Patent Application: US-59701505-A

Abstract:
the present invention provides a presentation system and method of use for quantifying a target moiety in a sample which may contain the target moiety , the method comprising using a specified concentration or varying the concentration of a presentation system in order to generate a comparison point or calibration curve which provides means for comparing a signal generated by the presentation system and a signal generated by a sample , wherein said presentation system comprises at least one copy of said target moiety or part thereof .

Description:
with regard to the i27 domain of titin , the cysteine residue c47 is the site for covalent attachment of epitope peptide . ( refer to example 2 , section “ production of an embodiment of the presentation system comprising a scaffolding protein comprising titin i27 domains ( denoted ( i27 ) 5 for details ). a synthetic gene encoding five copies of i27 in series was constructed . copies 1 , 2 , 4 & amp ; 5 of i27 lack cysteine residues , and copy 3 retains a single cys for peptide attachment . ( his ) 6 module is included for product purification . linker regions contain unique sequence & amp ; restriction sites ( see fig4 a ). a construct comprising five i27 domains from the human titin molecule was inserted downstream of the his6 tag of pet3d . domains i27 1 , i27 2 , i27 4 , i27 5 , have had their cysteine residues removed with the following mutations c47s and c67s . domain i273 has had the cysteine residue removed at position 63 ( c63s ) but retains the cysteine residue at position 47 ( see fig4 a ). construct was expressed in the blr ( de3 ) plyss cell line ( novagen ), cells being harvested 4 h post induction with 1 mm iptg . see fig4 a for a schematic representation of the ( i27 ) 5 construct . the unique restriction sites used for construction are shown in fig4 a . the term “ i39 ” is being used as the abbreviation for the image clone 3965951 which is the cdna clone provided by the image consortium of the mouse splicing factor 3 b , subunit 5 gene ( accession bc006603 ). the mouse splicing factor 3 b , subunit 5 gene product is a 10 kda protein that contains a single cysteine residue . pcr was employed to amplify the i39 cdna and to engineer the addition of flanking restriction enzyme recognition sites , bsshii ( 5 - prime ) and saci ( 3 - prime ). using these two restriction enzymes the central i27 3 domain of the ( i27 ) 5 construct ( fig4 a .) was replaced by the i39cdna sequence , creating the i39 ( i27 ) 4 construct ( fig4 b ). fig4 b shows the unique restriction sites used for construction . the i39 ( i27 ) 4 construct was expressed in the blr ( de3 ) plyss cell line ( novagen ), cells being harvested 1 h post induction with 1 mm iptg . construct c :— i39pet14b . the term “ i39 ” is being used as the abbreviation for the image clone 3965951 which is the cdna clone provided by the image consortium of the mouse splicing factor 3 b , subunit 5 gene ( accession bc006603 ), as discussed above . pcr was employed to amplify the i39 cdna and to engineer the addition of flanking restriction enzyme recognition sites , bamill ( 5 - prime ) and blpi ( 3 - prime ). using these two restriction enzymes the pcr product was cloned into reciprocal sites in the expression vector pet14b ( novagen ). for construction purposes the c - terminal residue of the protein was altered , n111t . the i39pet14b construct was expressed in the rosetta - gami b ( de3 ) plyss cell line ( novagen ), cells being harvested 4 h post induction with 1 mm iptg . the method of the present invention was used to detect the protein serca2a in a sample . a presentation system ( calibration standard ) for antibody α - clepaile , which recognises the c - terminus of serca2a , was constructed as described for calibration serca - ps38 by reacting 0 . 1 micromole peptide ylepaile ( single letter codes ) with 5 micromole sulfosuccinimidyl 4 -( n - maleimidomethyl )- cyclohexane - 1 - carboxylate ( sulfo - smcc ); purification of peptide - cross - link complex by gel filtration chromatography ; and incubation of the peptide - cross - link product with ( i27 ) 5 ( 0 . 02 micromoles ) in the presence of 9m urea . product was dialysed against water and the protein concentration determined using a bca assay and mass spectrometry as detailed in example 2 under the section “ serca ps - 38 recognition of a calibration standard ( presentation system )”. cardiac sarcoplasmic reticulum ( 10 μg ) and calibration - clepaile standards ( 15 - 0 . 