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WO1991000872A1 - Anitcorps monoclonaux contre la proteine reactive c - Google Patents

Anitcorps monoclonaux contre la proteine reactive c Download PDF

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Publication number
WO1991000872A1
WO1991000872A1 PCT/US1990/003487 US9003487W WO9100872A1 WO 1991000872 A1 WO1991000872 A1 WO 1991000872A1 US 9003487 W US9003487 W US 9003487W WO 9100872 A1 WO9100872 A1 WO 9100872A1
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Prior art keywords
crp
react
native
modified
mab
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PCT/US1990/003487
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English (en)
Inventor
Joan N. Siegel
Lawrence A. Potempa
Henry Gewurz
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Rush-Presbyterian-St. Luke's Medical Centre
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Publication date
Priority claimed from US07/374,166 external-priority patent/US5272258A/en
Application filed by Rush-Presbyterian-St. Luke's Medical Centre filed Critical Rush-Presbyterian-St. Luke's Medical Centre
Priority to CA002057058A priority Critical patent/CA2057058C/fr
Publication of WO1991000872A1 publication Critical patent/WO1991000872A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • CRP C-reactive protein
  • CRP and/or CRP complexes can activate the complement system, Kaplan and Volanakis, J. Immunol, 112, 2135 (1974); Siegel et al., J. Exp. Medicine, 140, 631 (1974). They can also bind to and promote the activation of neutrophils and macrophages and enhance the respiratory burst response of these cells to certain stimuli in vitro, Mortensen et al., J. Immunol., 117, 774 (1976); Kilpatrick and
  • CRP has the capacity to protect against pneumococcal infections, influence clearance reactions, modify antibody formation to certain antigens and inhibit the metastasis of certair, tumors in the mouse in vivo. Mold et al., J. Exp. Med., 154, 1703 (1981); Nakayama et al., J. Immunol. 128, 2435 (1982); Nakayama et al., J. Immunol., 54, 319 (1984); Deodhar et al., Cancer Res., 42, 5084 (1982). These properties suggest a significant biological role for
  • Native CRP is a cyclic pentamer composed of five identical, noncovalently-associated subunits, with its prototypic binding reactivity directed to phosphorylcholine (PC) in the presence of calcium. Volanakis and Kaplan, Proc. Soc. Exp. Bio. Med., 136, 612 (1971). Recently in Potempa et al., Molec. Immunol., 20, 1165 (1983) and Potempa et al., Molec. Immunol., 24, 531
  • neo-CRP antigenicity is expressed by native CRP modified by urea-chelation, acid treatment, heating, or direct immobilization on polystyrene plates.
  • the neo-CRP antigenicty is also expressed on the intact CRP subunits and on the in vitro translation product of the CRP gene produced by hosts transformed with the gene by recombinant DNA techniques, Mantzouranis et al., Pediatric Res.,
  • CRP CRP-binding protein
  • CRP expressing neo-CRP epitopes have the ability to activate and modulate the functional responses of platelets, polymorphonuclear leukocytes and monocytes, Potempa et al., Inflammation, 12, 391 (1988). Further, polyclonal antiserum to neo-CRP has been used to look for neo-CRP determinants in serum and tissue. Neo-CRP antigenicity has been found on the surface of human natural killer (NK) cells and B lymphocytes. Bray et al., J. Immunol , 140, 4271 (1988), as well as in human skeletal muscle tissue, Rees et al.,
  • neo-CRP epitopes in necrotic tissue was attributed to in situ acute phase modification of the CRP molecule. These properties suggest a significant biological role for neo-CRP, as well as native CRP.
  • Monoclonal antibodies to human CRP have been prepared which react with either native CRP, modified CRP or both forms of the molecule.
  • Modified CRP as used herein means CRP modified by immobilization on solid surfaces, heating, urea-chelation, sodium dodecyl sulfate treatment, acid treatment, other denaturation methods or other methods which can result in a conformational change in a protein.
  • the term also means any form of CRP that expresses neo-CRP antigenicity.
  • the mAbs of the invention were divided into four groups according to their binding characteristics to various CRP preparations using a combination of ELISA, dot blot and Western blot assays.
  • the mAbs were further characterized based upon their reactivity with CRP in the presence of calcium, their inhibition by phosphorylcholine (PC), their reactivity with serum amyloid P component and their reactivity with rabbit CRP.
  • PC phosphorylcholine
  • the first group of mAb have the following specificities:
  • Further reactivities of the mAb of this first group define at least four epitopes on native human CRP: 1) a calcium-dependent, PC-inhibitable idiotope; 2) a calcium-dependent, non-PC-inhibitable epitope; 3) a calcium-influenced, EDTA-enhanced epitope; and 4) a calcium-independent epitope which also displays a unique cross reactivity with rabbit CRP.
  • the second group of mAb react with native human CRP and with modified human CRP, thus identifying a fifth native CRP epitope. These mAbs display significantly greater reactivity with native than with modified CRP.
  • the mAb of this second group have the following additional specificities:
  • the third group of mAb have the following specificities:
  • the fourth group of mAb have the following specificities:
  • This peptide is an octapeptide identical to the carboxyl-terminal sequence of human CRP.
  • the invention also comprises hybridomas capable of producing mAbs having the specificities outlined above.
  • the invention further provides immunoassays for detecting or quantitating native CRP or modified CRP comprising contacting the native CRP or modified CRP with a mAb of the invention having an appropriate specificity.
  • kits for detecting or quantitating native CRP or modified CRP comprising a container of a mAb according to the invention having the proper specificity.
  • FIGURES 1A-1F Binding of mAb representative of each major group of mAb (the properties of which are further described below) by ELISA analysis to CRP captured by immobilized PC-KLH in the presence of calcium ( ⁇ ) and CRP directly immobilized in the absence of calcium ( O ) ( Figures 1A -1D).
  • Figures IE and IF show the reactivities of polyclonal goat antisera predominantly reactive with native- and neo-CRP epitopes, respectively.
  • FIG. 2 Western blot analysis of CRP in SDS-PAGE using mAb, Protein blots in the first three lanes (lane 1, standards; lane 2, CRP; and lane 3, Pronase-treated CRP-) were stained with Amido Black directly with no
  • Lane 4 was incubated with mAb 1D6; lane 5 with mAb 2C10; lane 6 with mAb
  • FIG. 3 Inhibition of the binding of mAb
  • FIG. 4 Phosphorylcholine (PC) inhibition of the binding of biotinylated CRP to mAb.
  • the mAb were used at concentrations giving 25 - 50% maximal color change.
  • Biotinylated CRP was added at a concentration of 1 ⁇ 10 -9 M.
  • the effects of increasing concentrations of PC on mAb 4H2 ( ⁇ ), JEV-EA4-1, ( ⁇ ), 15-2C10 ( ⁇ ) and JEV-HD2-4 ( ⁇ ) are shown.
  • mAbs to native CRP and modified CRP may be prepared using any of these known techniques. Briefly, immunoglobulin secreting cells from animals immunized with native CRP or modified CRP are fused to cells of an immortal cell line such as myeloma cells. The resultant hybrid cells (“hybridomas”) are cloned and
  • the mAbs of the invention may be used in immunoassays to detect or quantitate native CRP and modified CRP. Any known immunoassay technique may be used, except that certain additional considerations are necessary when performing solid phase immunoassays for native
  • Kits for detecting or quantitating native CRP or modified CRP are also part of the invention.
  • a suitable kit comprises a container holding a mAb according to the present invention of the proper specificity.
  • the mAb may be labeled or unlabeled.
  • the applications describe a method of removing aggregated immunoglobulins or immune complexes from a fluid, such as serum, plasma or a diagnostic or therapeutic reagent fluid.
  • the method comprises: contacting the fluid with modified CRP so that the aggregat immunoglobulins or immune complexes bind to the modified CRP; and separating the fluid from the aggregated immunoglobulin or immune complexes bound to the modified CRP.
  • immunoglobulin may naturally contain modified CRP, or may be reacted with modified CRP before, or simultaneously with, being contacted with the antibody to
  • modified CRP so that they may be removed from the fluid by the antibody.
  • Also described is a method of detecting or quantitating immune complexes comprising: contacting
  • the labeled component may be antibody to modified CRP.
  • immune complexes or aggregated immunoglobulin may naturally contain modified CRP, or may be reacted with modified CRP before, or simultaneously with, being contacted with the antibody to modified CRP.
  • a suitable device may comprise modified CRP bound to a solid surface and a means for encasing the solid surface or, alternatively, may comprise antibody to modified CRP bound to a solid surface and a means for encasing the solid surface.
  • the solid surface on which the modified CRP or antibody to modified CRP is immobilized and the encasing means of the device may be any biocompatible material.
  • the solid surface may be a membraneous surface, agarose-based beads or hollow fibers coated with modified CRP or antibody to modified CRP.
  • the device may be a column packed with beads, a hollow fiber membrane encased in a cylinder like those used in renal dialysis, a microtiter plate containing wells, or any suitable surface, coated with modified CRP or antibody to modified CRP.
  • the device may also include appropriate tubing for connecting it to a patient and a pump to aid the passage of the fluid through the device and to prevent air from entering the system.
  • the device may be sterilized for therapeutic use, and sterilization may be accomplished in conventional ways such as purging with ethylene oxide or by irradiating the device.
  • kits for detecting or quantitating immune complexes comprising a container holding antibody to modified CRP.
  • Another suitable kit comprises a container holding modified CRP and, optionally, a container of a labeled component that binds to immune complexes or modified CRP to allow the immune complexes to be detected or quantitated.
  • the labeled component may be antibody to modified CRP.
