Patent Application: US-201013381230-A

Abstract:
the present invention relates to a method for diagnosing a cardiovascular or cancer disease by detecting igfbp - 4 fragments in a patient sample . antibodies specifically recognizing novel epitopes originated by enzyme - dependent cleavage of igfbp - 4 are also disclosed .

Description:
the invention describes n - and c - terminal proteolytic fragments of igfbp - 4 as the biomarkers that are presented at significantly higher levels in the plasma sample of acs or some cancer patients in comparison with healthy donors &# 39 ; plasma . immunoassays for igfbp - 4 fragments could be used for early detection of acs or cancer , or to determine the degree of the risk of disease development . standard protocols were followed for the development of monoclonal antibodies specific to the igfbp - 4 peptides as well as intact igfbp - 4 . synthetic peptides used for animal immunization to obtain monoclonal antibodies , specific igfbp - 4 fragments , were corresponding to igfbp - 4 proteolytic fragments at the site of the papp - a - dependent cleavage . synthetic peptides contained additional terminal cysteines ( opposite to putative proteolytic site ) for coupling purposes . the sequences were verified by mass spectroscopy analysis and peptides were conjugated to carrier proteins . the resulting conjugates were used as antigens for mice immunization . peptide - binding monoclonal antibodies are prepared according to standard technology ( 19 - 22 ) known to those skilled in the art . after several cycles of animal immunization mouse splenocytes are fused with cells of myeloma cell line . such protocol includes also the stage of screening of formed hybridoma clones for desired specificity of produced antibodies . to obtain antibodies of the present invention , hybridoma clones were screened to specific binding to igfbp - 4 peptides , corresponding to igfbp - 4 proteolytic fragments , and at the same time not reacting to intact igfbp - 4 . this approach enabled to find out several hybridoma clones producing monoclonal antibodies specific to novel epitopes of igfbp - 4 , which were formed in the process of papp - a - dependent cleavage of the protein ( fig2 a ). in the experiments made a group of monoclonal antibodies specific to proteolytic fragments of igfbp - 4 , produced by papp - a - dependent cleavage , and having cross - reactivity to intact igfbp - 4 less than 5 % was selected . however , it should be noted that the cross - reactivity percentage obtained depends on , for instance , the method used , and 5 % should not be considered to be a restrictive value for low cross - reactivity . obtained peptide - specific antibodies were tested in sandwich immunoassay with monoclonal antibodies specific to the intact ( full - size ) igfbp - 4 in order to find two - site antibodies combinations , suitable for the development of sandwich immunoassays for specific determination of igfbp - 4 proteolytic fragments regardless of the presence of intact full - length igfbp - 4 . monoclonal antibodies specific to intact igfbp - 4 were used as capture antibodies , whereas monoclonal antibodies specific to proteolytic fragments of igfbp - 4 were used as detection antibodies . in some embodiments the opposite configuration of antibodies is also possible . sandwich immunoassays described in the present invention were highly specific to proteolytic fragments of igfbp - 4 ( fig2 b ). in the present invention the detection antibodies of developed sandwich immunoassay methods were labeled by stable eu3 + chelate . in various other embodiments detection antibody could be labeled by different types of labels able to generate different types of signals that could be visualized or detected using a variety of standard procedures , such as detection of luminescence , chemiluminescence , fluorescence , absorbance , radioactivity , or by microscopy , imaging , etc . immunoassays may include immunohistochemistry , enzyme - linked immunosorbent assay ( elisa ), western blotting , nephelometry , turbidimetry , immunoradiometric assay , lateral flow , immunohisto / cyto - chemistry and other