Patent Application: US-3684598-A

Abstract:
a method of assaying the growth hormone status of an individual by immuno - assay for the 150 kda insulin - like growth factor ternary complex . alternatively , a binary complex may be assayed . the method involves capturing the igfbp complex with a first antibody coupled to a solid phase and detecting the complex with a second antibody coupled to a label . a set of monoclonal and polyclonal antibodies usefutl for the assay is also provided .

Description:
the capture - detection igfbp complex assay is exemplified herein in two different formats . however , a variety of antibodies are described and mapped and various combinations of antibody pairs or triplets may be employed in the basic capture - detection assay format . the following examples describe the assays , epitope mapping and antibodies of the invention . edta - plasma samples from subjects with untreated gbrd ( 5 males age 1 - 32 yr , mean age 17 ± 14 . 5 , and 6 females age 2 . 3 - 65 yr , mean age 29 . 9 ± 25 ) and age - matched normal controls ( 8 males age 1 - 27 yr , mean age 13 . 3 ± 10 . 4 , and 8 females age 2 - 67 yr , mean age 36 . 6 ± 23 . 7 ) were obtained from dr . jaime guevara - aguirre , institute of endocrinology , metabolism and reproduction , quito , ecuador . ecuadorian ghrd patients were selected for this study since they have extremely low levels of igf - i and igfbp - 3 ( 20 ). the sample collection protocol was approved by the ethics committee of the institute of endocrinology , metabolism and reproduction ( quito - ecuador ) in compliance with the laws and regulations of the united states and ecuador . all subjects and / or guardians signed spanish versions of the approved informed consent forms . these samples were stored at − 70 ° c . until use . serum samples from consenting adults with acromegaly ( n = 8 ) and gh deficiency ( n = 5 ) were kindly provided by dr . john miell , department of medicine , kings college school of medicine , london , england . randomly selected fresh serum samples ( n = 42 ) were obtained from clinical laboratories in canada . these samples were residuals from routine clinical test samples and were from an adult population . upon collection , blood samples were allowed to clot , separated and , after clinical testing , the residuals stored at 4 ° c . were used for these studies within 48 hours . recombinant human igf - i and igf - ii were obtained from gropep , pty , ltd . ( adelaide , australia ) and recombinant non - glycosylated igfbp - 3 , was obtained from celtrix pharmaceutical , inc . ( santa clara , calif .). recombinant human igfbp - 2 , and igfbp - 4 through igfbp - 6 were purchased from austral biologicals ( san roman , calif .). igfbp - 1 was purified from human amniotic fluid and calibrated against pure recombinant human igfbp - 1 obtained from dsl ( webster , tex .). other materials and chemicals were obtained as described ( 40 , 41 ). purified human als was obtained from dsl ( webster , tex .) as described elsewhere ( 15 ). 125 i - igfbp - 3 ( 5 × 10 6 cpm / ml of 0 . 1 m napo4 , ph 7 . 4 ) was obtained from dsl ( webster , tex .). hrp - labelled streptavidin was purchased from amersham international ( buckinghamshire , england ). horseradish peroxidase ( ip ) was obtained from scripps laboratories ( san diego , calif .). tetramethylbenzidine peroxidase ( tmb / h 2 0 2 ) substrate system was from kirkegaard and perry laboratories , inc . ( gaithersburg , md .). sulfosucci nimidyl - 6 -( biotinamido ) hexanoate ( nhs - lc - biotin ) was from pierce chemical co . ( rockford , ill .). all other chemical reagents were of highest quality and were obtained from sigma chemical co . ( st . louis , mo . ), or amresco , inc . ( solon , ohio ). micro - titration strips and frames were products of costar ( cambridge , mass .). the igfbp - 3 / igf - i ( elisa - 1 ) and igfbp - 3 / als ( elisa - 2 ) assays described below are based on similar principles and identical components except for the antibody combination and selection of the assay buffer . the buffers were as follows : elisa - 1 assay buffer — 0 . 05 molar tris - maleate , ph 7 . 0 , 9 g / l nacl , 5 g / l bsa , 0 . 001 molar sodium edta , 0 . 5 ml / l tween 20 , 0 . 1 g / l thimerosal . elisa - 2 assay buffer — 0 . 05 molar tris - maleate , ph 7 . 0 , 0 . 9 g / l nacl , 20 g / l bsa , 0 . 5 g / l bovine gama globulin , 25 ml / l normal goat serum , 100 mg / l pmsf , 0 . 5 ml / l tween 20 , 0 . 1 g / l thimerosal . zero standard matrix buffer — 0 . 05 molar sodium phosphate , ph 7 . 4 , 9 g / l nacl , 1 g / l bsa , 0 . 005 molar sodium edta , 2 . 5 ml / l trasylol , 0 . 