05 pmol ) were separated in individual lanes on a 10 % sds - page gel . the samples were transferred to pvdf membrane and probed with an antibody specific for the serca2a sequence lepaile ( 1 : 5000 dilution ). antibody binding to its epitope was detected using goat anti - rabbit igg - peroxidase and a commercial chemiluminescent substrate preparation ( pierce ). chemiluminescence was detected using a ccd camera ( fig3 ). quantitation of the amount of serca2a in the sample can be achieved by analysis the band intensity of samples and calibration standards by densitometry . a plot of optical density ( corrected for background signal ) against quantity of the presentation system ( calibration standard ) should be prepared . this plot is considered to be a calibration standard curve . the serca2a protein content of the sample can be calculated using the calibration standard curve , by converting the optical density signal ( corrected for background ) of that sample to pmoles of epitope from this plot . the above example shows a way in which the presentation system and the method of the present invention may be used to positively identify a target moiety in a sample and to quantify the amount of target moiety in a sample . example 2 shows a way in which the presentation system and the present method may be used to negatively identify a target moiety in a sample i . e . wherein the target moiety is not present in the sample . the method of the present invention was used to detect phosphorylation of sacroplasmic reticulum ca2 +- atpase serca2a ) on serine - 38 . a standard western blot approach had failed to detect serine - 38 phosphorylated ca2 +- atpase in either kinase treated sarcoplasmic reticulum vesicles and isolated rat ventricular myocytes . the phosphorylation of the cardiac muscle isoform of the sarcoplasmic reticulum ca2 +- atpase ( serca2a ) on serine - 38 has been described as a regulatory event capable of very significant enhancement of enzyme activity . independent confirmation of these observations has not been forthcoming . a polyclonal antibody , wholly specific for the phosphorylated serine - 38 epitope on the ca2 +- atpase , was utilised to evaluate the phosphorylation of serca2a in isolated sarcoplasmic reticulum vesicles and isolated rat ventricular myocytes . a quantitative western blot approach failed to detect serine - 38 phosphorylated ca2 +- atpase in either kinase treated sarcoplasmic reticulum vesicles , or suitably stimulated cardiac myocytes . the presentation system of the present invention confirmed that the detection sensitivity of assays ( 0 . 03 - 0 . 1 pmol ) was adequate to detect phosphorylation ofjust 1 % of ca2 +- atpase molecules on serine - 38 . although a phosphoprotein of 100 kda was evident in rabbit cardiac sr preparations , it was not recognised by the phospho - serine - 38 specific antibody ( 2 ). phosphorylated ca 2 + - atpase peptide on ser - 38 residue ( 31 klkerwgs ( p04 ) nel 41 ) was prepared by the camkii phosphorylation of peptide 31 klkrwgsnel 41 . the phosphopeptide was purified to homogeneity by reverse phase high performance liquid chromatography . peptide was conjugated to keyhole limpet haemocyanin ( klh ) using carbodiimide cross linkage ( 3 ) and dialysed extensively against buffer ( 50 mm tris - hcl ph 7 . 2 , 150 mm nacl ). adult new zealand white rabbits were immunised with ˜ 150 μg klh and attached peptide at 6 weeks intervals and immune serum collected 11 days after immunisations . serum was prepared and stored at − 70 ° c . a polyclonal antiserum is described herein : serca ps - 38 raised to the phosphorylated peptide . production of an embodiment of the presentation system comprising a scaffolding protein comprising titin i27 domains ( denoted ( i27 ) 5 ). a gene encoding a concatamer of mutant forms of the i27 domain of titin was used . the construct differs from the one described in brockwell et al . ( 4 ) in that the two c - terminal cysteine residues have been deleted . it is referred to as ( i27 ) s throughout this study . ( i27 ) 5 was expressed and purified as described in brockwell et al . ( 4 ). purified phosphorylated ser - 38 peptide ( 31 klkerwgs ( po 4 ) nel 41 ) ( 0 . 1 μmol ) was mixed with an excess of sulfosuccinimidyl 4 -( n - maleimidomethyl )- cyclohexane - 1 - carboxylate ( sulfo - smcc ) cross - linker ( 5 μmol ) in buffer containing 0 . 1m sodium phosphate , 0 . 15 m nacl , ph 7 . 