methods known to those of skill in the art . immunoassays could be used to determine presence or absence of a biomarker in a sample as well as the amount of a biomarker in a sample . the amount of igfbp - 4 proteolytic fragments in the sample can be determined by comparison to ( or as a ratio to ) a reference or standard , such as an intact igfbp - 4 or different polypeptide known to be present in the sample . the amount of igfbp - 4 proteolytic fragments in the sample can also be determined by comparison to a reference or standard , such as the amount of the endogenous or recombinant or synthetic igfbp - 4 fragments in a reference or control sample . accordingly , the amount of a biomarker in a sample need not be quantified in absolute terms , but may be measured in relative terms with respect to a reference or control . in the present invention detection of proteolytic fragments of igfbp - 4 in the plasma samples of acs was carried out . significant increase of the fragments was revealed by using ibp521 - ibp30 sandwich pair ( fig3 ). various embodiments of this invention include detection of n - terminal or c - terminal , or simultaneous c - terminal and n - terminal fragments of igfbp - 4 in the patients &# 39 ; plasma samples for the assessment of acs development risk . a further embodiment of the invention is an immunoassay kit for the detection igfbp - 4 proteolytic fragments or measurement of igfbp - 4 proteolytic fragments amount in the sample . the kit may comprise ( i ) a monoclonal antibody specific to n - terminal or c - terminal igfbp - 4 proteolytic fragment , ( ii ) a second monoclonal detection antibody specific to any appropriate epitope of intact igfbp - 4 , and ( iii ) a standard or calibrator preparation of endogenous igfbp - 4 fragments or recombinant protein corresponding at least partially to the sequence of igfbp - 4 fragment . the second monoclonal antibody may be appropriately labeled for the detection of antibody — igfbp - 4 fragments complex . in some embodiments the kit for igfbp - 4 fragments competitive measurement may comprise ( i ) a monoclonal antibody specific to n - terminal or c - terminal igfbp - 4 proteolytic fragment , ( ii ) standard or calibrator preparation of endogenous igfbp - 4 fragments or recombinant protein corresponding at least partially to the sequence of igfbp - 4 fragment , labeled for the detection . the levels of igfbp - 4 fragments in the analyzing sample can be determined by the degree of competitive removal of the said labeled standard preparation of igfbp - 4 fragments . generation of mouse monoclonal antibodies specific to novel proteolysis - mediated epitopes of igfbp - 4 peptide - 1 and peptide - 2 were synthesized using solid - phase fmoc chemistry ( 23 ). peptides were prepared on p - alkoxybenzylalcohol resin . after cleavage from the resin , the crude peptide preparation was purified by reversed - phase high - pressure liquid chromatography . c18 preparative column was applied with a gradient of 0 . 1 % trifluoroacetic acid in water and 0 . 1 % trifluoroacetic acid in acetonitrile . the purity (& gt ; 95 %) was determined by analytical c18 high - pressure liquid chromatography and mass spectroscopy ( matrix - assisted laser desorption / ionization mass spectrometry with accuracy ± 0 . 5 dalton ). igfbp - 4 peptide - 1 ( seq id no . 1 ) contained the amino acid sequence identical to igfbp - 4 fragment 121 - 135 of seq id no : 3 , with one additional cysteine residue from the n - terminus . igfbp - 4 peptide - 2 ( seq id no . 2 ) contained the amino acid sequence identical to igfbp - 4 fragment 136 - 150 of seq id no : 3 with one additional cysteine residue from the c - terminus . sulphhydryl groups of these additional cysteine residues were used for the preparation of the peptide conjugates with carrier proteins . preparation of conjugates of the peptides with carrier proteins was performed by using sulfo - smcc obtained from pierce ( rockford , ill .) according to manufacturer &# 39 ; s instructions . for the conjugation 2 . 5 mg of carrier protein — bovine serum albumin , bsa ) or ovalbumin ( both obtained from sigma chemicals , st . louise , mo .) was dissolved in 10 mm khpo 4 , 150 mm nacl , ph 7 . 