  • modified CRP used in the method, kits and devices disclosed in these two applications is the same as the modified CRP described herein. Accordingly, the monoclonal antibodies of the present invention directed to epitopes on modified CRP may be used in the methods, kits and devices described in U.S. patent application Serial No. 07/176,923 and PCT application US/89/01247 wherever it is indicated that antibodies to modified CRP should be used.
  • the antibodies of the present invention may also be used to purify modified CRP for use in the assays described in these two applications which employ modified CRP.
  • the mAbs of the present invention may be useful in purifying modified CRP from cell culture supernatants of microorganisms genetically engineered to produced CRP, which as noted above expresses neo-CRP antigenicity.
  • the mAbs of the present invention heaving specificity for native CRP may be utilized to purify native CRP.
  • modified CRP or native CRP could be purified by contacting the modified or native CRP with an antibody according to the present invention of the proper specificity.
  • the antibody could be immobilized on a solid surface such as agarose beads and used as a column.
  • Modified or native CRP bound to antibody can be eluted using known means.
  • Immunoassays for detecting or quantitating native CRP, modified CRP or immune complexes are those known in the art, with the exception stated earlier that solid-phase assays for native CRP must be performed by the ligand capture technique.
  • suitable conventional immunoassays that may be used otherwise include competitive assays and immunometric assays. Examples of the latter type are radioimmunometric assays (IRMA) and enzyme-linked immunosorbent assays (ELISA).
  • IRMA radioimmunometric assays
  • ELISA enzyme-linked immunosorbent assays
  • the antigen is labelled with a detectable label. The sample containing the antigen is incubated with the antibody and the labelled antigen, and after formation of immune complexes, separation and detection, the level of antigen in the sample is determined.
  • the antigen is immobilized on a solid phase, e.g., on the surface of microtiter plate wells.
  • the antibody, or an antigen-binding fragment of the antibody is detectably labelled. Incubation of sample with labelled antibody leads to an immobilized antigen-antibody complex and, after separation of unbound antibody, the amount of label is proportional to the amount of antigen.
  • one antigen-binding antibody is detectably labelled. Another antibody binding the same antigen is immobilized on a solid phase. Incubation of antigen with labelled and immobilized antibody leads to a sandwich and, after separation of unbound antibody, the amount of label is proportional to the amount of antigen.
  • Immunometric assays can be carried out in forward, reverse or simultaneous modes, depending on the order of addition of the immobilized and/or labelled antibodies.
  • the antibody or the antigen-binding fragment thereof may be coupled with a detectable label such as an enzyme, radioactive isotope, fluorescent
  • One of the ways in which the antibody can be detectably labelled is by linking it to an enzyme.
  • This enzyme when later exposed to its substrate will react with the substrate in such a manner as to produce a chemical moiety which can be detected by, for example, spectrophotometric or fluorometric means (ELISA system).
  • ELISA system spectrophotometric or fluorometric means
  • enzymes that can be used as detectabl labels are horseradish peroxidase, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate
  • the pro cedures described can be modified using biotinylated antibody reacting with avidin-enzyme conjugates.
  • the amount of antigen can also be determined by labelling the antibody with a radioactive isotope.
  • radioactive isotope would then be determined by such means as the use of a gamma counter or a scintillation counter.
  • Isotopes which are particularly useful are 3 H, 125 I, 123 I, 32 P, 35 S, 14 C,
  • Determination of the antigen is also possible by labelling the antibody with a fluorescent compound.
  • fluorescently labelled molecule When the fluorescently labelled molecule is exposed to light of the proper wavelength, its presence can then be detected due to fluorescence of the dye.
  • fluorescent labelling compounds are most important fluorescent labelling compounds.
  • fluorescein isothiocyanate rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, and
  • Fluorescence emitting metal atoms such as Eu (europium), and other lanthanides, can also be used.
  • metal chelating groups such as DTPA or EDTA.
  • Another way in which the antibody can be detectably labelled is by coupling it to a chemiluminescent compound.
  • the presence of the chemiluminescent-tagged immunoglobulin is then determined by detecting the presenc of luminescence that arises during the course of a chemica reaction.
  • particularly useful chemiluminescen labelling compounds are luminol, isoluminol, aromatic acridinium ester, imidazole, acridinium salt, and oxalate ester.
  • bioluminescent compound may also be used as a label.
  • Bioluminescence is a special type of chemiluminescence which is found in biological systems and in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a biolurainescent molecule would be determined by detecting the presence of luminescence.