2 . after 1 h of incubation at room temperature , the maleimide - activated peptide was purified by gel filtration chromatography using a superdex peptide hr 10 / 30 column ( pharmacia biotech ). the chromatography was performing using 0 . 1m sodium phosphate , 0 . 15 m nacl , ph 7 . 2 and a flow rate of 0 . 25 ml / min . fractions of interest were pooled and urea was added to the fractions to make a final urea concentration of 9m . ( i27 ) 5 concatamer ( 0 . 1 μmol ) was added to the mixture and incubated for 2 h at room temperature . the conjugate was dialysed extensively against water . final product ( the presentation system according to the present invention ) was stored at − 20 ° c . the same procedure was followed to conjugate serca2a peptide ( ylepaile ) to ( i27 ) 5 concatarner to form an alternative presentation system . protein concentration was determined by a bca assay ( 5 ). myocardial proteins were separated by sds - page using 10 % and 15 % polyacrylamide gels as described by laemmli ( 8 ). following separation , proteins were transferred to pvdf membranes ( pall biosupport , portsmouth , uk ) by semi - dry blotting , and nonspecific binding sites were blocked for 2 - 4 h at room temperature using 5 % dried milk and tris - buffered saline ( ph 7 . 4 ), 0 . 1 % tween 20 . membranes were probed overnight at 4 ° c . with primary antibodies : pt - 17 ( 1 : 5000 ) for the thr - 17 phosphorylated form of phospholamban ( 6 ); α - clep ( 1 : 5000 ) for serca2a ( 16 ); and serca ps - 38 ( 1 : 5000 ) antiserum specific for the ser - 38 phosphorylated form of ca 2 + - atpase . a secondary horseradish peroxidase - labeled antibody raised in rabbit ( goat anti - rabbit igg ( h + l ); jackson immunochemicals ; lot number 38179 ) or protein a peroxidase ( sigma ) were used in combination with two enhanced chemiluminescent detection system ( supersignal west pico chemiluminescent substrate and supersignal west femto maximum sensitivity substrate , pierce ) to visualize the primary antibodies . data were captured using a fuji las - 1000 imaging system ccd camera ( aida software for analysis ). the phosphorylation of serca2 on ser - 38 has been described as a regulatory feature capable of very significant activation of ca2 +- atpase activity . this site , although unique to serca2 , is contained within a segment of the protein which is highly conserved between serca1 and serca2 particularly from residue 39 onwards . as such , the two proteins are likely to display comparable structures in this region . by analogy with serca1 , for which two high resolution structures exist the ser - 38 site of phosphorylation on serca2 is predicted to be in a surface exposed , highly mobile segment of the protein . this segment remains solvent exposed in both conformational extremes of the enzyme ( e1 , e2 ;), which would make it accessible to the kinase and phosphatase in these states . these properties also lend themselves to antibody binding to the site , as it is surface exposed , and highly mobile . we have produced a phosphorylation - site specific antibody to this feature in an effort to define the incidence and role of ser - 38 phosphorylation in cardiac muscle . a polyclonal antibody was produced to the sequence 31 klkerwgs ( po4 ) nel 41 , phosphorylated , as shown , at ser - 38 this polyclonal antiserum , serca ps - 38 , was wholly specific for the phosphorylated peptide , as the phosphopeptide was a potent inhibitor of antibody binding to antigen ( ic50 18 nm ), whereas the equivalent dephosphorylated peptide was unable to interfere with antibody : antigen recognition . having confirmed that polyclonal antibody serca ps - 38 was wholly specific for the phosphorylated ser - 38 epitope we examined the phosphorylation status of this residue in serca following exposure of cardiac sr vesicles to camkii . serca was not detected in these western blot experiments . it was important to establish the basis of this negative result , to ensure that it was providing information about the incidence of ser - 38 phosphorylation , rather than recording a technical failing of the antibody or experiment . to this end , we constructed a presentation system comprising a target moiety , in this case , the phosphopeptide epitope , attached to an inert scaffold protein of known molecular weight . the scaffolding protein was chosen as it contained a single site for peptide attachment ( fig1 a ) thus providing a uniform structure for the presentation of epitope peptide , ideal for accurate quantitation . the