4 ( pbs ) to the concentration 10 mg / ml . two milligrams of sulfo - smcc , dissolved in 0 . 1 ml dimethyl sulfoxide , were added to the protein solution . reaction of carrier protein activation was carried out for 2 hours at room temperature . excess of sulfo - smcc was removed by gel - filtration using nap - 5 columns ( obtained from ge healthcare life sciences , piscataway , n . j .). nap - 5 columns were pre - equilibrated with 10 mm khpo 4 , 150 mm nacl , ph 7 . 2 . then 2 mg of synthetic peptide - 1 or peptide - 2 were added to protein solution to start the conjugation . this reaction was carried out for 2 hours on ice with constant shacking . unreacted peptide fraction was removed from protein - peptide conjugate by using gel - filtration nap - 5 columns , pre - equilibrated with pbs . the conjugation of the peptides to appropriate carrier protein was confirmed by 3 - 5 kda increase in the protein molecular weight revealed by using sodium dodecyl sulphate polyacrylamide gel electrophoresis . conjugates were aliquoted and stored at − 20 ° c . until use . groups of five balb / c mice were immunized five times with peptide - protein conjugates . group 1 : first immunization : intraperitoneally 0 . 2 ml of 10 microg bsa - peptide - 1 in pbs with 60 % freund &# 39 ; s complete adjuvant ; second immunization : on day 30 , intraperitoneally 0 . 2 ml of 5 microg bsa - peptide - 1 in pbs with 60 % freund &# 39 ; s incomplete adjuvant ; third immunization : on day 60 , intraperitoneally 0 . 2 ml of 2 . 5 microg bsa - peptide - 1 in pbs . group 2 : first immunization : intraperitoneally 0 . 2 ml of 10 microg bsa - peptide - 2 in pbs with 60 % freund &# 39 ; s complete adjuvant ; second immunization : on day 30 , intraperitoneally 0 . 2 ml of 5 microg bsa - peptide - 2 in pbs with 60 % freund &# 39 ; s incomplete adjuvant ; third immunization : on day 60 , intraperitoneally 0 . 2 ml of 2 . 5 microg bsa - peptide - 2 in pbs . twenty days after third immunization mice with the highest titer of peptide - specific antibodies were selected for the last immunizations and hybridization . mice were intravenously injected with 0 . 2 ml of 10 microg bsa - peptide - 1 in pbs for group 1 , and with 0 . 2 ml of 10 microg bsa - peptide - 2 in pbs for group 2 . intravenous injections were repeated next day at the same protocol ( fifth immunization ). then two days after the fifth immunization , spleens of immunized mice were sterilely isolated and homogenized tissue was fused with the mouse myeloma cell line sp2 / 0 as described previously ( 19 - 22 ). conditioned culture of growing hybridomas was screened for antibodies by enzyme linked immunosorbent assay ( elisa ). hybridomas that produced antibodies specific to peptide - 1 or peptide - 2 were selected by elisa with ovalbumin - peptide - 1 or ovalbumin - peptide - 2 , respectively , used as presorbed antigens . human recombinant igfbp - 4 expressed in ns0 cell line ( obtained form sigma chemicals , st . louise , mo .) was used as well as a presorbed antigen for the additional test . for the assay 50 ng / 0 . 1 ml pbs per well of ovalbumin - peptide - 1 , or ovalbumin - peptide - 2 , or human recombinant igfbp - 4 were sorbed on the immunoassay polystyrene plates ( obtained from corning , cambridge , mass .). after 40 min of antigen sorption the plates were washed two times and blocked for 10 min with pbs , containing detergent tween 20 , 0 . 1 % ( pbst ). then the plates were incubated with 0 . 05 ml of conditioned media collected from growing hybridomas for 30 min and washed two times with pbst . mouse antibodies bound to presorbed antigens were revealed by 30 min incubation with secondary anti - mouse igg polyclonal antibodies , conjugated with hrp , 0 . 1 ml of 1 : 1000 dilution in pbst per well . secondary antibodies were from sigma chemicals , st . louise , mo . after the incubation with secondary antibodies the plates were washed with pbst six times and 3 , 3 ′, 5 , 5 ′- tetramethyl benzidine ( tmb ) peroxidase substrate , containing 0 . 