  • Important bioluminescent compounds for purposes of labelling are luciferin,
  • kits for the detection or quantitation of native CRP, modified CRP and immune complexes may comprise one or more containers such as bottles, vials, tubes, and the like, each of which contains one of the separate elements to be used in the desired immunoassay method.
  • the kit may comprise a container of unlabeled or detectably labelled mAb according to the present invention of proper specificity.
  • the antibody may be in lyophilized form or in solution or may be immobilized on a solid surface like those described above.
  • Other containers may comprise reagents necessary for determining the amount of labelled antibody or
  • ancillary reagents such as buffer solutions and standards.
  • CRP Human CRP was purified from pleural and ascites fluids by sequential affinity chromatography with PC substituted Biogel, ion exchange chromatography with DE52 and calcium-dependent adsorption chromatography with Biogel A 0.5 m to remove residual serum amyloid P component (SAP) as described in Potempa et al., Molec. Immunol., 20, 1165 (1983).
  • SAP serum amyloid P component
  • the purified protein was adjusted to 1 mg/ml, dialyzed against TBS-calcium (0.01 M Tris-HCl, 0.15 M NaCl and 0.002 M CaCl 2 , pH 7.3) with 0.02% (w/v) sodium azide, sterile-filtered and stored at 4oC.
  • CRP CRP at 1 mg/ml in TBS-calcium was chelated with 0.005 M EDTA and incubated in 8 M ultra-pure urea for 2 hr at 37°C. The urea was removed by dialysis against low ionic strength TBS (0.01 M
  • Tris-HCl and 0.05 M NaCl, pH 7.4), and the soluble CRP preparation obtained is referred to as "urea-CRP".
  • Heat-treated CRP was prepared by heating CRP at 63°C for 2 minutes in the absence of calcium to cause denaturation and modification of CRP.
  • mice Female 5-6 week old Balb/c mice were obtained from the Jackson Laboratories (Bar Harbor, ME) or Harlan Sprague Dawley Inc. (Indianapolis, IN).
  • mice Female 5-6 week old RBF/DN mice were obtained from the Jackson Laboratory (Bar Harbor, ME). All mice were housed and maintained in the facilities of the Comparative Research Center of Rush Medical College. Immunizations began at 8-9 weeks of age.
  • Hybridomas The mAb were produced by standard hybridoma technology using in vivo systemic immunization and polyethylene glycol (PEG) fusion as described in Kennett et al., "Methods for
  • mice were rested for 2-4 weeks and given a final i.p.
  • a mixture of urea- and heat-modified human CRP expressing neo-CRP determinants was used in Freund's adjuvant for immunization of Balb/c mice.
  • fusion 26 a combination of urea-CRP and heat-modified rabbit CRP immunogens in the RIBI adjuvant system were injected into Balb/c mice. All immunizations resulted in antibodies to both human native-CRP and neo-CRP
  • Splenic immune lymphocytes from the immunized mice were fused with the hypoxanthineguanine phosphoribosyltransferase-deficient myeloma FOX-NY (as described in Taggart and Samloff, Science, 219, 1228 (1983)) using PEG 1500 (Aldrich Chemical Co. Inc., Milwaukee, WI).
  • Hybridomas were selected in the appropriate defined medium (HB101 or HB102; Hana Biologies Inc., Berkeley, CA) containing hypoxanthine. aminopterin and thymidins (HAT; Sigma Chemical Co., St. Louis, MO) or adenosine, aminopterin and thymidine (AAT; Sigma Chemical Co.), respectively.
  • Antigen-specific clones were screened by direct enzyme linked immunosorbent assay (ELISA) (described below) and cloned at least twice by limiting dilution.
  • ELISA enzyme linked immunosorbent assay
  • Antigen-positive clones were retested for specificity by direct ELISA on plates coated with native CRP, modifie CRP (displaying the neo-CRP epitope) and a non-specific control protein (human transferrin, Sigma Chemical Co., or human IgG, Jackson ImmunoResearch Laboratories, Inc., Avondale, PA) and by a ligand capture assay using CRP bound to a PC-KLH coated plate (described below).
  • Stable clones of interest were expanded in vitro in medium supplemented with 2% FCS.
  • the mAb were isotyped by direct ELISA on antigen (i.e., CRP)-coated plates.
  • Hybridomas Stable hybridomas were cultured in HB101 or HB102 defined medium with appropriate supplements (Hana Biologies) containing 2% or 5% heat-inactivated fetal calf serum (FCS) (Biologos Inc., Naperville, IL), 0.002 M L-glutamine (GIBCO, Grand
  • Viable cells at 2 - 2.5 ⁇ 10 5 /ml were cultured in plastic culture flasks in a humidified atmosphere at 37°C in 5% pCO 2 .
  • cells (5 ⁇ 10 6 /ml/vial) were washed, resuspended in HB basal medium containing 20% FCS and 10% dimethyl sulfoxide (DMSO; Fisher Scientific Co., Fairlawn NJ) and slow-frozen over liquid nitrogen.
  • DMSO dimethyl sulfoxide
  • Immunoglobulin fractions of the mAb were prepared from culture supernatants by precipitation with 50% saturated ammonium sulfate as described in Kennett et al., in Monoclonal Antibodies. Hybridomas: A New Dimension In Biological Analysis p. 363 (1980), followed by immediate dialysis into PBS, pH 7.2 (Bacto-FA buffer, Difco Latoratories, Detroit, MI). All immunoglobulin preparations were clarified by centrifugation in a Beckman microfuge, made to 0.02% with sodium azide, stored at -70°C and quantitated using the extinction coefficient of mouse immunoglobulin.
  • the mAbs produced in Example 1 were characterize by. ELISA, dot blot and Western blot assays.