1 g / l thimerosal . antibody - hrp conjugate concentrate buffer — 0 . 02 molar sodium phosphate , ph 7 . 0 , 9 g / l nacl , 1 g / l cacl 2 , 5 g / l bsa , 0 . 1 g / l thimerosal . methods for preparation of both monoclonal antibodies and polyclonal antibodies are now well established ( 49 ). for this invention , the monoclonal antibodies were generated in mice and the polyclonal antibodies were raised in goats . the antibodies could have been raised in various species however , including but not limited to , mouse , rat , rabbit , goat , sheep , donkey , horse , etc . the anti - igf and anti - igfbp - 3 were raised against unglycosylated recombinant human igf - i , igf - ii or igfbp - 3 ( see table 1 for antibody names and characteristics ). anti - als antibodies were raised against synthetic unique n - and c - terminal regions of human als ( 15 ). the als peptides were conjugated to ovalbumin using glutaraldehyde , mixed with complete freund &# 39 ; s adjuvant and injected into goats ( 0 . 1 mg / injection ) using a monthly boost and bleed schedule . the als peptides were prepared using a model 430a applied biosystems ( foster city , calif .) peptide synthesizer ( synpep corp , dublin , calif .) and purified by high - performance reverse phase liquid chromatography ( hplc ). igf - i , igf - ii and igfbp - 3 antigens were injected as above without modification . for the development of monoclonal antibodies , splenocytes from appropriately immunized balb / c mice were fused with myeloma cells by the polyethylene glycol method . the viable hybridomas were selected , screened and propagated . the supernatants from selected clones were screened against the corresponding analyte by elisa technique . the positive hybridomas were cloned by limiting dilution and clones secreting specific antibodies were used for ascites production . all monoclonal antibodies were affinity purified on protein a columns and appropriately screened for specificity using well established western - immunoblot methods ( 50 ). polyclonal antibodies were first purified by affinity chromatography over gel columns containing the immobilized corresponding immunogen and the antibody fractions fuirther purified by chromatography over protein - a columns . as outlined below , the specificity of the antibodies was further substantiated by immunoassay cross - reactivity studies . all antibodies were produced , characterized and purified by dsl , inc . ( webster , tex .). the anti - igf - i , anti - igf - ii , anti - als and the anti - igfbp - 3 polyclonal antibodies are highly specific for the corresponding analyte ( 15 , 37 , 38 , 39 ) and have been employed in corresponding elisas manufactured by dsl inc ., ( webster , tex .). the anti - igfbp - 3 monoclonal antibodies are also highly specific for igfbp - 3 and are currently under evaluation for development of additional novel igfbp - 3 elisas . antibody coating to micro - titre wells was performed at a concentration of 2 . 5 - 30 mg / l , unless otherwise indicated , using previously published methods ( 40 ). in brief , 0 . 1 - 0 . 2 ml of the antibody solution ( 5 - 10 mg / l ) was added to each micro - titre well and allowed to incubate overnight at room temperature . the wells were then washed once with the wash solution and 0 . 2 ml / well of the blocking solution was added and allowed to incubate for 1 hour as above . the wells were washed once prior to use , or stored for up to 1 week in the blocking buffer at 4 ° c . igfbp - 3 coating to micro - titre wells was performed as described above , except that igfbp - 3 was coated at a concentration of 0 . 25 - 2 mg / l . antibody coupling to hrp was performed as previously described ( 41 ). the coupling reaction involved initial activation of the enzyme with sulfosuccinimidyl 4 -( n - maleimidomethyl ) cylcohexane - 1 - carboxylate ( smcc ) and its subsequent conjugation to 2 - iminothiolane activated antibody . the stock hrp - conjugated antibody solution was stored at 4 ° c . in the dark and appropriately diluted ( at least 1000 - fold ) in the appropriate assay buffer prior to use . antibody coupling to biotin was performed as previously described ( 42 ). biotinylation was performed at about a 150 - fold molar excess of nhs - lc - biotin added to 0 . 5 mg / ml of the antibody solution . the unconjugated biotin was removed by dialysis at 4 ° c . for 24 hours against several changes of 0 . 1 molar sodium bicarbonate , ph 8 . 3 , containing 9 g / l of nacl and 0 . 25 g / l of sodium azide . the stock antibody - biotin conjugate solution stored at 4 ° c . and appropriately diluted in 0 . 05 molar napo 4 , ph 7 . 2 , containing 9 g / l of nacl , 2 g / l of bsa and 1 . 