03 % hydrogen peroxide , was added . the reaction was stopped after 15 minutes of incubation by adding 0 . 1 ml of 0 . 5 m phosphoric acid and absorbance in wells was measured at 450 nm . the measurement of the absorbance was performed with the labsystems multiscan microplate reader ( labsystems , finland ). hybridomas producing antibodies specific to appropriate peptide , conjugated with ovalbumin ( absorbance at described above conditions at 450 nm & gt ; 0 . 5 over background ), and at the same time not reacting with human recombinant igfbp - 4 ( absorbance at 450 nm & lt ; 0 . 025 over background ), were selected for further work . such hybridomas were cloned by limiting dilution . hybridoma clones secreting the monoclonal antibodies of interest were grown in dulbecco &# 39 ; s modified eagle &# 39 ; s medium ( dmem ), containing 10 % fetal bovine serum ( hyclone laboratories , logan , utah ). monoclonal antibodies were raised in mouse ascitic fluid after intraperitoneal injection of selected hybridoma clones . antibodies were purified from ascitic fluid by using protein a affinity chromatography . the resin was from ge healthcare life sciences ( piscataway , n . j . ), and purification was carried out according to manufacturer &# 39 ; s instructions . purified monoclonal antibodies were stored as suspensions in 50 % ammonium sulfate at 4 ° c . to confirm the specificity of selected monoclonal antibodies igfbp - 4 proteolytic fragments were obtained . papp - a - dependent proteolytic reaction was performed according to conditions described earlier ( 14 ). two microg of human recombinant igfbp - 4 was incubated in 0 . 23 ml of 50 mm tris - hcl , ph 7 . 5 , in the presence of 2 mm cacl 2 , 1 . 8 microg igf - ii ( obtained from sigma chemicals , st . louise , mo .). 40 ng of human recombinant papp - a ( hytest , turku , finland ), and 2 microliters protease inhibitors cocktail ( obtained from sigma chemicals , st . louise , mo ). the reaction was carried out for 15 hours at 37 ° c ., and was stopped by freezing the sample at − 20 ° c . the degree of papp - a - dependent cleavage of igfbp - 4 was determined by western blotting by using 1 microg / ml specific rabbit polyclonal antibodies obtained from abcam ( cambridge , mass .) ( fig1 ). specificity studies of selected monoclonal antibodies to igfbp - 4 proteolytic fragments were performed in indirect elisa using affinity - purified antibodies ( fig2 a ). ten ng of full - length recombinant igfbp - 4 or igfbp - 4 fragments produced by papp - a - dependent cleavage ( preparation described above ) were sorbed on polystyrene plate . after 40 min of incubation the plates were washed two times and blocked for 10 min with pbs , containing detergent tween 20 , 0 . 1 % ( pbst ). then selected mabs ( 10 microg / ml ) were incubated for 30 min at room temperature with shaking and after that washed two times with pbst . specifically bound antibodies were detected by anti - mouse igg polyclonal antibodies , conjugated with hrp , 0 . 1 ml of 1 : 1000 dilution in pbst per well . secondary antibodies were from sigma chemicals , st . louise , mo . after incubation with secondary antibodies the plates were washed with pbst six times and 3 , 3 ′, 5 , 5 ′- tetramethyl benzidine ( tmb )— containing peroxidase substrate , supplemented with 0 . 03 % hydrogen peroxide , was added . the reaction was stopped after 15 minutes of incubation by adding 0 . 1 ml of 0 . 5 m phosphoric acid and absorbance was measured at 450 nm . the group of monoclonal antibodies specific to proteolytic fragments of igfbp - 4 , produced by papp - a - dependent cleavage , and having cross - reactivity to intact igfbp - 4 less than 5 % was finally selected : ibp28 , ibp27 , ibp12 , ibp3 , ibp4 , ibp7 , ibp13 , ibp18 , ibp19 , ibp20 , ibp30 , ibp167 , ibp166 , ibp168 , ibp174 , ibp160 , ibp161 , ibp164 , ibp171 . all monoclonal antibodies were of igg isotype , except that ibp30 was of igm isotype . specificity of affinity - purified monoclonal antibodies was also checked in sandwich immunoassays ( fig2 b ). several sandwich assays utilizing one monoclonal antibody specific to proteolytic fragment of igfbp - 4 ( n - or c - terminal ; cross - reaction with full - length molecule less than 5 % in indirect elisa ) and another mab , recognizing any epitope of intact igfbp - 4 , were developed . generation of mouse monoclonal antibodies specific to intact igfbp - 4 is described in example 2 . to perform sandwich fluorescent immunoassays , we used detection mabs labeled with stable eu3 + chelate as described by hyytiä et al . ( 24 ). capture antibodies in this assay were specific to intact igfbp - 4 , whereas detection antibodies were specific to proteolytic neo - epitopes of igfbp - 4 . capture antibodies ( ibp513 , ibp521 ), 2 μg per well in 100 μl of phosphate buffer saline , were incubated in 96 - well immunoassay plates for 30 min at room temperature upon constant shaking . the plates were washed with 10 mm tris - hcl ( ph 7 . 8 ) buffer , supplemented by 0 . 15 m nacl , 0 . 025 % tween 20 and 0 . 5 g / l nan 3 ( buffer a ). after washing 0 . 1 ml of assay buffer ( 50 mm tris - hcl buffer , ph 7 . 7 , 9 g / l nacl , 0 . 01 % tween 40 , 0 . 5 % bsa and 0 . 5 g / l nan 3 ), containing 100 ng / ml of full - length human recombinant igfbp - 4 or igfbp - 4 fragments produced by papp - a - dependent cleavage were added to the plates . the plates were incubated for 30 min at room temperature with constant shaking . after washing with buffer a 0 . 1 ml of the solution ( 1 mg / l ) of detection antibodies ( ibp12 , ibp27 and ibp30 ) in the assay buffer were added . the plates were incubated for 30 min at room temperature with constant shaking . after washing with buffer a , 0 . 2 ml of enhancement solution ( 1 . 75 m nascn , 1 m nacl , 5 % glycerol , 20 % 1 - propanol , 5 mm na 2 co 3 , 50 mm glycine - naoh , ph 10 . 0 ) per well ware added and incubated for 3 min at room temperature with gentle shaking . fluorescence of eu3 + was measured on a victor 1420 multilabel counter ( wallac - perkin elmer ). the fluorescence was expressed in counts per second ( cps ). developed sandwich immunoassays were able to detect only igfbp - 4 fragments produced by papp - a - dependent cleavage and had no crossreaction ( or very low — less than 1 %) with full - length igfbp - 4 . five balb / c mice were immunized five times with human recombinant igfbp - 4 expressed in mammalian ns0 cell line . the protein was obtained from sigma chemicals , st . louise , mo . first immunization : intraperitoneally 0 . 2 ml of 5 microg igfbp - 4 in pbs with 60 % freund &# 39 ; s complete adjuvant . second immunization : on day 30 , intraperitoneally 0 . 2 ml of 2 microg igfbp - 4 in pbs with 60 % freund &# 39 ; s incomplete adjuvant . third immunization : on day 60 , intraperitoneally 0 . 2 ml of 2 microg igfbp - 4 in pbs . twenty days after third immunization mice with the highest titer of protein - specific antibodies were selected for the following immunizations and hybridization . mice were intravenously injected for a fourth time with 0 . 2 ml of 2 microg igfbp - 4 in pbs . the last intravenous injection was performed on the next day according to the same protocol ( fifth immunization ). two days later , spleen of immunized mice was sterilely isolated and homogenized tissue was fused with the mouse myeloma cell line sp2 / 0 as described previously ( 19 - 22 ). conditioned media of growing hybridomas was screened for igfbp - 4 - specific antibodies using elisa method . hybridomas producing antibodies specific to intact igfbp - 4 were selected by means of indirect elisa . for the assay 50 ng / 0 . 1 ml pbs per well of full - length human recombinant igfbp - 4 were sorbed on the immunoassay polystyrene plates . after 40 min of incubation the plates were washed two times and blocked for 10 min with pbs , containing detergent tween 20 , 0 . 1 % ( pbst ). then the plates were incubated for 30 min with 0 . 05 ml of conditioned media collected from wells containing growing hybridomas . after incubation the plates were washed two times with pbst . after washing the plates were incubated with 0 . 1 ml of per well of secondary anti - mouse igg polyclonal antibodies , conjugated with hrp ( 1 : 1000 dilution in pbst ) for 30 min . after incubation with secondary antibodies the plates were washed with pbst six times and peroxidase substrate , containing tmb and 0 . 