  • Reference Antibodies Polyclonal antibodies used in ELISA, dot blot and Western blot assays included unconjugated and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG plus IgM (H + L) and
  • HRP-conjugated rabbit anti-goat IgG obtained from Pel Freez Biologicals (Rogers, AK), and HRP-conjugated avidin obtained from Boehringer Mannheim Biochemicals (Indianapolis, IN).
  • the mAb were isotyped using rabbit antiheavy and -light chain isotype-specific reagents obtained from Miles Scientific (Naperville, IL), and HRP-conjugated goat anti-rabbit IgG (H + L) obtained from Kirkegaard and Perry Laboratories, Inc. (Gaithersburg, MD).
  • HD2-4 obtained from the American Type Culture Collection and described in Kilpatrick et al., Molec Immunol., 19, 1159 (1982); EA4-1 obtained from Dr. John E. Volanakis, Dept. of Medicine, University of Alabama in Birmingham (Birmingham, AL) and described in Kilpatrick et al., Molec. Immunol., 19, 1159 (1982); and BB-9 from Dr. Richard F. Mortensen, Dept. of Microbiology, Ohio State University (Columbus, OH) and describ in Tseng et al., Hybridoma, 7 , 185 (1988).
  • PC-KLH Phosphorylcholine (PC) substituted Keyhole Limpet hemocyanin (PC-KLH) was prepared by incubating KLH (Sigma Chemical Co., St.
  • Biotinylation of CRP Purified human CRP at 1 mg/ml was incubated with 1/8 volume of NHSLC-Biotin (Pierce, Rockford, IL) at 1 mg/ml for 4 hr at room temperature with occasional mixing. Biotinylated protein was dialyzed at 4 C for 24 hr against 8 liters of TBS-calcium buffer. The biotinylated CRP preparation showed calcium-dependent binding to PC-KLH, and reacted comparably to unlabeled CRP in the ELISA assays involving anti-native-CRP and anti-neo-CRP mAb.
  • CRP peptides 1, 2 and 4 synthesized as described in Lindner and Robey, Int. J. Pept. Protein Res., 30, 794 (1987) were generously provided by Dr. F.A. Robey, National Institute of Dental Research, (Bethesda, MD).
  • Peptide 1 (identical to residues 23-30 of human CRP), peptide 2 (identical to residues 109-123) and peptide 4 (identical to residues 199-206) were prepared to include the tuftsin-like sequences previously identified in the human CRP molecule as described in Osmand et al., Proc. Nat'l. Acad. Sci. USA, 74, 1214 (1977); Robey et al., J. Biol. Chem..
  • the anti-CRP mAb were serially diluted with wash buffer and 50 ⁇ l aliguots added to the wells for 30 min at 37°C followed by washing.
  • Peroxidase-conjugated goat anti-mouse IgG and IgM (H+L) in wash buffer was added to the wells for 30 min at 37°C. After washing, 50 ⁇ l ABTS substrate
  • the ligand capture assay identifying the calcium dependence and PC inhibitability of epitope recognition utilized wells coated with polyclonal goat anti-mouse IgG (50 ⁇ l at 15 ⁇ l/ml in bicarbonate buffer, pH 9.6) and blocked as before. After washing, mAb (50 ⁇ l) at a concentration producing half-maximal capture were added and incubated for 30 min at 37°C. Biotinylated CRP
  • Substrate solution (4-chloro-1-naphtho l in 10 mM TBS containing methanol and H 2 O 2 ) prepared as directed- (Bio-Rad, Richmond, CA), was added to the wells, and incubation was continued for 30 min at RT for color development.
  • Protein was transferred to the nitrocellulose membrane using the BioRad Transblot system. The procedure for performing the immunoblot assays was carried out as described for the dot blot assays.
  • Results of ELISA Seventeen stable mAb were generated against human CRP and first characterized by ELISA for reactivities both with CRP bound to PC-KLH in the presence of calcium (i.e., with "native” CRP epitopes) and with urea-modified CRP in the absence of calcium (i.e., with "neo-CRP” epitopes). Table 1 shows the relative avidity of mAb to these two forms of the molecule on polystyrene plates.
  • Figures 1A-1D show the ELISA binding curves of representative mAb of each group, and Figures IE and IF show the reactivities of polyclonal antisera to native and modified CRP, respectively, for comparison.
  • the mAb assigned to group I did not react with modified CRP even at concentrations greater than those listed (in general >20 ⁇ l/ml), and since as little as 0.07 ⁇ l/ml yielded half-maximal activity with native CRP, we concluded that these antibodies had binding specificities exclusively for native CRP epitopes.
  • the group II mAb could bind to both native and modified
  • the identification of a native or neo-CRP specificity reflected a >7-fold preferential reactivity with the appropriate native or modified form of CRP.
  • the significantly lower preferential reactivitie of mAb 3A1 (group I) and 6A5 (group IV) may reflect technical complications resulting from the use of high concentrations of immunoglobulin in the ELISA assays, a lower affinity of interaction, or a lower stability of antibody in the salting-out procedure.
  • Fragment A consists of residues 1-146 and/or an additional breakdown product 10 residues shorter (this mixture is referred to as “fragment A” or “residues 1-146" herein).
  • Fragment B consists of residues 147-206 (Kinoshita et al., FASEB J. , 2 , 1149a (1988); Kinoshita et al. submitted for publication) .
  • the slowest migrating band corresponded to the intact CRP subunit (apparent molecular weight 23 kDa), the second band to fragment A and the third band to fragment B (Fig. 2, top to bottom).