0 ml / l of the antibacterial / antifungal preservative proclin 300 ( sigma ) prior to use . a pool of fresh serum samples was assigned 100 arbitrary units per litre ( au / l ) of igf - binding protein complex ( igfbp - 3 complex ) and used for elisa - 1 and elisa - 2 standardization . standards were prepared by appropriately diluting the serum pool in the zero standard matrix buffer to give reference standard values of 0 . 78 , 1 . 56 , 6 . 25 , 25 and 50 au / l of igfbp - 3 / igf - i complex , and 3 . 13 , 6 . 25 , 12 . 5 , 25 , and 50 au / l of igfbp - 3 / als complex for use in elisa - i and elisa - 2 , respectively . the standards were stable for up to 24 hours at 4 ° c . and greater than 2 months at − 20 ° c . or lower . the quality control samples used were also appropriately diluted serum pools . the nominal concentrations of the control samples were established by analysing them in igfbp - 3 complex elisa - 1 and elisa - 2 . a . s imultaneous b inding of a nti - igfbp a ntibodies the anti - igfbp - 3 antibodies ( b1 to b10 ) were evaluated for simultaneous binding ( pairing ) to igfbp - 3 . in brief each antibody was immobilized onto micro - titre wells at 500 ng / 100 μl / well and reacted with igfbp - 3 at 0 . 0 to 100 μg / l ( 25 μl / well standards plus 100 μl / well assay buffer ). after 1 hour shaking ( 500 - 700 rpm ) incubation at room temperature ( rt ), wells were washed four times and reacted with each remaining hrp4abelled anti - igfbp - 3 antibody as above for 30 minutes . stock hrp - antibodies were diluted about 10 , 000 - fold in the assay buffer and used at 100 μl / well ( about 5 ng antibody ). after washing , the reaction was developed by 10 minute incubation of the wells with the tmb / h 2 o 2 substrate solution ( 100 μl / well ) and addition of the stopping solution as described below . maximum increase in optical density ( od ) of ≦ 3 × background ( zero standard signal ) between 3 × background to 1 od , between 1 od to ≦ 2 od , and ≧ 2 od were ranked as indication of no simultaneous binding ( pairing ) to igfbp - 3 , weak pairing , moderate pairing and strong pairing , respectively . b . c ompetitive b inding of a nti - igfbp a ntibodies the anti - igfbp - 3 antibodies ( b1 to b10 ) were also evaluated for competitive binding to igfbp - 3 . in brief , each biotinylated antibody at a predetermined dilution was mixed with increasing concentrations of each remaining urdabelled antibody ( 0 - 50 μg / ml ) and added ( 50 μl antibody plus 100 μl assay buffer ) to triplicate wells pre - coated with igfbp - 3 ( about 75 ng / well ). after 2 hours shaking incubation , the wells were washed and developed by 30 minute reaction with hrp - labelled streptavidin ( 100 μl / well at 2000 - fold dilution in the assay buffer ) followed by tmb / h 2 o 2 and addition of the stopping solution . decrease in od of ≦ 20 %, 20 - 60 %, and ≧ 60 % were ranked as indicative of non - interfering , moderately interfering and strongly interfering binding of the antibody pair to igfbp - 3 . c . e ffects of igf o ccupancy on a nti - igfbp b inding the effect of igf occupancy of igebp - 3 on binding of anti - igfbp - 3 antibodies ( b1 to b10 ) was also evaluated . in brief , 125 i - igfbp - 3 ( 1 × 10 6 cpm / ml ) was mixed with 0 . 0 to 0 . 75 μg / ml igf - i or igf - ii ( in elisa 1 assay buffer ), incubated for 2 hours at rt , and 100 μl / well were added in triplicate to anti - igfbp - 3 antibody coated wells . after 2 hours rt incubation , wells were washed three times with dh 2 o and counted for bound radioactivity in a packard riastar gamma counter from packard canada ( mississauga , ontario ). an antibody binds at or near the igf binding site when its igfbp - 3 binding signal ( cpm ) is decreased by at least 30 % in response to igfbp - 3 pre - incubation with igfs . d . s imultaneous b inding of a nti - igfbp and a nti - igf or - als a ntibodies native serum igfbp - 3 complex was also evaluated for simultaneous binding to anti - igfbp - 3 antibodies ( b1 - b10 ) in combination with anti - igf4 antibodies ( i - 1 , i - 2 ), anti - igf - ii antibodies ( pi - 3 ) or anti - als antibodies ( pa - 1 , pa - 2 ) in pair - wise “ mix - antibody ” sandwich elisa . in brief , a pool of human serum was prepared by mixing aliquots from 15 different serum samples . micro - titration strips coated with each antibody were incubated , in quadruplicates , with 50 μl / well of the serum pool or standard matrix buffer and 100 μl / well of the appropriate assay buffer for 2 hours at rt as above . after washing , each quadruplicate set of serum pool / zero standard matrix buffer treated wells were then incubated with 100 