at 200 rpm for 1 hour at 37 ° c . cells were harvested by centrifugation at 4000 rpm for 10 minutes at 4 ° c . library plates were prepared by resuspending the cell pellet in 600 μl of fresh 2 × ty media and by spread - plating on tye plates containing 1 % ( w / v ) glucose and 100 μg / ml carbenicillin . plates were incubated overnight at 37 ° c . for each successive round of biopanning using competitive elution , 500 μl of the neu5gc - bsa and neu5gc - paa conjugates in 1 % ( w / v ) bsa 1 × pbst ( ph 7 . 2 ) were added to the immuno - tubes ™ at the following concentrations : round 2 , 500 μg / ml of neu5gc - bsa and 40 μg / ml neu5gc - paa , round 3 , 300 μg / ml of neu5gc - bsa and 20 μg / ml neu5gc - paa , round 4 , 200 μg / ml of neu5gc - bsa and 20 μg / ml neu5gc - paa , and round 5 , 100 μg / ml of neu5gc - bsa and 10 μg / ml neu5gc - paa . the immuno - tubes ™ coating concentration ( 50 μg / ml ) and washing ( three times 1 × pbst ( ph 7 . 2 ) and 1 × pbs ( ph 7 . 2 ) was kept the same for all rounds of competitive elution panning . for the determination of affinity maturation , a polyclonal phage elisa was performed . a 96 - well plate was coated overnight at 4 ° c . with 100 μl of 10 μg / ml neu5gc - bsa . the plate was then blocked for 1 hour at 37 ° c . with 3 % ( w / v ) bsa in 1 × pbs ( ph 7 . 2 ). after blocking , the plate was washed three times with 1 × pbst ( ph 7 . 2 ) and 1 × pbs ( ph 7 . 2 ). 100 μl of phage particles from each round of panning ( diluted 1 : 10 in 1 % ( w / v ) bsa 1 × pbst , ph 7 . 2 ) were assayed in triplicate . plates were washed three times with 1 × pbst ( ph 7 . 2 ) and 1 × pbs ( ph 7 . 2 ) and 100 μl of a 1 : 5000 dilution of hrp - conjugated mouse anti - m13 monoclonal antibody ( ge healthcare , u . k .) in 1 % ( w / v ) bsa 1 × pbst ( ph 7 . 2 ) was added for 1 hour at room temperature . the plate was washed three times with 1 × pbst ( ph 7 . 2 ) and 1 × pbs ( ph 7 . 2 ). subsequently , 100 μl of tmb substrate ( sigma - aldrich , u . k .) solution ( 2 mg / ml tmb in citrate 0 . 05m phosphate - citrate buffer , ph 5 . 0 , sigma - aldrich , u . k .) was added to each well . the plate was incubated at room temperature to allow chromophore development , after which the reaction was stopped by the addition of 100 μl of 10 % ( v / v ) hcl . the optical density ( o . d .) was determined at 450 nm with a tecan safire plate reader ( tecan , u . k .). good affinity maturation for both libraries was seen when assayed on neu5gc - bsa . antibody fragments without the piii protein were produced by infecting phagemid dna from rounds 3 and 4 of panning into e . coli top 10f ′ cells ( stratagene , usa ) at mid - logarithmic growth phase . after incubation for 30 minutes at 37 ° c ., serial dilutions were prepared in 2 × ty ( 10 − 2 to 10 − 10 ), and plated on tye plates containing 1 % ( w / v ) glucose and 100 μg / ml carbenicillin . single colonies were inoculated into individual wells of a 96 - well elisa plate containing 200 μl of 2 × ty in the presence of carbenicillin ( 100 μg / ml ) and glucose ( 1 . 0 % ( w / v )). after an overnight incubation at 37 ° c ., master plates of the original clones were prepared by adding glycerol ( 20 % ( w / v )) and storing at − 80 ° c . these plates were used as a backup stock for each putative clone of interest . twenty μl from the overnight subculture plates were inoculated into fresh 2 × ty media ( 180 μl ) containing 1 × 505 medium ( 0 . 5 % ( v / v ) glycerol , 0 . 05 % ( v / v ) glucose final concentration ), 1 mm mgso 4 and 100 μg / ml carbenicillin . the sterile 96 well plates were propagated at 37 ° c . at 180 rpm until a cell density of ˜ 0 . 