  • Table 2 The results are summarized in Table 2, with the Western blot patterns of representative mAb from each of the groups shown in Figure 2.
  • the group I mAb did not react with any band on the Western blots.
  • the group II mAb reacted with the intact CRP subunit, but not with either fragment A or B.
  • the group III mAb reacted, with the intact CRP subunit and fragment A (residues 1-146), while the group mAb reacted with the. CRP subunit and fragment B (residues 147-206).
  • the group II mAb together with the determinant recognized by the group II mAb, there are at least three epitopes (i.e., two neo-CRP and one native CRP determinant) expressed on the modified CRP molecule.
  • polyclonal antibody to native CRP did not react with any band on the SDS gels while polyclonal anti-neo-CRP reacted with all three bands, emphasizing that this latter antibody is comprised of at least two different anti-neo-CRP specificities.
  • group I mAb (1D6, 8D8, 3G12, 4H2 and 3A1 ) reacted only with native CRP and not with the modified forms (Table 3).
  • Group II mAb (2C10 and 1A8) reacted with both native and modified CRP.
  • the remaining mAb (groups III and IV) reacted only with urea- or SDS-modified CRP but not with native CRP either in the presence or absence of calcium.
  • the mAb were used at concentrations at least 5-fold greater than those required for Maximal reactivity in the ELISA assays.
  • Figure 3 shows the inhibition curve of the binding of a representative group IV mAb (3H12) by peptide 4, and the lack of inhibition by peptides 1-3. Whether the inability to demonstrate a reactivity of mAb 6A5 with peptide 4 by direct binding or peptide inhibition reflected a real specificity difference or another factor, such as its apparent lower titer or a low affinity, is not yet clear.
  • HD2-4 displayed anti-native CRP reactivities indistinguishable from those of the group I mAb, while BB-9 and EA4-1 exhibited both anti-native- and anti-neo-CRP reactivities in a pattern characteristic of group II antibodies.
  • JEV-EA4-1 0.918 ⁇ 0.002 -100%
  • Ouchterlony analyses co identify, three antigenic determinants on human CRP: one determinant found only on human CRP; one found on human and monkey, but not on rabbit CRP; and one shared by human, monkey and rabbit proteins. See Nilsson, Int. Arch. Allergy, 32, 545
  • the group III and IV mAb did not react with native CRP either in the presence of absence of calcium, but reacted strongly with the modified forms of CRP. These results suggest that epitopes recognized by group I mAb are conformational and discontinuous in amino acid sequence, while epitopes recognized by group II, III and IV mAb probably are comprised of continuous amino acid sequences which are not exposed or expressed until the molecule is unfolded or modified in certain other ways.
  • CRP is a calcium-binding protein with one or two calcium binding sites per subunit (Gotschlich and Edelman, Proc. Natl. Acad. Sci. USA, 57, 706 (1967).
  • a summary of the data with our mAb suggest that there are at least four epitopes detectable on native human CRP by the group I mAb: 1) a calcium-dependent, PC-inhibitable idiotope identified by mAb 4H2; 2) a calcium-dependent, non-PC-inhibitable epitope identified by mAb 1D6; 3) a calcium-influenced, EDTA-enhanced epitope identified by mAb 3A1; and 4) a calciumindependent epitope identified by mAb 8D8 which also displays a unique cross reactivity with rabbit CRP.
  • the group II mAb (i.e., 2C10 and 1A8) recognize a fifth native CRP epitope, which unlike the other four, is retained as the molecule is altered by the various treatments.
  • Neo-CRP epitopes were defined using CRP modified in the absence of calcium by immobilization onto plastic, by urea or SDS treatments or by limited digestion with Pronase, and further localized utilizing synthetic CRP peptides. Although neo-CRP epitope expression had been shown in previous studies principally to require a modification in the absence of calcium (see Potempa et al., Molec. Immunol., 20, 1165 (1983); Potempa et al., Molec. Immunol., 24, 53, (1987)), immobilization of CRP on polystyrene ELISA plates in the presence or absence of calcium resulted in equivalent expression of antineo-CRP epitopes. In addition, the antibody capture assay showed that chelation of fluid phase CRP alone did not lead to the expression of neo-CRP epitopes.
  • neo-CRP expression itself does not appear to be dependent on either the presence or absence of calcium.
  • the group III anti-neo-CRP reactivity was localized to residues 1-146 of the CRP subunit (fragment A of the Pronase digest), while reactivity of the group IV mAb was localized to residues 147-206 of the CRP subunit (fragment B of the Pronase digest) and predominantly to the C-terminal octapeptide of CRP.
  • the data indicate that there are minimally two neo-CRP epitopes expressed only on modified CRP conformations and at least one epitope seen on native CRP which is retained after the various modifications.
  • mAb defined herein will be important tools for identifying neo-CRP epitopes in vivo and for structure-function mapping of complement- and cell-activating properties of the CRP molecule.
  • Hybridomas Four hybridomas, the preferred one from each of the four groups identified herein, were deposited at the American Type Culture