μl / well of each of hrp - labelled anti - igf - i , anti - igf - ii , or anti - als antibodies for 30 min at rt . after washing , the reaction was developed by 10 min incubation with tmb / h 2 o 2 substrate and addition of the stopping solution . increase in od of ≦ 3 × background ( zero standard signal ), between 3 × background to 1 od , and between 1 -≧ 2 od indicates no binding , moderate binding or strong simultaneous binding of the two antibodies to complexed serum igfbp - 3 , respectively . mix - antibody combinations showing the strongest signal were selected for igfbp - 3 complex elisa development as described below . detailed information on epitopes recognized by igfbp - 3 monoclonal antibodies was obtained by evaluating all possible twosite ( capture - detection ) combinations in pair - wise sandwich elisa . the spacial distribution of epitopes recognized by each antibody in relation to others was then evaluated in pair - wise competitive elisa which evaluates binding of a given antibody to igfbp - 3 in the presence of excess amounts of each of the remaining antibodies . the latter provided information on whether an epitope recognized by one antibody was distinct enough to allow non - interfering ( independent ) binding of a second antibody or whether the epitope recognized were completely or partially overlapping , resulting in binding interferences . in the third series of experiments , interference in antibody binding to igfbp - 3 by igf - i or igf - ii was evaluated . this identified antigenic domains at or near the igf binding site and was assessed by monitoring binding of solid - phase antibodies to igfbp - 3 before and after pre - incubation with radio - labelled igfs . finally , the ability of igfbp - 3 antibodies for binding to serum igfbp - 3 complex in pair - wise combination with anti - igf - i , igf - ii and als antibodies was evaluated in “ mix antibody ” sandwich elisa . in similar experiments , simultaneous binding of complexed igfbp - 3 to anti - igfs paired with anti - als antibodies was also examined . as shown in table 2 , assessment of the 10 igfbp - 3 antibodies ( b1 to b10 ) in sandwich elisa identified 31 of the possible 100 combinations . the binding patterns appear to cluster into four antigenic regions based on reacting antibody combinations and the strength of pairing signal generated . the antibodies were grouped as follows : group i included b5 , b6 and b8 ; group ii included b1 , b2 , b4 , and b7 ; group iii included b3 ; and group iv included b9 . antibodies in the same group did not bind simultaneously to igfbp - 3 in sandwich elisa , but demonstrated weak to strong pairing with antibodies in other groups . pairing of antibodies in group i and group ii appear to depend , to some extend , on whether a given antibody was used for coating or detection . this is presumably due to suboptimal antibody concentrations and / or epitope conformational changes induced by binding of the first antibody to igfbp - 3 . the strongest two - site binding signals were generated between antibodies in group i and group iii ( b3 ). b9 and b10 were unable to form sandwich with each other or with remaining antibodies . b9 demonstrated strong binding to igfbp - 3 only when used as the detection - antibody in combination with b3 . a clearer pictured emerged when antibodies were evaluated for binding to solid - phase igfbp - 3 in pair - wise competitive elisa ( table 3 ). in these experiments , non - pairing , moderately pairing and strongly pairing antibodies identified in table 2 , appeared to compete with one another strongly , moderately or not - at all , respectively . only combinations of group i with group iii antibodies could bind simultaneously to igfbp - 3 without any interference . again , b9 could also bind strongly to igfbp - 3 when b3 was present . binding of group i antibodies to igfbp - 3 were significantly inhibited by pre - incubation of igfbp - 3 with igf - i or igf - ii . this positioned group 1 recognition epitopes at or near igf binding site . in repeat experiments , binding of group i antibodies to igfs / igfbp - 3 complexes decreased by more than 50 %, whereas activity of group ii , and group iii antibodies as well as the igfbp - 3 polyclonal antibody ( pb11 ) remained relatively unchanged ( table 4 , fig2 ). binding of b9 was about 2 - fold higher than that of an irrelevant antibody ( anti - psa ) and was not affected by igf binding to igfbp - 3 . b10 was non - reactive as it generated similar signal to that of the anti - psa antibody . in two - site “ mix - antibody ” elisa ( anti - igfbp paired with anti - als or anti - igf ), antibodies in group ii and group iii