Method for detection of IGFBP-4 fragments
09964549 ยท 2018-05-08
Assignee
Inventors
- Alexey G. Katrukha (Turku, FI)
- Alexander B. Postnikov (Moscow, RU)
- Tatiana I. Solovyeva (Moscow, RU)
- Alexey V. Kharitonov (Moscow, RU)
Cpc classification
G01N2800/324
PHYSICS
International classification
G01N33/50
PHYSICS
Abstract
Disclosed is a method for detecting IGFBP-4 (Insulin-like Growth Factor Binding Protein-4) fragments. The method uses antibodies or antigen binding fragments that specifically recognize epitopes on IGFBP-4 fragments. The IGFBP-4 fragments include the N-terminal and C-terminal fragments resulting from PAPP-A dependent cleavage of IGFBP-4. Assay and diagnostic methods using detection of IGFBP-4 fragments are also disclosed.
Claims
1. A method for detecting IGFBP-4 fragments in a sample, said method comprising the steps of: contacting an isolated antibody or an antigen-binding fragment thereof with at least one IGFBP-4 fragment, wherein the isolated antibody or an antigen-binding fragment thereof binds an epitope located within an IGFBP-4 protein fragment consisting of amino acid residues 121-135 of SEQ ID NO: 3 or an epitope located within an IGFBP-4 protein fragment consisting of amino acid residues 136-150 of SEQ ID NO: 3 wherein the antibody or antigen binding fragment has a cross-reactivity to intact IGFBP-4 of less than 5%; and performing an immunoassay.
2. The method according to claim 1, wherein the antibody is a monoclonal antibody or a recombinant antibody.
3. The method according to claim 1, further comprising the steps of: a) providing a sample containing the IGFBP-4 fragment as a sample IGFBP-4 fragment; b) preparing a calibration curve using a standard IGFBP-4 fragment; and c) comparing the sample IGFBP-4 fragment detected by the isolated antibody or antigen-binding fragment to the calibration curve to determine the quantity of the sample IGFBP-4 fragment in the sample.
4. The method of claim 1, wherein the immunoassay is one or more selected from the group consisting of sandwich immunoassay, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), Western blotting, nephelometry, turbidimetry, immunoradiometric assay, lateral flow, and immunohisto/cyto-chemistry.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1)
(2) IGFBP-4 (200 ng per lane) was treated by:
(3) Lane 1, recombinant PAPP-A;
(4) Lane 2, atherosclerotic tissue PAPP-A;
(5) Lane 3, without PAPP-A;
(6) Lane 4, Molecular weight standards; shown in kDa.
(7) Rabbit anti-IGFBP-4 polyclonal antibodies were used for immunostaining.
(8)
(9) 10 ng of full-length recombinant IGFBP-4 or IGFBP-4 fragments produced by PAPP-A dependent cleavage were adsorbed on polystyrene plate. After washing fragment-specific MAbs IBP3, IBP12, IBP27, IBP13 and IBP28 (10 microg/ml) were incubated in wells during 30 min with shaking. Specifically bound antibodies were detected by anti-mouse IgG polyclonal antibodies, conjugate with horseradish peroxidase (substrate: TMB).
(10)
(11) Immunoassays:
(12) Capture MAbs: IBP513, IBP521 (200 ng/well).
(13) Detection MAbs: IBP12, IBP27, and IBP30 (1 microg/ml) labeled with stable Eu3+ chelate.
(14) Antigens: 100 ng/ml of full-length recombinant IGFBP-4 or IGFBP-4 fragments produced by PAPP-A dependent cleavage.
(15) Incubation volume 0.1 ml.
(16) Incubation time: 30 min at room temperature.
(17) CPScounts per second; AU (450 nm)absorbance units at 450 nm.
(18)
(19) Immunoassay:
(20) Capture MAb: IBP521 (200 ng/well).
(21) Detection MAb: IBP30 (1 microg/ml) labeled by stable Eu(3+) chelate.
(22) MAB IBP521 recognizes full-size IGFBP-4 as well as its C-terminal fragment generated by PAPP-A-mediated proteolysis
(23) MAb IBP30, specifically recognizes C-terminal fragment of IGFBP-4 only and does not recognize full-size IGFBP-4.
(24) Incubation volume 0.1 ml. Incubation time: 30 min at room temperature. CPScounts per second.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
(25) 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' 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.
(26) 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.
(27) 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 (
(28) 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.
(29) 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 (
(30) 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.
(31) 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.
(32) 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 (
(33) 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 antibodyIGFBP-4 fragments complex.
(34) 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.
EXAMPLE 1
Generation of Mouse Monoclonal Antibodies Specific to Novel Proteolysis-mediated Epitopes of IGFBP-4
(35) Synthetic peptides obtained for mice immunization:
(36) Peptide-1 (SEQ ID NO. 1)
(37) Peptide-2 (SEQ ID NO. 2)
(38) 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 (>95%) was determined by analytical C18 high-pressure liquid chromatography and mass spectroscopy (matrix-assisted laser desorption/ionization mass spectrometry with accuracy0.5 Dalton).