600 was achieved . a final concentration of 1 mm isopropyl - β - d - thiogalactoside ( iptg ) was added to each individual well and the plates were induced overnight at 180 rpm at 30 ° c . the overnight cultures were frozen at − 80 ° c . the periplasmic scfv was extracted from the cells by three cycles of freeze - thaw . cell extracts were cleared by centrifugation ( 4000 rpm , 10 minutes ) and the lysates were diluted 1 : 5 in 1 % ( w / v ) bsa 1 × pbs ( ph 7 . 2 ). elisa - based analysis was performed as follows . a 96 - well plate was coated overnight at 4 ° c . with 100 μl of 10 μg / ml neu5gc - bsa . the plate was then blocked for 1 hour at 37 ° c . with 3 % ( w / v ) bsa in 1 × pbs ( ph 7 . 2 ). after blocking , the plate was washed three times with 1 × pbst ( ph 7 . 2 ) and 1 × pbs ( ph 7 . 2 ). 100 μl of the periplasmic scfv cell extracts ( diluted 1 : 5 in 1 % ( w / v ) bsa 1 × pbst , ph 7 . 2 ) were assayed in triplicate . plates were washed three times with 1 × pbst ( ph 7 . 2 ) and 1 × pbs ( ph 7 . 2 ) and 100 μl of a 1 : 2000 dilution ( 1 % ( w / v ) bsa 1 × pbst ( ph 7 . 2 ) of rat anti - ha monoclonal antibody conjugated with peroxidase ( roche diagnostics , usa ) was added for 1 hour at 37 ° c . the plate was washed three times with 1 × pbst ( ph 7 . 2 ) and 1 × pbs ( ph 7 . 2 ). subsequently , 100 μl of tmb substrate ( sigma - aldrich , u . k .) solution ( 2 mg / ml tmb in citrate 0 . 05m phosphate - citrate buffer , ph 5 . 0 , sigma - aldrich , u . k .) was added to each well . the plate was incubated at room temperature to allow chromophore development , after which the reaction was stopped by the addition of 100 μl of 10 % ( v / v ) hcl . the optical density ( o . d .) was determined at 450 nm with a tecan safire plate reader ( tecan , u . k .). results for competitively - eluted and trypsin eluted scfvs are shown in fig3 a and 3 b , respectively . 2 . 7 . assessing the ability of the soluble anti - sialic clones to recognise neu5gc in the context of a polyacrylamide backbone . in order to identify scfvs that could not only bind neu5gc - bsa but also recognise this monosaccharide in the context of an alternative backbone , positive clones identified from monoclonal scfv elisa - analysis , were tested against neu5gc - paa . a 96 well nunc maxisorb plate was coated overnight at 4 ° c . with 5 μg / ml of neutravidin in coating buffer 1 × pbs ( ph 7 . 2 ). the plate was washed three times with 1 × pbs ( ph 7 . 2 ) and 1 × pbst ( ph 7 . 2 ). 100 μl of biotinylated - paa - neu5gc ( 25 μg / ml ) was added and the plate was incubated for one hour at 37 ° c . the plate was blocked with 3 % ( w / v ) bsa solution in 1 × pbs ( ph 7 . 2 ) for 1 hour at 37 ° c . after washing three times with 1 × pbs ( ph 7 . 2 ) and 1 × pbst ( ph 7 . 2 ), 100 μl of soluble - expressed scfv ( diluted 1 : 5 in 1 % ( w / v ) bsa 1 × pbst , ph 7 . 2 ) were added to the plate . after a 1 hour incubation at 37 ° c ., the plate was washed three times with 1 × pbs ( ph 7 . 2 ) and 1 × pbst ( ph 7 . 2 ) and 100 μl of 1 : 2000 dilution ( 1 % ( w / v ) bsa 1 × pbst ( ph 7 . 2 )) of an anti - ha rat monoclonal antibody conjugated with peroxidase was added . the plate was incubated for 1 hour , washed 3 times with 1 × pbs ( ph 7 . 2 ) and 1 × pbst ( ph 7 . 2 ). subsequently , 100 μl of tmb substrate ( sigma - aldrich , u . k .) solution ( 2 mg / ml tmb in citrate 0 . 05m phosphate - citrate buffer , ph 5 . 0 , sigma - aldrich , u . k .) was added to each well . the plate was incubated at room temperature to allow chromophore development , after which the reaction was stopped by the addition of 100 μl of 10 % ( v / v ) hcl . the optical density ( o . d .) was determined at 450 nm with a tecan safire plate reader ( tecan , u . k .). results for this assay are illustrated in fig3 c . 2 . 7 . 