demonstrated simultaneous binding to the naturally occurring ( native ) serum igfbp - 3 complex in combination with anti - igf - i ( i1 ) or an anti - als ( pa - 1 ) antibodies used for detection . the strongest pairing signal was observed with the b2 / pa - 1 antibody pair and the b3 / i1 antibody pair . no simultaneous binding to serum igfbp - 3 complex was observed when the anti - igf - i antibodies were evaluated against anti - als antibodies in all possible mix - antibody combinations . bindings of other possible mix - antibody combinations were similar to that involving the negative control anti - psa antibody ( table 5 ). group i : igfbp antibodies ( b5 , b6 and b8 ) this group of antibodies recognized epitopes that mapped at or near the igfbp - 3 ligand binding site as defined by their binding inhibition in response to igfbp - 3 pre - incubation with the igfs . these antibodies could not be distinguished from each other as they did not bind simultaneously to igfbp - 3 in sandwich elisa and strongly competed with each other in competitive binding assays . by western immunoblot of various igfbp - 3 fragments , two of group i antibodies ( b5 and b8 ) have been recently shown to recognize epitopes at both n - terminal ( igfbp - 3 1 - 97 ) as well as c - termninal ( igfbp - 3 ˜ 200 - 264 ) regions of igfbp - 3 , whereas b6 was found to react only with the n - terminal ( igfbp - 3 1 - 97 ) fragment ( 44 ). the result of the present study seems to indicate binding of b6 to the c - terminal as well as n - terminal regions of igfbp - 3 . b6 strongly inhibited binding of b5 and b8 to igfbp - 3 in pair - wise competitive elisa , but as with b5 and b8 , demonstrated strong “ non - overlapping ” sandwich formation with b3 . it is also possible that b6 binds to n - terminal sequences that forms part of the igfbp - 3 ligand ( igf ) binding site . as the igf binding site of igfbp - 3 is thought to involve both n - as well as c - terminal sequences , antibodies that bind to such conformational epitopes could compete strongly for binding to the native molecule , but differently to denatured fragments in western immunoblot analysis . however , our findings of ligand binding site specificity for antibodies that reportedly bind to both n - and c - terminal of igfbp - 3 ( 44 ) is consistent with the notion that both n - and c - terminal sequences of igfbp - 3 contribute to the formation of igf - binding site ( 44 , 45 ). group ii : igfbp antibodies ( b1 , b2 , b4 , b7 )— although epitopes recognized by group ii antibodies overlapped with those specified by group - i antibodies , the immuno - reactivity of group ii antibodies was not affected by igf binding to igfbp - 3 . the antigenic cluster recognized by group ii antibodies was therefore , physically mapped to an area on the molecule distant from the igfbp - 3 ligand binding site . again group ii antibodies could not be distinguished from each other as they appeared to bind to overlapping epitopes . consistent with our findings is the reported specificity of group ii antibodies for the intermediate sequences of igfbp - 3 ( igfbp - 3 98 - 159 ) as evaluated by western immunoblot analysis ( 44 ). group iii : igfbp antibody ( b3 ) only one antibody defined a third epitope . the determinant for this antibody also overlapped with those of group ii , but was distinct from group i antibodies . b3 formed strong non - competing sandwich assays with antibodies in group - i . interestingly , this antibody have been found to recognize an epitope within the n - terminal region of igfbp - 3 ( igfbp - 3 1 - 97 ) ( 44 ), which according to our findings must not overlap with those of n - terminal igf - binding region . group iv : igfbp antibody ( b9 ) a fourth antigenic epitope recognized by b9 appeared to be a distinct conformational epitope that was accessible only after binding of b3 to igfbp - 3 . in capture - detection - antibody binding elisa , group ii and group iii antibodies could bind simultaneously to the native serum igfbp - 3 complexes in combination with the i - 1 or pa - 1 antibody used for detection . the strongest pairing signal was obtained for the b3 / i - 1 ( elisa - 1 ) and b2 / pa - 1 ( elisa - 2 ) combinations . this indicates that even in the native igfbp - 3 ternary complexes , antigenic domains on igf - i as well as als are accessible for antibody binding and can participate in two - site “ capture - detection ” antibody formation . consistent with these findings , are the recent observations that binding of rigf - i to solid - phase igfbp - 3 was detectable by c - terminal specific ( d - domain , residues 63 - 70 ) anti - igf - i mouse monoclonal antibodies in