03 % hydrogen peroxide , was added . the reaction was stopped after 15 minutes of incubation by adding 0 . 1 ml of 0 . 5 m phosphoric acid and the absorbance in wells was measured at 450 nm . hybridomas producing antibodies specific to full - length igfbp - 4 ( absorbance at described above conditions at 450 nm & gt ; 0 . 5 over background ) were cloned by limiting dilution method . hybridoma clones secreting the monoclonal antibodies of interest were cultivated in dmem , containing 10 % fetal bovine serum . monoclonal antibodies specific to full - length igfbp - 4 were raised in mouse ascitic fluid after intraperitoneal injection of selected hybridoma clones . antibodies were purified from ascitic fluid by using protein a affinity chromatography . the resin was from ge healthcare life sciences ( piscataway , n . j . ), and purification was carried out according to manufacturer &# 39 ; s instructions . purified monoclonal antibodies were stored as suspensions in 50 % ammonium sulfate at 4 ° c . samples of human atherosclerotic coronary vessels were stored at − 70 ° c . until used . papp - a was extracted from atherosclerotic coronary arteries after tissue homogenization in 50 mm tris - hcl ( ph 7 . 8 ) buffer , containing 0 . 15 m nacl , 0 . 5 % triton x100 , and protease inhibitors cocktail . extracted papp - a was purified by means of affinity chromatography . affinity matrix used for papp - a purification was prepared utilizing papp - a - specific monoclonal antibody 4g11 ( obtained from hytest , turku , finland ). to confirm identity of purified protein to papp - a , western blotting analysis with several papp - a - specific monoclonal antibodies and liquid chromatography / tandem mass spectrometry analysis were used . for proteolytic activity analysis of atherosclerotic papp - a 2 microg of human recombinant igfbp - 4 was incubated in 0 . 23 ml of 50 mm tris - hcl , ph 7 . 5 , in the presence of 2 mm cacl 2 , 1 . 8 microg igf - ii ( obtained from sigma chemicals , st . louise , mo . ), 40 ng of atherosclerotic papp - a , and 2 microliters protease inhibitors cocktail ( obtained from sigma chemicals , st . louise , mo .). the reaction was carried out for 15 hours at 37 ° c ., and was stopped by freezing of the sample at − 20 ° c . the degree of papp - a - dependent cleavage of igfbp - 4 was determined by western blotting using igfbp - 4 - specific rabbit polyclonal antibodies ( obtained from abcam , cambridge , mass .) ( fig1 ). atherosclerotic papp - a was shown to cleave igfbp - 4 with the same efficiency as recombinant papp - a . thus , for the first time it was shown that endogenous papp - a expressed in human plaques is an active protease that is able to cleave igfbp - 4 in the presence of igf - ii . blood of 43 patients with acs ( with st - segment elevation ) as well as plasma samples from 34 healthy donors were tested by fragment - specific sandwich immunoassays . all plasma samples were collected from the patients in the presence of edta and were stored at − 70 ° c . before measurements . for the sandwich immunoassay measurements capture antibody ibp521 , 2 microg per well in 0 . 1 ml of phosphate buffer saline , was incubated in 96 - well immunoassay plates for 30 min at room temperature upon constant shaking . after washing with buffer a , 0 . 1 ml of patients &# 39 ; plasma samples diluted 1 : 1 with the assay buffer were added to the plates . plates were incubated for 30 min at room temperature with constant shaking . after washing with buffer a 0 . 1 ml detection antibody ibp30 ( 1 mg / l ) in the assay buffer was added . the plates were incubated for 30 min at room temperature with constant shaking . after washing with buffer a , 0 . 2 ml of enhancement solution per well was added and incubated for 3 min at room temperature with gentle shaking . fluorescence of eu3 + was measured using a victor 1420 multilabel counter ( wallac - perkin elmer ). the level of igfbp - 4 fragments in the plasma of acs patients was 3 . 2 - fold higher ( p & lt ; 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