1 . cross reaction - analysis of the soluble anti - neu5gc clones with other mono and disaccharides . the capacity of the anti - neu5gc clones to cross - react with other carbohydrate elements was assessed by analysis against the following structures : neu5ac - paa , ( neu5ac ) 2 - paa , neu5gc - dope , glucose - paa and galactose - paa ( fig4 ). a 96 well nunc maxisorb plate was coated overnight at 4 ° c . with 5 μg / ml of neutravidin in coating buffer 1 × pbs ( ph 7 . 2 ). the plate was washed with three times with 1 × pbs ( ph 7 . 2 ) and 1 × pbst ( ph 7 . 2 ). 100 μl of biotinylated - paa - conjugate ( neu5ac - paa , ( neu5ac ) 2 - paa , neu5gc - dope , glucose - paa and galactose - paa ) at 25 μg / ml was added and the plate was incubated for one hour at 37 ° c . the plate was blocked with 3 % ( w / v ) bsa solution in 1 × pbs ( ph 7 . 2 ) for 1 hour at 37 ° c . after washing three times with 1 × pbs ( ph 7 . 2 ) and 1 × pbst ( ph 7 . 2 ), 100 μl of soluble - expressed scfv ( diluted 1 : 5 in 1 % ( w / v ) bsa 1 × pbst , ph 7 . 2 ) were added to the plate . after a 1 hour incubation at 37 ° c ., the plate was washed three times with 1 × pbs ( ph 7 . 2 ) and 1 × pbst ( ph 7 . 2 ) and 100 μl of 1 : 2000 dilution ( 1 % ( w / v ) bsa 1 × pbst ( ph 7 . 2 )) of an anti - ha rat monoclonal antibody conjugated with peroxidase was added . the plate was incubated for 1 hour , washed 3 times with 1 × pbs ( ph 7 . 2 ) and 1 × pbst ( ph 7 . 2 ). subsequently , 100 μl of tmb substrate ( sigma - aldrich , u . k .) solution ( 2 mg / ml tmb in citrate 0 . 05m phosphate - citrate buffer , ph 5 . 0 , sigma - aldrich , u . k .) was added to each well . the plate was incubated at room temperature to allow chromophore development , after which the reaction was stopped by the addition of 100 μl of 10 % ( v / v ) hcl . the optical density ( o . d .) was determined at 450 nm with a tecan safire plate reader ( tecan , u . k .). to ensure the fidelity of the sequence data , three different samples ( stab culture , plasmid prep and unpurified pcr products ) of the same clone were sent for sequencing . double stranded dna sequencing of all clones was performed by eurofins mwg operon ( ebersberg , germany ). a panel of anti - sialic acid clones were grown in 1 . 5 ml eppendorf stab cultures . in addition , purified plasmid was obtained from each clone using the wizard ® plus sv minipreps dna purification system in accordance with the manufacturer &# 39 ; s instructions . furthermore , the scfv gene fragment was also amplified using colony pick pcr . for colony pick pcr , a 50 μl pcr reaction contained the following : 2 μl of an overnight culture , 60 pmole of csc - f and csc - b , 5 × pcr buffer , 1 . 5 mm mgcl 2 , 200 μm dntps and 0 . 25 μl gotaq ® dna polymerase . touchdown pcr was performed with the following cycling conditions : 10 minutes at 94 ° c . ( initial denaturation ), followed by 30 cycles of 30 sec at 94 ° c . ( denaturation ), 30 sec at 56 ° c . ( annealing )— the annealing temperature of each cycle was decreased by 0 . 1 ° c ., 1 minute at 72 ° c . ( extension ) and the reaction was terminated after 10 minutes at 72 ° c . ( final extension ). the sequences of the cdlr1 , cdlr2 , cdlr3 , cdhr1 , cdhr2 and cdhr3 regions for the ae8 , ag9 , cd3 , and cc1 clones are shown in fig5 . the full sequences of the variable heavy and light chains of the clones , and the sequences of the full clones ( including the spacer which is underlined ) are provided below : for further analysis by hplc and surface plasmon resonance ( spr ), the ae8 clone was purified by immobilised metal affinity chromatography ( imac ). a single colony of the ae8 clone was sub - cultured into 5 mls of 2 × ty containing 100 μg / ml carbenicillin and 1 % ( w / v ) glucose and grown overnight at 37 ° c . five hundred uls of the overnight culture was inoculated into 500 mls of terrific - broth ( tb ; tryptone 13 . 3 g / l , yeast extract 26 . 