sequential binding experiments ( 46 ). the lack of simultaneous “ two - site ” binding of the above i - 1 and pa - 1 to serum igfbp - 3 complex suggests close binding proximity of the n - terminal part of als and igf binding site of igfbp - 3 ( fig1 ). this observation may provide an explanation for the reported finding that als binding to igfbp - 3 is an important modulator of igfbp - 3 affinity for the igf peptides ( 47 ). similar to the effect of antibody b3 on the immuno - reactivity of antibody b9 described above , proximal als binding to igfbp - 3 blgand binding site could cause conformational changes , leading to enhanced igf binding affinity of igfbp - 3 . among possible anti - igfbp - 3 / anti - igf - i combinations , the anti - igfbp - 3 b3 and anti - igf - i i - 1 generated strongest binding signal to serum igf - binding protein complex when used as capture and detection - antibodies , respectively . the elisa protocol was optimized as described previously ( 37 , 41 ). in the assay , standards or serum samples ( 0 . 025 ml of 10 - 40 fold diluted in the assay zero standard matrix buffer ) were added in duplicate to antibody coated wells , followed by addition of the elisa 1 assay buffer ( 0 . 10 ml ) and 1 hour incubation at room temperature with continuous shaking . the wells were washed five times and incubated with 0 . 1 ml / well of the anti - igf - i - hrp conjugate ( diluted in the assay buffer to approximately 0 . 1 - 0 . 25 mg / l ) for 1 hour at room temperature . the wells were washed five times with the wash solution , 0 . 1 ml of the tmb / h 2 o 2 substrate solution added for an additional 10 min incubation at room temperature . stopping solution ( 0 . 1 ml ) was then added and absorbance measured by dual wavelength measurement at 450 nm with background wavelength correction set at 620 nm . elisa data were analysed using data reduction packages included in the lab systems microplate reader with cubic spline ( smoothed ) curve fit . other statistical analyses were performed using the microsoft excel 97 statistical package by microsoft corporation ( usa ) on an ibm clone pentium computer . descriptive data are presented as the mean and sd unless otherwise specified . linear - regression analysis was performed by the least - squares method and correlation coefficients were determined by the pearson method . among possible anti - igfbp - 3 / anti - als combinations , the anti - igfbp - 3 b2 and anti - als pa - 1 generated the strongest binding signal to serum igf - binding protein complex when used as capture and detection - antbodies , respectively . the assay protocol was exactly as described above , except that the optimal sample and assay buffer volumes were 25 μl and 50 μl / well , respectively . for validation of igfbp - 3 complex elisa - 1 and elisa - 2 , appropriately diluted fresh serum samples were used . in both assays , the lower limit of detection ( sensitivity ) was determined by interpolating the mean plus 2sd of 12 replicate measurements of the negative control ( zero standard matrix buffer ). the intra - assay correlation coefficients ( cvs ) were determined by replicate analysis ( n = 12 ) of 3 samples at igfbp - 3 / igf - i levels of 8 . 6 - 24 . 3 au / l , and igfbp - 3 / als levels of 6 . 7 - 18 . 7 au / l ; interassay cvs by duplicate measurement of appropriate samples in 7 - 9 separate runs . recovery was assessed by adding 50 μl of samples containing different igfbp - 3 complex levels to 450 μl of 10 - fold diluted serum samples followed by analysis . percent recovery was determined by comparison of the amount of added igfbp - 3 complex to the amount measured after subtracting the endogenous igfbp - 3 complex level . linearity was tested by analysing serum samples diluted first 10 - fold , then serially 2 - 16 fold in the zero standard matrix buffer . igfbp - 3 complex elisa specificity was analysed by assaying igf - i ( up to 300 μg / l ), igf - ii ( up to 3000 μg / l ), igfbp - 1 , 2 , 4 - 6 up to 500 μg / l , and igfbp - 3 up to 4 . 