presentation system employed comprised a concatamer of five copies of a domain from titin ( i27 ), mutated to remove all but one cysteine residue from the concatamer sequence ( fig1 a ). the target moiety , in this case purified phosphoepitope peptide , was conjugated to the ( i27 ) 5 concatamer via the only cysteine residue in the protein ( c47 in i27 domain 3 , presented schematically in fig1 a ), and the stoichiometry of covalent attachment of the peptide was evaluated by mass spectrometry . a low stoichiometry of peptide attachment to the concatamer ( final product mass 54124 da , labelled calibration - 38 ; fig1 b ) was observed on this occasion , which comprises 5 . 4 % of the total preparation . nevertheless , this low level of conjugation to the concatamer proved sufficient for immunodetection ( fig1 c ). fig1 c shows that antibody serca ps - 38 recognised the concatamer product decorated with the relevant phosphopeptide ( calibration - 38 ), but did not recognise the same concatamer ( i27 ) 5 decorated with an irrelevant peptide ( calibration - αclep ) even when 60 pmol of concatamer was presented . the calibration standard migrates as a single molecular species of ˜ 60 kda in sds - page . furthermore , the phosphorylated epitope was detected by antibody serca ps - 38 with high sensitivity , down to a limit of 0 . 1 pmol epitope peptide using standard ( supersignal west pico , pierce ) ecl substrate ( fig1 c ). the calibration standard ( calibration - 38 ) contained some minor contaminants . a contaminant of 51230 da , seen on the mass spectrum , does not appear to accept peptide ( not detected in western blot experiments , fig1 c ). this material was included in the calculation of percentage product ( calibration - 38 ) as it made an appreciable contribution to total protein . a second contaminant of the ( i27 ) 5 preparation is covalently labelled by peptide . it underlies the immunostaining of a complex of high mr (˜ 250 kda , fig1 c ). this product was undetectable in the mass spectrum and therefore is present in low amounts in the calibration - 38 preparation . it does not make an appreciable contribution to total protein and was excluded from consideration in the quantification performed in this study . thus we conclude that serca phosphorylation , if occurring at all , results in the generation of less than 0 . 03 pmol ser - 38 phosphoprotein in the cells studied ( 10 , 000 viable myocytes ). in previous studies , ser - 16 phosphorylation of phospholamban in myocytes following similar interventions was quantified at 8 . 5 pmol / 1 , 000 cells ( 7 ) indicating the presence of at least 85 pmol phospholamban in the 10 , 000 cells of the present study . as phospholamban and serca are expressed in similar amounts in cardiac muscle ( 2 phospholamban per serca , ( 1 ), we might expect 42 pmol of serca in the experiments performed . our failure to detect ser - 38 phosphoprotein with the antibody described herein suggests that less than 0 . 1 % of serca is phosphorylated in rat cardiac myocytes treated with camkii stimulants . the present study has described a polyclonal antibody wholly specific for a phosphorylated ser - 38 epitope on serca2 . the antibody was able to detect the phosphorylated epitope in a calibration standard with high sensitivity ( 0 . 03 - 0 . 1 pmol ). however , it failed to recognise serca2 in cardiac sr samples from a variety of animal species , despite the presentation of large amounts of serca ( 10 - 60 pmol ) and the presence of a phosphoprotein of 100 kda . this indicates that either serca is not phosphorylated on ser - 38 , or that only a minor fraction of serca molecules ( i . e less than ˜ 1 %) are phosphorylated on ser - 38 . camkii activation in isolated cardiac myocytes was achieved using four independent stimuli resulting in phospholamban phosphorylation on thr - 17 . none of these resulted in detectable ser - 38 phosphorylation of serca using this antibody , despite immunodetection of 0 . 03 pmol of the calibration standard in the same experiment . this indicates that between 0 % and 0 . 1 % of serca molecules become phosphorylated on ser - 38 in response to camkii activating stimuli in intact cardiac myocytes . this study does not provide evidence that ser - 38 phosphorylation of serca2a is a significant event in cardiac myocytes or cardiac sr preparations . as shown in fig5 , three different peptides ( ps - 38 , pt17 and a1 ) were conjugated to the free thiol group on the identical and non - identical constructs via their thiol specific malemide reactive group . ( a )=( i27 ) 5 + ps - 38 , pt17 or a1 . ( b )= i39 -( i27 ) 4 + ps - 38 , pt17 or a1 . ( m = markers , kda = kilodaltons ). conjugation reactions are performed by mixing a 1 : 100 molar ratio of concatamer ( identical and non - identical ) ( dissolved in conjugation buffer containing 20 mm napo 4 , 0 . 