(39) 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 IN 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.
(40) Preparation of conjugates of the peptides with carrier proteins was performed by using sulfo-SMCC obtained from Pierce (Rockford, Ill.) according to manufacturer's instructions. For the conjugation 2.5 mg of carrier proteinbovine serum albumin, BSA) or ovalbumin (both obtained from Sigma Chemicals, St. Louis, Mo.) was dissolved in 10 mM KHPO.sub.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.sub.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.
(41) Immunization of Mice with of Peptide-(Carrier Protein) Conjugates
(42) Groups of five BALB/c mice were immunized five times with peptide-protein conjugates.
(43) Group 1: First immunization: intraperitoneally 0.2 ml of 10 microg BSA-Peptide-1 in PBS with 60% Freund's complete adjuvant; Second immunization: on day 30, intraperitoneally 0.2 ml of 5 microg BSA-Peptide-1 in PBS with 60% Freund's incomplete adjuvant; Third immunization: on day 60, intraperitoneally 0.2 ml of 2.5 microg BSA-Peptide-1 in PBS.
(44) Group 2: First immunization: intraperitoneally 0.2 ml of 10 microg BSA-Peptide-2 in PBS with 60% Freund's complete adjuvant; Second immunization: on day 30, intraperitoneally 0.2 ml of 5 microg BSA-Peptide-2 in PBS with 60% Freund's incomplete adjuvant; Third immunization: on day 60, intraperitoneally 0.2 ml of 2.5 microg BSA-Peptide-2 in PBS.
(45) 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).
(46) 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. Louis, 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. Louis, 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).
(47) Hybridomas producing antibodies specific to appropriate peptide, conjugated with ovalbumin (Absorbance at described above conditions at 450 nm>0.5 over background), and at the same time not reacting with human recombinant IGFBP-4 (Absorbance at 450 nm<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's modified Eagle's medium (DMEM), containing 10% fetal bovine serum (HyClone Laboratories, Logan, Utah).
(48) Affinity Purification of Antibodies
(49) 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's instructions. Purified monoclonal antibodies were stored as suspensions in 50% ammonium sulfate at 4 C.
(50) Investigation of Specificity of Monoclonal Antibodies
(51) 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.sub.2, 1.8 microg IGF-II (obtained from Sigma Chemicals, St. Louis, Mo.). 40 ng of human recombinant PAPP-A (HyTest, Turku, Finland), and 2 microliters protease inhibitors cocktail (obtained from Sigma Chemicals, St. Louis, 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.) (
(52) Specificity studies of selected monoclonal antibodies to IGFBP-4 proteolytic fragments were performed in indirect ELISA using affinity-purified antibodies (
(53) Design of Sandwich Immunoassays for Quantification of IGFBP-4 Fragments
(54) Specificity of affinity-purified monoclonal antibodies was also checked in sandwich immunoassays (
(55) To perform sandwich fluorescent immunoassays, we used detection MAbs labeled with stable Eu3+ chelate as described by Hyyti 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 g per well in 100 L 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/1 NaN.sub.3 (buffer A). After washing 0.1 ml of assay buffer (50 mM Tris-HCl buffer, pH 7.7, 9 g/1 NaCl, 0.01% Tween 40, 0.5% BSA and 0.5 g/l NaN.sub.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.sub.2CO.sub.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).
(56) Developed sandwich immunoassays were able to detect only IGFBP-4 fragments produced by PAPP-A-dependent cleavage and had no crossreaction (or very lowless than 1%) with full-length IGFBP-4.
EXAMPLE 2
Generation of Mouse Monoclonal Antibodies Specific to Intact IGFBP-4
(57) Immunization of Mice
(58) 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. Louis, Mo. First immunization: intraperitoneally 0.2 ml of 5 microg IGFBP-4 in PBS with 60% Freund's complete adjuvant. Second immunization: on day 30, intraperitoneally 0.2 ml of 2 microg IGFBP-4 in PBS with 60% Freund's incomplete adjuvant. Third immunization: on day 60, intraperitoneally 0.2 ml of 2 microg IGFBP-4 in PBS.
(59) 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).
(60) 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.
(61) Hybridomas producing antibodies specific to full-length IGFBP-4 (absorbance at described above conditions at 450 nm>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.
(62) Affinity Purification of Antibodies
(63) 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's instructions. Purified monoclonal antibodies were stored as suspensions in 50% ammonium sulfate at 4 C.
EXAMPLE 3
Proteolytic Activity of Atherosclerotic PAPP-A
(64) 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.
(65) 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.sub.2, 1.8 microg IGF-II (obtained from Sigma Chemicals, St. Louis, Mo.), 40 ng of atherosclerotic PAPP-A, and 2 microliters protease inhibitors cocktail (obtained from Sigma Chemicals, St. Louis, 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 Abeam, Cambridge, Mass.) (
EXAMPLE 4
Measurement of IGFBP-4 Fragments in ACS Patients' Plasma Samples
(66) 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.
(67) 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' 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<0.0005) than in plasma of healthy donors (
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