3 mg / l . to evaluate igfbp - 3 complex stability , and thus practical use of the assay , aliquots of fresh serum samples ( n = 3 ) were stored at rt , 4 ° c ., and − 20 ° c . and then analysed on day 0 , 2 and 3 of storage by elisa - 1 and elisa - 2 after 10 - fold dilution in the assay zero standard matrix buffer . on the day of analysis , samples were assayed against a new set of standards freshly prepared from a frozen ( at − 20 ° c .) aliquot of the stock standard serum pool . aliquots of a set of standards were also stored at the above temperatures and similarly analysed . elisa - 1 and - 2 results were compared with results obtained from prior art assays . igf - i , igf - ii , igfbp - 3 and als were analysed by immunoassay kits manufactured by dsl ( webster , tex .). these assays are based on non - competitive elisa principles performed in antibody coated micro - wells ( 15 , 37 , 38 , 39 ) and horseradish peroxidase ( hrp ) labelled detection - antibodies as described ( 41 ). igf - i and igf - ii were assayed by the activ ™ non - extraction igf - i and igf - ii elisa ( dsl , webster ) which incorporates a sample pre - treatment step to dissociate igf - i from igfbps prior to analysis ( 43 ). the sample pre - treatment step involves mixing 20 μl of sample with 1 . 0 ml of igf - i acidification buffer followed by 30 min room temperature incubation , and addition of 1 . 0 ml of the neutralization buffer . the final sample preparation dilution factor was 101 - fold and 20 μl of the treated sample is used for igf analysis . the igf elisa kits each have a total incubation time of less than 3 hours and an overall imprecision of less than 10 %. the active ™ igfbp - 3 elisa ( dsl , webster ) incorporates a 101 - fold sample pre - dilution and uses 25 μl of the pre - diluted sample for igfbp - 3 analysis . the assay has a total incubation time of about 3 hours , a standard range of 2 - 100 μg / l ( 0 . 20 - 10 mg / l after correction for sample dilution factor ) and an overall imprecision of less than 10 % ( 39 ). the active ™ total als elisa ( dsl , webster ) incorporates a 101 - fold sample pre - treatment step that would result in unfolding of both complexed and uncomplexed als , thus allowing measurement of the total als levels ( 15 ). the sample pre - treatment step involves mixing of 10 μl of sample with 1 . 0 ml of the sample pre - treatment buffer followed by 30 minute room temperature incubation . the final sample preparation dilution factor was 101 - fold and 20 μl of the treated sample is used for total als analysis . the assay has a total incubation time of about 2 hours , a standard range of 6 - 600 μg / l ( 0 . 6 - 60 mg / l after correction for sample pre - treatment dilution factor ) and an overall imprecision of less than 10 %. absorbance of elisas were measured with the labsystems multiskan multioft microplate reader by labsystems , ( helsinki , finland ). based on the above epitope mapping , novel immunoassays for quantisation of circulating igfbp - 3 complexes were developed . both the igfbp - 3 / igf - i elisa - 1 and the igfbp - 3 / als elisa - 2 involve a two - site noncompetitive ( sequential ) immunoreaction and are based on solid - phase anti - igfbp - 3 b3 or b2 antibody paired with detection anti - igf - i i - i or anti - als pa - 1 antibodies , respectively . optimized protocols were established by evaluating the effects of various technical manipulations on the analytical performance of the assays as previously described ( 37 , 41 ). a coating antibody concentration of 10 mg / l , a detection - antibody concentration of about 0 . 1 - 0 . 25 mg / l , a 60 minute first and second step room temperature incubations , and a 10 minute substrate development step were selected . among the variables examined , composition of the assay buffer and coating antibody concentrations had the most obvious effect on sensitivity and dynamic range of the assays . a typical standard curve and performance characteristics of elisa - 1 and elisa - 2 are summarized in fig3 a - 3b and table 6 . addition of igf - i ( up to 300 μg / l ), igf - ii ( up to 3000 μg / l ), igfbp - 2 and igfbp - 4 - 6 ( up to 500 μg / l ), and igfbp - 3 ( up to 4 . 2 mg / l ) to the zero standard matrix buffer did not show any cross - reactivity . serum igfbp - 3 complexes were analysed in replicate aliquot of samples stored at rt , 4 ° c . and − 20 ° c . as measured by both elisa - 1 and elisa - 2 , igfbp - 3 complexes demonstrated high stability at all temperatures for up to 3 days of storage , and recoveries at 4 ° c . and − 20 ° c . were at least 85 % of the day 0 values ( fig4 a - 4c , 5 a - 5 c ). serum - based standards stored and analysed as above demonstrated similar stability , and igfbp - 3 complexes in a pool of fresh sera stored at − 20 ° c . demonstrated at least 2 months stability . despite the complexity of their design , the elisa - 1 and elisa - 2 assays for igfbp complex demonstrated acceptable analytical performance characteristics . the finding of relatively high stability of the igfbp - 3 complexes at various temperature and even in diluted form was unexpected and was instrumental in the success of the development of the assays . the demonstrated linearity of the assays in response to sample dilution may suggest measurement of only the