5 m nacl and 8 m urea ) to gmbs - modified peptide ( dissolved in h 2 o ). gmbs is a heterobifunctional cross - linker . as used here , it contains a thiol directed reactive group . the amine reactive group of gmbs was used to attach the molecule to the n - terminus of the peptide during chemical synthesis of the peptide . the mixtures are left at room temperature for 2 hours . samples of each reaction are run out on a 12 % sds - page gel and transferred overnight for western blot . specific antibodies for each peptide are used to detect the conjugates : ps - 38 primary antibody = rabbit anti - ps - 38 polyclonal ( 1 in 5000 dilution ). pt17 primary antibody = rabbit anti - pt17 polyclonal ( 1 in 5000 dilution ). a1 primary antibody = muse anti - a1 monoclonal ( 1 in 5000 dilution ). a secondary antibody is used to amplify the signal of the primary antibody . for a rabbit primary antibody , a goat - anti - rabbit hrp ( horse radish peroxidase ) secondary antibody is used ( 1 in 5000 dilution ). for a mouse primary antibody , a goat - anti - mouse hrp secondary antibody is used ( 1 in 5000 dilution ). blots are imaged using perbio supersignal west pico chemiluminescent kit ( see above for reaction details ). samples of ( i27 ) 5 and i39 -( i27 ) 4 conjugated to either a1 , ps - 38 or pt17 peptide ( as in example 3 ), and naked ( unconjugated ) concatamer backbone were put through a 12 % sds - page gel in triplicate . the gel was then transferred to membrane , for western blot , overnight . the membrane is then split into three and probed with antibodies specific to an epitope on each peptide . therefore , the antibodies should only recognise their specific epitope and not the epitope on a different peptide , or any part of the concatamer itself . we have generated a western blot ( data not shown ) showing that each antibody recognises only its specific epitope on the peptide it was raised to and not the epitope of any other peptide . also , the antibodies do not recognise and bind to the naked concatamer ( presentation system without target moiety ) itself . conjugates of ( i27 ) 5 and i39 -( i27 ) 4 to the a1 peptide , and of ( i27 ) 1 to the a1 peptide , produced as described in example 3 were blended together ( a1 -( i27 ) 5 + a1 -( i27 ) 1 and a1 - i39 -( i27 ) 4 + a1 -( i27 ) 1 separately ) to show that a mixture of at least two different sized conjugates in the same sample can be run out , clearly separated and probed . this blend is not limited to conjugates based upon the same concatamer domain , but can work when one of the conjugates has a different functional domain ( i39 -( i27 ) 4 ). see fig6 . see fig7 and fig8 for results . samples of ps - 38 -( i27 ) 5 , pt17 -( i27 ) 5 and a1 -( i27 ) 5 were run out on a 12 % sds - page gel and stained with a phosphoprotein specific dye called pro - q diamond ( molecular probes ). only ps - 38 -( i27 ) 5 and pt17 -( i27 ) 5 should be detected since only ps - 38 and pt17 are phosphorylated . a1 is not phosphorylated , therefore , a1 -( i27 ) 5 will not be detected . all samples and procedures were done in accordance with the dye manufacturer &# 39 ; s instructions . the markers used are peppermintstick phosphoprotein molecular weight standards ( molecular probes ), which also contain positive controls at 45 ( ovalbumin = phosphorylated ) and 23 . 6 kda ( β - casein = phosphorylated ). ps - 38 -( i27 ) 5 was loaded at two different volumes of 40 and 20 μl respectively . the pro - q stained gel ( left hand side of fig7 ) shows the preferential detection of phosphorylated conjugates specifically from nonphosphorylated conjugates or proteins . this can be seen by the increased optical density of phosphorylated proteins over nonphosphorylated proteins , which do have some background fluorescence ( also seen with non - phosphoprotein components of the peppermintstick phosphoprotein using a fluorophore ( alexa fluor 488 ( molecular probes )) it was possible to determine the exact amount of conjugate ( presentation system ) produced in a conjugation experiment . ( i27 ) 5 and ( i27 ) 1 , were reacted with a 100 times molar excess of alexa