6 g / l and glycerol 0 . 44 %, ( v / v )) that contained 1 × 505 medium ( 0 . 5 % ( v / v ) glycerol , 0 . 05 % ( v / v ) glucose final concentration ), 50 mls potassium phosphate solution ( kh 2 po 4 2 . 31 g / l , k 2 hpo 4 12 . 54 g / l ), 1 mm mgso 4 and 100 μg / ml carbenicillin . the culture was incubated at 37 ° c . at 240 rpm until an approximate od 600 of 0 . 6 was reached . the culture was then induced with 1 mm iptg and incubated at 30 ° c . overnight at 240 rpm . the following day , the culture was centrifuged at 4000 rpm for 10 minutes at 4 ° c . and the pellet was completely re - suspended in 30 mls of ice - cold sonication buffer ( 1 × pbs , 0 . 5m nacl and 20 mm imidazole ) and then aliquoted ( 1 ml ) into 1 . 5 ml eppendorf tubes . each individual sample was sonicated on ice for 45 seconds ( 40 % amplitude ) with 6 sec pulses for 3 minutes . the samples were then centrifuged at 14 , 000 rpm for 10 minutes at 4 ° c . the lysates were pooled , filtered through a 0 . 2 μm filter and stored at 4 ° c . all imac purifications were performed using pd - 10 columns ( ge healthcare , u . k .). two millilitres of ni - nta resin ( qiagen , usa ) were added to the column and allowed to form a packed bed . after equilibration with 30 mls of running buffer ( sonication buffer containing 1 % ( v / v ) tween ), the pooled lysate was then added to the column and the flow - through was collected and stored at 4 ° c . the column was subsequently washed with 30 mls of running buffer and the bound scfv was eluted by adding 20 mls of 100 mm sodium acetate ( ph 4 . 4 ). 400 μl volumes of eluent were added to 50 μl of 10 × pbs ( ph 7 . 2 ) and 50 μl of 100 mm naoh before mixing . individual fractions were tested for the presence of protein by quantification at 280 nm with the nanodrop nd1000 spectrophotometer . those fractions that contained the eluted scfv were pooled and concentrated using a 5000 da molecular weight cut - off ( mwco ) buffer exchange column ( sartorius , germany ). the scfv - containing sample was concentrated to a volume of 500 μl by centrifugation ( 4000 rpm ) at 4 ° c . five mls of 1 × pbs were subsequently added to the column and , after an overnight incubation at 4 ° c ., the sample was buffer exchanged and re - concentrated by centrifugation until the final volume was approximately 200 μl . protein concentration was determined by quantification at 280 nm using a nanodrop nd1000 spectrophotometer ( labtech international , u . k .). hplc size exclusion chromatography ( sec - hplc ) was used to determine the species composition , apparent molecular weight and to purify the monomeric fraction of the recombinant ae8 scfv . a shimadzu lc system ( shimadzu corporation , japan ), equipped with a shimadzu cbm - 20a controller , shimadzu lc - 20ab pumps , shimadzu spd - 20a uv - vis spectrophotometric detector , shimadzu sil - 20a autosampler , shimadzu frc - 10a fraction collector , shimadzu cto - 20ac column oven and shimadzu &# 39 ; s lcsolution software for data handling . the experiments were carried out using the size exclusion bio - sep - sec - 52000 column ( phenomenex ; 300 × 7 . 8 mm ) protected with a guard column ( phenomenex ; 35 × 7 . 8 mm ). the hplc system was operated isocratically at room temperature using filtered and degassed 1 × pbs ( ph 7 . 2 ) as the mobile phase . prior to sample analysis , the column was equilibrated for 45 minutes by gradually increasing the flow rate in increments of 0 . 1 ml / minutes . all samples ( 20 μl ) were diluted in 1 × pbs ( ph 7 . 2 ) and assayed at a flow rate of 0 . 5 mls / minutes with uv detection ( 280 nm ). the following protein standards ( agilent , usa ) were used : bovine thyroglobulin ( 670 kd ), human gamma globulin ( igg ; 150 kd ), ovalbumin ( 44 kd ) and myoglobin ( 17 kd ). samples were interspersed with water blanks to ensure that all residual protein was eluted . the monomeric ae8 scfv was isolated with the bio - sep - sec - 52000 hplc column by the collection of several fractions between 17 . 