tightly bound igfbp - 3 ternary complexes . antibody binding and sample dilution may induce igf dissociation and , thus removal of loosely bound complexes . plasma samples from subjects with untreated ghrd ( n = 11 ) and age - matched normal subjects ( n = 16 ), and serum samples from adults with acromegaly ( n = 8 ) or ghd ( n = 5 ) were simultaneously analysed by elisa - 1 and elisa - 2 and for igf - i , igf - ii , igfbp - 3 and total als levels . regression analysis of data showed high degree of correlations between igfbp - 3 complex elisa - 1 and - 2 versus igfs , igfbp - 3 and total als levels ( table 7 , and fig6 a - 9 b ). the best overall correlations were observed in comparisons with igf - i levels , while correlation against igf - ii levels were relatively poor . the flattened “ low - end ” appearance of correlation graphs are due to significant differences in the relative levels of the various analytes as a function of age ( fig1 ) as well as clinical conditions , and not necessarily due to poor low end correlations . in fact , correlations in the ghrd range was the same as those involving all sample values . although the number of normal samples in various age groups were small , the overall pattern of igfs , igfbp - 3 and als levels versus age were similar to those previously reported , with levels showing significant rise during pubertal age ( 21 , 34 , 35 ). as expected , igfbp - 3 complex levels were similarly affected by age . however , igf - i based complexes measured by elisa - 1 showed the widest variations , being lowest in the youngest and oldest age groups and highest during pubertal age ( fig1 ). in plots of mean analyte levels as a percentage of the corresponding mean of normal values , the mean of igfbp - 3 complex levels measured in ghrd and ghd subjects by elisa - 1 were less than 8 % and 2 % of the mean of normal values , respectively . the next largest differences were observed for igf - i levels ; the mean igf - i levels in ghrd and ghd subjects were less than 13 % and less than 24 % of the mean of normal values ( fig1 ). in comparative distribution plots of the individual values , igfbp - 3 complex levels by elisa - 1 demonstrated similar if not better discrimination between the various sample groups , particularly between ghrd , normals and ghd subjects ( fig1 a - 12 e ). in several experiments involving randomly selected samples , the igfbp complex elisas showed significant correlation with igf - i , igfbp - 3 and als levels . this was subsequently confirmed by the preliminary clinical evaluations involving samples from subjects with ghrd , their age - matched normal controls , and specimens from acromegalic and ghd adults . overall , both elisa - i and elisa - 2 demonstrated high correlation with igf - i , igfbp - 3 and als levels . this supports the reported observations that most of circulating igfs and igfbp - 3 are primarily present in the ternary protein complex and that igf availability may be a key determinant of ternary complex formation ( 4 - 12 ). of interest was the observation that the igfbp complexes measured by elisa - 1 showed the widest relative variations as a function of age . the igfbp complex levels of the individual samples were lowest in the younger age group and highest in the older , pubertal range subjects ( fig1 ). similarly , in comparative distribution plots of the individual sample values , elisa - 1 demonstrated a similar , if not better discrimination between the various sample groups ( fig1 a - 13 e ). the relatively lower igfbp complex measured in younger age group by elisa - 1 may suggest availability of proportionally higher amount of igf - i in the free ( or dissociable ) form in this age group , as has been recently reported for early infancy ( 48 ). the data presented here establish that the antibodies described herein can be selected on the basis of their epitope maps and employed in a successful capture - detection elisa assay format . additional antibody mapping can be performed in the manner described here . in particular , it would be beneficial to identify anti - igfbp antibodies whose binding is unaffected by igf - i or als binding . such antibodies would allow the assay of total igfbp - 3 without regard for the degree of complex formation . in addition to the two antibody pairs exemplified above ( elisa - 1 and - 2 ), other antibody pairs or even triplets can be optimized in a manner similar to that described herein . hence , the above examples are not to be construed as limiting , but rather as illustrative of the many antibody combinations that can be employed in a capture - 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