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  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Toxicology (AREA)
  • Immunology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne des anticorps monoclonaux réagissant avec la protéine réactive C (PRC) native et la PRC modifiée dont les spécificités sont décrites dans l'invention. L'invention concerne également les hybridomes utilisés pour produire ces anticorps. On utilise l'anticorps monoclonal 3H12 afin d'illustrer la liaison d'inhibition ainsi qu'une absence de liaison d'inhibition à l'aide de peptides. On peut utiliser les anticorps de l'invention afin de détecter ou de quantifier la PRC native et la PRC modifiée, des kits permettant de procéder à ces analyses font partie de l'invention.
PCT/US1990/003487 1989-06-27 1990-06-20 Anitcorps monoclonaux contre la proteine reactive c WO1991000872A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CA002057058A CA2057058C (fr) 1989-06-27 1990-06-20 Anticorps monoclonaux contre les proteines c-reactives

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US374,166 1982-05-03
US37244289A 1989-06-27 1989-06-27
US372,442 1989-06-27
US07/374,166 US5272258A (en) 1989-06-29 1989-06-29 Monoclonal antibodies to C-reactive protein

Publications (1)

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WO1991000872A1 true WO1991000872A1 (fr) 1991-01-24

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EP (1) EP0479922A4 (fr)
JP (1) JPH04505857A (fr)
AU (1) AU642430B2 (fr)
CA (1) CA2057058C (fr)
WO (1) WO1991000872A1 (fr)

Cited By (15)

* Cited by examiner, † Cited by third party
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US5874238A (en) * 1993-02-26 1999-02-23 Immtech International Inc. Mutant protein and methods and materials for making and using it
US6251624B1 (en) 1999-03-12 2001-06-26 Akzo Nobel N.V. Apparatus and method for detecting, quantifying and characterizing microorganisms
EP1182213A1 (fr) * 2000-06-12 2002-02-27 Wako Pure Chemical Industries, Ltd. Une enzyme hybride et son utilisation
EP1139101A3 (fr) * 2000-03-31 2002-05-15 Ortho-Clinical Diagnostics, Inc. Immunoessai pour la protéine C-réactive
US6429017B1 (en) 1999-02-04 2002-08-06 Biomerieux Method for predicting the presence of haemostatic dysfunction in a patient sample
US6502040B2 (en) 1997-12-31 2002-12-31 Biomerieux, Inc. Method for presenting thrombosis and hemostasis assay data
US6564153B2 (en) 1995-06-07 2003-05-13 Biomerieux Method and apparatus for predicting the presence of an abnormal level of one or more proteins in the clotting cascade
US6703219B1 (en) 1993-02-26 2004-03-09 Immtech International Inc. Mutant protein and methods and materials for making and using it
EP0589348B1 (fr) * 1992-09-25 2005-01-19 Chiron Behring Gmbh & Co. Procédé pour quantification immunologique des antigènes inactivés
US6898532B1 (en) 1995-06-07 2005-05-24 Biomerieux, Inc. Method and apparatus for predicting the presence of haemostatic dysfunction in a patient sample
WO2005058359A3 (fr) * 2003-12-15 2005-12-15 Theralogic Gmbh & Co Kg Procede de neutralisation des effets de la crp en vue d'augmenter les reactions immunitaires contre le vih
US7179612B2 (en) 2000-06-09 2007-02-20 Biomerieux, Inc. Method for detecting a lipoprotein-acute phase protein complex and predicting an increased risk of system failure or mortality
US7211438B2 (en) 1999-02-04 2007-05-01 Biomerieux, Inc. Method and apparatus for predicting the presence of haemostatic dysfunction in a patient sample
WO2009107170A1 (fr) * 2008-02-29 2009-09-03 学校法人日本大学 Anticorps anti-crp et son utilisation
CN118344476A (zh) * 2024-05-16 2024-07-16 武汉爱博泰克生物科技有限公司 人c反应蛋白单克隆抗体、抗体对和检测试剂或试剂盒及其应用

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US5266477A (en) * 1990-02-02 1993-11-30 Pitman-Moore, Inc. Monoclonal antibodies which differentiate between native and modified porcine somatotropins
JP3871677B2 (ja) * 2001-12-27 2007-01-24 松下電器産業株式会社 免疫反応測定方法及びそれに用いる免疫反応測定用試薬キット
JP5807300B2 (ja) * 2008-11-18 2015-11-10 株式会社シノテスト 試料中のｃ反応性蛋白質の測定方法及び測定試薬
CN103238071B (zh) * 2009-03-30 2016-07-06 北欧生物科技公司 纤维化生物标志物的测定
EP4292612A4 (fr) 2021-02-15 2025-02-19 Canon Medical Systems Corporation Anticorps neutralisant spécifique de la crp dénaturée humaine, et médicament et agent anti-inflammatoire contenant ledit anticorps