4 and 18 . 2 minutes at a flow rate of 0 . 5 ml / minute ( fig6 ). fast protein liquid chromatography ( fplc ) was used to estimate the molecular weight of the ae8 protein . the äkta ™ explorer 100 system ( ge healthcare , usa ) equipped with a uv - 900 monitor , monitor ph / c - 900 , sample pump , fraction collector frac - 950 and unicorn ™ software for data handling was used for protein analysis . 100 μl of the ae8 sample was applied to a hiload ™ 16 / 60 superdex ™ 200 prep - grade fplc column using filtered and degassed 1 × pbs ( ph 7 . 2 ) at a flow rate of 1 ml / min . the following protein standards ( agilent , usa ): bovine thyroglobulin ( 670 kd ), human gamma globulin ( igg ; 150 kd ), ovalbumin ( 44 kd ) and myoglobin ( 17 kd ) were used for the molecular weight estimation of the scfv ( fig7 ). 3 . 0 surface plasmon resonance analysis of the ae8 clone using the biacore ® 3000 biosensor analysis of the binding and kinetic properties of the ae8 clone was performed using the biacore ® 3000 biosensor which monitors ‘ label - free ’ biomolecular interactions in ‘ real - time ’ using the phenomenon of surface plasmon resonance ( spr ). the basic assay format for ae8 spr analysis was as follows : neutravidin was immobilised on the dextran surface of a biacore ® cm5 chip , biotinylated polyacrylamide neu5gc conjugate was then passed over and captured by the neutravidin , after which the scfv was passed over the surface to check for binding to the sugar . as a negative control , the scfv was also passed over a neutravidin surface which had no biotinylated sugar . 3 . 1 pre - concentration , immobilisation of neutravidin on a carboxy - methylated dextran chip and capture of biotinylated - neu5gc polyacrylamide ( paa ). for all biacore ® 3000 ( ge healthcare , sweden ) experiments , the running buffer used was filtered and degassed hepes buffered saline ph 7 . 4 ( hbs : 50 mm nacl , 10 mm hepes , 3 . 4 mm edta and 0 . 05 % ( v / v ) tween - 20 ). 50 μg / ml solutions of neutravidin ( thermo fisher scientific , usa ) were prepared in 10 mm sodium acetate ( sigma - aldrich , usa ) buffers that had been adjusted with 10 % ( v / v ) acetic acid ( sigma - aldrich , usa ) to ph values 4 . 0 , 4 . 2 , 4 . 4 , 4 . 6 , 4 . 8 , and 5 . 0 . 20 μl of protein at each respective ph was sequentially passed over the underivatised carboxy - methylated dextran sensor chip surface ( cm5 , ge healthcare , sweden ) at a flow - rate of 10 μl / minute . a ph of 4 . 6 was determined to be the optimal ph for neutravidin immobilisation as this yielded the largest change in response units ( ru ). neutravidin was immobilised on the cm5 chip with the following protocol : 70 μl of 400 mm of 1 - ethyl - 3 -[ 3 - dimethylaminopropyl ] carbodiimide hydrochloride ( edc ) ( ge healthcare , sweden ) was mixed with 70 μl of 100 mm n - hydroxysuccinimide ( nhs ) ( ge healthcare , sweden ) and injected over the sensor chip surface for 10 minutes at a flowrate of 10 μl / minute . a 50 μg / ml solution of neutravidin was prepared in 10 mm sodium acetate ( sigma - aldrich , usa ), ph 4 . 6 and injected over the activated chip surface for 24 minutes at a flow - rate of 10 μl / minute . unreacted nhs ester groups were capped and loose , non - covalently attached proteins were removed by injection of 1m ethanolamine hydrochloride ( ge healthcare , sweden ), ph 8 . 