fluor 488 , which is thiol reactive , according to the manufacturers instructions . see fig1 . the conjugation mixture was dialysed against water to remove excess unbound fluorophore . the degree of conjugation was determined using a fluorimeter ( to measure fluorescence emitted by the conjugate ), spectrophotometer ( a 280 nm to measure total protein content , a 493 nm to measure alexa fluor 488 content ) and formulas provided by the manufacturer of the fluorophore ( molecular probes ). it was calculated that there was 100 % conjugation between ( i27 ) 5 and alexa fluor 488 ( producing alexa -( i27 ) 5 ), and 12 % conjugation between ( i27 ) 1 and alexa fluor 488 ( producing alexa -( i27 ) 1 ). now the degree of labelling had been determined , an accurate and true calibration of conjugate could be produced . a calibration curve of alexa -( i27 ) 5 conjugate was loaded in triplicate on a 15 % sds - page gel , transferred for western blot and then probed using an anti - alexa fluor ( rabbit polyclonal igg fraction ) primary antibody ( 1 in 3000 dilution ) and then a goat - anti - rabbit hrp secondary antibody ( 1 in 5000 dilution ). the blot was then developed as previously described . also loaded on the gel was three different blends of alexa -( i27 ) 5 and alexa -( i27 ) 1 showing that the blending capabilities of the conjugates is not influenced or determined by the entity bound to the concatamer . the western blot ( not shown ) demonstrates that the moiety conjugated to the concatamer is not limited to peptides . seven different quantities of calibrant make up the calibration line : 5 pmols , 2 . 5 pmols , 1 . 25 pmols , 0 . 63 pmols , 0 . 31 pmols , 0 . 16 pmols and 0 . 08 pmols of alexa -( i27 ) 5 , loaded in triplicate . of alexa -( i27 ) 5 and alexa -( i27 ) 1 showing that the blending capabilities of the conjugates is not influenced or determined by the entity bound to the concatamer . the western blot ( not shown ) demonstrates that the moiety conjugated to the concatamer is not limited to peptides . seven different quantities of calibrant make up the calibration line : 5 pmols , 2 . 5 pmols , 1 . 25 pmols , 0 . 63 pmols , 0 . 31 pmols , 0 . 16 pmols and 0 . 08 pmols of alexa -( i27 ) 5 , loaded in triplicate . the optical density of each band , for each amount of alexa -( i27 ) 5 , was measured and like quantities were averaged and plotted in excel to produce a calibration line ( fig1 , mean +/− standard deviation ). such a calibration line can then be used to determine the previously unknown quantity of a sample . using a calibration line of a1 -( i27 ) 5 to determine the quantity of phospholamban in canine sarcoplasmic reticulum . a calibration line of 5 pmols , 2 . 5 pmols , 1 . 25 pmols , 0 . 63 pmols , 0 . 31 pmols , 0 . 16 pmols and 0 . 08 pmols of a1 -( i27 ) 5 was loaded in triplicate on a 15 % sds - page , transferred for western blot and probed for the a1 epitope as previously described . the a1 epitope is from a protein called phospholamban ( plb ), which is present in the sarcoplasmic reticulum ( sr ) membrane . three samples of canine sarcoplasmic reticulum ( csr ) were also loaded on the gel / blot and the quantity of plb in the samples was determined using the anti - a1 ( mouse monoclonal ) primary antibody , which recognises the a1 epitope of plb in csr ( the same a1 epitope that is present on the a1 peptide used to produce a1 -( i27 ) 5 conjugate ). seven different quantities of calibrant make up the calibration line ( fig1 ): 5 pmols , 2 . 5 pmols , 1 . 25 pmols , 0 . 63 pmols , 0 . 31 pmols , 0 . 16 pmols and 0 . 08 pmols of a1 -( i27 ) 5 , loaded in triplicate . the optical density of each band , for each amount of a1 -( i27 ) 5 , was measured and like quantities were averaged and plotted in excel ( see fig1 , mean ± standard deviation ) to produce a calibration line . this calibration line can was used to determine the previously unknown quantity plb in each csr sample . using the line in fig1 , the pmol quantity of plb in each csr sample was calculated . : detection of ps - 38 -( i27 ) 5 construct using two different detection methods and two different recognition epitopes . with regard to fig1 , lanes 1 to 4 represent loadings of 20 , 15 , 10 and 5 pmols of ps - 38 -( i27 ) 5 conjugate . in panel a , the blot was probed with the perbio ™ india hisprobe ™- hrp probe . following stripping , the blot was re - probed with a polyclonal antibody specific to the ps - 38 conjugated peptide , panel b . the blot was