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Adv. Intern. Med., Volume 27, issued 1982, GEWURZ, H. et al., "C-Reactive Protein and the Acute Phase Response", pages 345-372, see the entire document. *
Hospital Practice, issued June 1982, GEWURZ, H., "Biology of C-Reactive Protein and the Acute Phase Response", pages 67-81, see the entire document. *
Hybridoma, Volume 7, No.2, issued 1988, TSENG, J et al., "Monoclonal Antibodies to Human C-Reactive Protein (CRP) That Recognize Epitopes in Functional Regions", pages 185-191, see entire document. *
J. Allergy Clin. Immunol., issued 1986, POTEMPA, R.T. et al., "Identification and Quantification of A Neo-Antigen of C-Reactive Protein (Neo-CRP) in Inflammatory Disease", Abstract No. 135a, page 77, see Abstract. *
Molecular Immunology, Volume 19, No. 9, issued 1982, KILPATRICK, J.M. et al., "Demonstration of Calcium-Induced Conformational Change (8) in C-Reactive Protein by Using Monoclonal Antibodies", pages 1159-1165, see entire document. *
See also references of EP0479922A4 *
Seventy second Annual Meeting of the Federation Am. Soc. for Experimental Biology, Volume J 2 (5), issued 1988, KINOSHITA, C.M. et al., "The Effect of Calcium-Inhibited Partial Proteolysis of Human C-Reactive Protein on Calcium-Dependent Binding", Abstract No. 4959, page A1149, see Abstract. *

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0589348B1 (fr) * 1992-09-25 2005-01-19 Chiron Behring Gmbh & Co. Procédé pour quantification immunologique des antigènes inactivés
US6703219B1 (en) 1993-02-26 2004-03-09 Immtech International Inc. Mutant protein and methods and materials for making and using it
US5874238A (en) * 1993-02-26 1999-02-23 Immtech International Inc. Mutant protein and methods and materials for making and using it
US6898532B1 (en) 1995-06-07 2005-05-24 Biomerieux, Inc. Method and apparatus for predicting the presence of haemostatic dysfunction in a patient sample
US6564153B2 (en) 1995-06-07 2003-05-13 Biomerieux Method and apparatus for predicting the presence of an abnormal level of one or more proteins in the clotting cascade
US6502040B2 (en) 1997-12-31 2002-12-31 Biomerieux, Inc. Method for presenting thrombosis and hemostasis assay data
US7211438B2 (en) 1999-02-04 2007-05-01 Biomerieux, Inc. Method and apparatus for predicting the presence of haemostatic dysfunction in a patient sample
US6429017B1 (en) 1999-02-04 2002-08-06 Biomerieux Method for predicting the presence of haemostatic dysfunction in a patient sample
US6251624B1 (en) 1999-03-12 2001-06-26 Akzo Nobel N.V. Apparatus and method for detecting, quantifying and characterizing microorganisms
US6838250B2 (en) 2000-03-31 2005-01-04 Ortho-Clinical Diagnostics, Inc. Immunoassay for C-reactive protein
EP1139101A3 (fr) * 2000-03-31 2002-05-15 Ortho-Clinical Diagnostics, Inc. Immunoessai pour la protéine C-réactive
US7179612B2 (en) 2000-06-09 2007-02-20 Biomerieux, Inc. Method for detecting a lipoprotein-acute phase protein complex and predicting an increased risk of system failure or mortality
EP1182213A1 (fr) * 2000-06-12 2002-02-27 Wako Pure Chemical Industries, Ltd. Une enzyme hybride et son utilisation
WO2005058359A3 (fr) * 2003-12-15 2005-12-15 Theralogic Gmbh & Co Kg Procede de neutralisation des effets de la crp en vue d'augmenter les reactions immunitaires contre le vih
WO2009107170A1 (fr) * 2008-02-29 2009-09-03 学校法人日本大学 Anticorps anti-crp et son utilisation
JPWO2009107170A1 (ja) * 2008-02-29 2011-06-30 学校法人日本大学 抗ｃｒｐ抗体及びその利用
CN118344476A (zh) * 2024-05-16 2024-07-16 武汉爱博泰克生物科技有限公司 人c反应蛋白单克隆抗体、抗体对和检测试剂或试剂盒及其应用

Also Published As

Publication number Publication date
AU642430B2 (en) 1993-10-21
CA2057058C (fr) 2000-09-05
EP0479922A4 (en) 1992-06-17
CA2057058A1 (fr) 1990-12-28
JPH04505857A (ja) 1992-10-15
EP0479922A1 (fr) 1992-04-15
AU6352490A (en) 1991-02-06

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