5 , for 11 minutes . four 30 second sequential pulses of 5 mm naoh at a flow - rate of 10 μl / minute were used to remove any other loosely bound material . after neutravidin immobilisation ( fig8 b ), a 100 μg / ml solution of biotinylated - neu5gc - paa in hbs was passed over the chip surface at 10 μl / min for 20 minutes ( fig8 c ). the sialic acid binding ability of the ae8 clone was assessed with the previously prepared neu5gc sensor chip . a 1 in 100 dilution of the imac purified ae8 clone in fibs was simultaneously passed over flow cells 1 and 2 of the sensor chip at a flow rate of 10 μl / min for 7 minutes . following on - line reference subtraction ( 2 - 1 ), the sensorgram indicated a response increase of 1 , 077 . 4 ru above baseline . bound antibody was dissociated with a 30 second pulse of 10 mm naoh and the baseline was restored with the injection of hbs running buffer over the chip surface . the experiment was repeated five times and , on each occasion , the ae8 neu5gc binding response was greater than 1000 ru . to assess the ability of the anti - sialic acid scfv to bind the sialic acid conjugate in solution - phase , an inhibition binding assay was performed . the purified ae8 scfv was diluted 1 in 2000 in hbs buffer ( ph 7 . 4 ). the neu5gc - bsa conjugate was also diluted in hbs buffer ( ph 7 . 4 ) to the following concentrations : 2000 ng / ml , 1000 ng / ml , 500 ng / ml , 250 ng / ml , 125 ng / ml and 62 . 5 ng / ml . 100 μl of the ae8 sample was mixed with 100 μl of each of the neu5gc - bsa conjugate dilutions to yield the following free conjugate working concentrations : 1000 ng / ml , 500 ng / ml , 250 ng / ml , 125 ng / ml , 62 . 5 ng / ml and 31 . 25 ng / ml . the zero conjugate sample contained 100 μl of 1 in 2000 dilution of the purified ae8 scfv in hbs buffer ( ph 7 . 4 ) and 100 μl of hbs buffer ( ph 7 . 4 ). samples were incubated for 1 hour at 37 ° c . and then injected ( 40 μl ), in random order , over flow cells 1 and 2 of the neu5gc chip at a flow rate of 10 μl / minute for 4 minutes and the change in response recorded . bound antibody was removed by injection of 5 μl of 5 mm naoh at a flow rate of 10 μl / minute for 30 seconds . the amount of free antigen necessary to cause 50 % displacement of antibody ( ic 50 ) was 5 . 7 ng / ml . spr was used to determine the association and dissociation rate constants of the anti - sialic acid scfv . the rate constants were fitted with a pre - defined fitting algorithm using the biaevaluation 4 . 1 software . to avoid mass - transfer limited binding , a smaller quantity ( 1 μg / ml ) of neutravidin (& lt ; 10 , 000 ru ) was immobilised ( see section 3 . 1 ) on the sensor chip surface . subsequently , for the capture step , a 40 ng / ml solution of biotinylated - neu5gc - paa in ems buffer ( ph 7 . 4 ) was passed over the chip surface at 10 μl / min for 1 minute . a final level of 28 . 6 ru of captured biotinylated - neu5gc - paa was achieved . furthermore , to rule out the contribution of avidity in the determination of the rate constants , only the monomeric hplc - purified fraction of ae8 was used for biacore kinetic analysis . the rate constants were calculated using different concentrations ( 6 . 67 μg / ml , 4 . 44 μg / ml , 2 . 96 μg / ml , 1 . 98 μg / ml , 1 . 32 μg / ml , 0 . 88 μg / ml , 0 . 59 μg / ml and 0 μg / ml ) of monomeric scfv diluted in hbs buffer ( ph 7 . 4 ). the kinject command was used to inject 90 μl of each sample over flow cells 1 and 2 of the neu5gc sensor chip , at a flow rate of 30 μl / minute for 3 minutes with a dissociation time of 12 minutes . the zero scfv sample was analysed twice and all samples were run in random order . to reflect the pbs composition of the hplc eluted monomeric scfv , the zero scfv sample contained 1 × pbs ( ph 7 . 2 ) diluted 1 in 10 in hbs buffer ( ph 7 . 4 ). bound antibody was removed by injection of 5 μl of 1 . 25 mm naoh at a flow rate of 30 μl / minute for 10 seconds . all sensorgrams were reference - 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