stripped again and fmally probed using a monoclonal antibody ( novagen ) against the his - 6 tag , panel c . panel a of fig1 shows the detection of the ps - 38 -( i27 ) 5 construct using the non - antibody mediated perbio ™ india hisprobe ™- hrp probe recognising the his6 tag epitope . thus , the his - 6 tag is considered one of the target moieties in this embodiment . panel b of fig1 shows the detection of the ps - 38 -( i27 ) 5 construct using a primary antibody raised against the ps - 38 peptide . ( the additional band at approximately 150 kda in panel b is thought to be an oligomer of the construct , but could alternatively be a contaminant ). panel c of fig1 shows the detection of the ps - 38 -( i27 ) 5 conjugate using a primary antibody raised against the his6 tag . thus , the primary antibody is considered a specific binding partner and the his6 tag is a target moiety . panels a , b and c indicate that the presentation system can be recognised by different detection methods and using different binding partners , i . e by modified enzymes ( panel a ) and antibodies ( panels b and c ). furthermore , different target moieties on the same presentation system may be used for detection : his 6 epitope ( panels a and c ) and epitopes of the ps - 38 peptide ( panel b ). with regard to fig1 , there is shown an ( i39 ) 1 domain that contains no i27 modules . conjugation was done with a 100 molar excess of gmbs - pt17 to ( i39 ) 1 at room temperature . samples were run out on a 15 % sds - page gel , transferred for western blot and probed with anti - pt17 antibody as previuosly described . the blot shows detection of pt17 -( i39 ) 1 after conjugation at room temperature . a1 -( i27 ) 1 conjugate was inmunoprecipitated with anit - a1 specific monoclonal antibody , using a standard protocol . by analysing the recovered sample by western blot ( in different quantities ) it is possible to determine the amount recovered using a calibration line of a1 -( i27 ) 1 . hmnunoprecipitation was unsuccessful in this experiment , with 0 % of a1 -( i27 ) 1 recovered ( data not shown ). the a1 -( i27 ) 5 conjugate was precipitated with anti - a1 specific monoclonal antibody , using a standard protocol . by analysis of recovered sample by western blot ( in different quantities ) it is possible to determine the amount recovered using a calibration curve . fig1 shows the western blot of a1 -( i27 ) 5 ip samples and a1 -( i27 ) 5 calibrant . seven different quantities of calibrant make up the calibration line : 5 pmols , 2 . 5 pmols , 1 . 25 pmols , 0 . 63 pmols and 0 . 31 pmols of a1 -( i27 ) 5 . the optical density of each band for each amount of a1 -( i27 ) 5 calibrant , was measured and plotted in excel to produce the line of fig1 . this calibration line was used to determine recovered quantities of a1 -( i27 ) 5 after ip . using the graph , the pmol quantity of recovered a1 -( i27 ) 5 after ip was calculated below . a1 -( i27 ) s was clearly immunoprecipitated ( lane a1 recovery sample 2 ) whereas little was precipitated by protein a beads alone ( control recovery ). densitometry of the calibration curve samples and the immunoprecipitate samples permitted the calculation of 0 . 53 pmol a1 -( i27 ) 5 in the immunoprecipitate . if 100 % efficient , 30 pmol a1 -( i27 ) 5 would have been recovered , therefore the process was 1 . 77 % efficient . the data also permit identification of where in the process inefficiency was introduced . quantification of the samples control supernatant and a1 supernatant allowed description of a1 -( i27 ) 5 material removed by the a1 antibody at the end of the immunoprecipitation process . this was 50 pmol of a possible 100 pmol , i . e . 50 % efficient . the various wash steps post - immunoprecipitation were responsible for dramatic losses in product leading to a fmal yield ( or efficiency ) of 1 . 77 %. this quantitative approach hereby allows description of the effectiveness of individual steps in an experimental ( or industrial ) process . thus it will be appreciated that in this example the presentation system and method of the invention may also be used to monitor efficiency of ip techniques during all stages so to assess at what point in the process is least / most effective and could benefit from improvements . it is also believed that the presentation system and method of the invention may also be used to monitor efficiency of other techniques . 1 . colyer , j ., wang , j . h . 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