WO1998045710A1 - PROCEDE D'ANALYSE IMMUNOLOGIQUE UTILISANT UN ANTICORPS MONOCLONAL REAGISSANT A L'ENZYME iNOS D'ORIGINE HUMAINE - Google Patents

PROCEDE D'ANALYSE IMMUNOLOGIQUE UTILISANT UN ANTICORPS MONOCLONAL REAGISSANT A L'ENZYME iNOS D'ORIGINE HUMAINE Download PDF

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WO1998045710A1
WO1998045710A1 PCT/US1997/006500 US9706500W WO9845710A1 WO 1998045710 A1 WO1998045710 A1 WO 1998045710A1 US 9706500 W US9706500 W US 9706500W WO 9845710 A1 WO9845710 A1 WO 9845710A1
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human
human inos
inos
monoclonal antibodies
peptide
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PCT/US1997/006500
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Robert Webber
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Robert Webber
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Priority to PCT/US1997/006500 priority Critical patent/WO1998045710A1/fr
Priority to CA002285408A priority patent/CA2285408A1/fr
Priority to DE69739091T priority patent/DE69739091D1/de
Priority to AU27350/97A priority patent/AU2735097A/en
Priority to EP97921260A priority patent/EP1012605B1/fr
Priority to JP54272298A priority patent/JP4117911B2/ja
Priority to BR9714657-9A priority patent/BR9714657A/pt
Publication of WO1998045710A1 publication Critical patent/WO1998045710A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes

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  • the present invention relates to a novel and useful panel of monoclonal antibodies which may be employed in immunoassays and other procedures for detection and/or quantitation of human iNOS.
  • Nitric oxide has recently been recognized as an effector and/or regulator molecule.
  • a recent field of investigation focused on the activity of NO upon the activation of soluble guanylate cyclase, which is responsible for endothelial dependent relaxation in the vasculature.
  • An article entitled "Immunohistochemical Demonstration of a Paracrine Role of Nitric Oxide in Bronchial Function" by Rangass y et al . , American Physiological Society (1994) recognizes this effect with respect to bronchial blood vessels.
  • NO acts as a new neurotransmitter in the central and peripheral nervous system.
  • activated macrophage cytotoxicity was found to be activated in host defense mechanisms based on the presence of NO.
  • NO is now considered the smallest biosynthetically derived effector molecule secreted in mammalian systems. Reference is made to an article entitled “The Molecule of the Year", Science Magazine, Volume 258 (December 1992) , by Koshland, which elaborates on the physiological importance of NO.
  • nNOS neuronal NOS
  • eNOS endothelial
  • NO biosynthetic pathway has been extensively examined recently. It is now recognized that there is a family of isozymes which produce NO. An article entitled "The Nitric Oxide Synthase Family of Proteins", by Sessa, J. Vase. Res. (1994) recognizes the trio of NOS isozymes. All three NOS isozymes catalyze the conversion of L- arginine and oxygen to citrulline and NO. In addition, five co-factors have also been found to be required for this catalytic conversion. These are calmodulin, NADPH, FAD, FMN, and tetrahydrobiopterin.
  • nNOS NO synthase
  • iNOS type 2
  • eNOS type 3
  • nNOS and eNOS are constitutively expressed in the cells in which they are found.
  • iNOS is not constitutively expressed, but rather is induced by a number of cytokines and lypopolysaccarides (LPS) .
  • LPS lypopolysaccarides
  • vascular tone and hemodynamic control has been linked to eNOS.
  • the three (3) isoforms of the NOS enzyme fall in the category of true isozymes since they share approximately 60% sequence homology.
  • iNOS has been specifically implicated in certain pathological diseased states.
  • An article entitled “Expression and Preferential Inhibition of Inducible Nitric Oxide Synthase in Aortas of Endotoxemic Rats", by Weigert et al., Journal of the American Society of Nephrology, Volume 5, No. 12 (1995) discusses the functional importance of iNOS with respect to septic shock.
  • cytokines and LPS from gram negative bacteria potentially can induce the expression of iNOS in monocytes, macrophages, neutrophils, hepatocytes, or other cell types, which leads to the overproduction of NO. This in turn leads to the deleterious effects associated with sepsis and septic shock due to extensive systemic vasodilation.
  • a brochure entitled "Isostrip” by Boehringer Mannheim Corporation illustrates a simplified mouse monoclonal antibody isotyping kit which uses treated strips to detect mouse immunoglobulin subclasses, and kappa or lambda light chains.
  • a novel and useful panel of monoclonal antibodies specific to human iNOS have been developed and have been demonstrated to be useful in immunoassays that are specific for human iNOS. These monoclonal antibodies have been characterized by a number of different standard techniques.
  • assays have been developed using the monoclonal antibodies which are reactive to human iNOS, that also bind to specific linear synthetic peptide analogues of the protein.
  • assays of the competitive binding ELISA, immunofluorescent assay (IFA) types have been developed.
  • Western blot, dip stick, fluorescent polarization, enzyme capture, and radioimmunoassay (RIA) may also be employed in this regard.
  • the assays were employed with mouse models of human septic shock and human samples of septic shock, and validated under controlled circumstances.
  • a number of specific regions of human iNOS were employed in the present invention. Namely, the A3, A4 , A3+A4, F6, Gil and/or the HI loci of human iNOS were targeted. Specific binding pairs and specific binding reagent molecules were also developed in conjunction with these loci.
  • the monoclonal antibodies above mentioned, served as specific binding entities.
  • polyclonal antibodies, oligonucleotides, polymers imprinted as artificial antibodies, phage display binding sites, and the like may also be used in this regard. Further, combinations of peptides may be incorporated into the assays of the present invention.
  • Peptide and peptide analogues at the iNOS regions above identified can be used with specific binding pair partners or with specific binding reagent molecules for direct, indirect, captured, competitive binding, displacement, and other types of assays and assay kits. Any one of such assays or assay kits may be used to detect or measure iNOS quantitatively or qualitatively. Such assays may be of the clinical diagnostic type.
  • the peptide and peptide analogues also include an active region or active portion which will produce satisfactory results.
  • epitope mapping was performed for the clones identified as 21C10-1D10, 2D2-B2, and 5B3-E6.
  • synthetic peptides, recombinant peptides, recombinant proteins, fusion proteins, fusion peptides, phage displayed proteins, phage displayed peptides, peptide libraries, peptide analog libraries, and the like may be employed, where the active component is a mimic to A3, A4, A3+A4 , F6 , Gil, and/or HI loci of human iNOS.
  • regions of human iNOS may be combined in whole or in part in the present assay. For example, region F6 may be combined with a small portion of region HI of human iNOS in an assay regent.
  • the assays of the present invention may detect or measure human iNOS in cells and tissues for various pathophysiological conditions such as sepsis, septic shock in humans and in mouse models, yocardial infarction, rejection of tissue in organs following transplantation, monitoring "flare ups" in certain autoimmune diseases such as lupus, psoriasis, multiple sclerosis, and the like.
  • IFA and competitive binding ELISA assays tested several of such tissues.
  • other assay methods may also be employed using the binding entities of the present invention.
  • the sensitivity of certain assays, such as competitive binding ELISA were increased by using combination of peptides.
  • the peptide to A3+A4 was deemed to increase the sensitivity of the assay to A4 alone, by 4 times.
  • Amplification with avidin-biotin complex increased the assay sensitivity 12 to 15 times.
  • the two combined increased sensitivity 48 to 60 times.
  • An object of the present invention is the development of immunoassays which can be used as clinical tests for hiNOS utilizing monoclonal antibodies specific to hiNOS.
  • Another object of the present invention is to develop a separate panel of polyclonal rabbit anti-peptide antibodies, which are specific for the three (3) isoforms of hiNOS.
  • Yet another object of the present invention is to produce peptide sequences which mimic regions of hiNOS , and that bind to the monoclonal antibodies of the present invention.
  • a further object of the present invention is to provide a method to carry out immunoassays which utiliz specific binding entities which are reactive to human iNOS protein and which reveal the presence of the same by any number of immunoassay formats.
  • Another object of the present invention is to provide immunoassay method which utilizes a specific binding entity reactive to mimics of human iNOS protein to reveal the presence of human iNOS protein in a sample.
  • a further object of the present invention is to provide truncated peptide sequences which mimic regions of hiNOS and that bind to the monoclonal antibodies of the present invention.
  • Another object of the present invention is to provide homolog peptides from proteins other than human iNOS to test the specificity characteristics of the monoclonal antibodies.
  • Yet another object of the present invention is to characterize the panel of monoclonal antibodies of the present invention to ascertain their individual utility in various assays and procedures.
  • FIG. 1 is a listing of five amino acid sequences representing regions of hiNOS to which various monoclonal antibodies, from the overall panel of monoclonal antibodies, of the present invention have bound.
  • FIGS. 2-6 are photos of positively tested microtiter plates using the monoclonal antibodies of the present invention, as described in Example 3.
  • FIGS. 7A-D are a listing of the peptide sequences usable for epitope mapping of the monoclonal antibodies of the present invention.
  • FIG. 8 is a listing of the peptide sequences usable to determine specificity characterization of the monoclonal antibodies of the present invention.
  • FIG. 9 is a graph representing a sandwich ELISA that measures hiNOS using polyclonal rabbit anti-peptide antibodies and four mouse monoclonal antibodies.
  • FIG. 10 is a graph representing a sandwich ELISA that measures hiNOS using mouse IgG2b monoclonal antibody 21C10-1D10 and two mouse IgG 1 monoclonal antibodies.
  • FIG. 11 is a graph representing a sandwich ELISA that measures hiNOS using mouse IgM monoclonal antibody 7D8-B3 and three mouse IgG monoclonal antibodies.
  • FIG. 12 is a photograph of a western immunoblot of hiNOS using four different primary monoclonal antibodies and HRP-conjugated goat anti-mouse IgG secondary antibody.
  • FIG. 13 is a graph representing a sandwich ELISA that measures hiNOS in non-induced and induced A-172 cell lysates using a mouse IgM monoclonal catch antibody, 7D8- B3 , and a mouse IgG., monoclonal detection antibody, 1E8-B8.
  • FIG. 14 is a graph representing a sandwich ELISA that measures iNOS in non-induced and induced RAW 264.7 cell lysates using mouse IgM monoclonal catch antibody, 7D8-B3, and mouse IgG 1 monoclonal detection antibody, 1E8- B8.
  • FIG. 15 is a photograph showing the indirect immunofluorescent staining of induced A-172 cells with mouse IgG 1 monoclonal antibody 1E8-B8 magnified 1600 x.
  • FIG. 16 is a photograph showing the indirect immunofluorescent staining of induced A-172 cells with mouse IgG 1 monoclonal antibody 2A12-A4 magnified 1600 x.
  • FIG. 17 is a photograph showing the indirect immunofluorescent staining of induced A-172 cells with mouse IgM monoclonal antibody 2H11-D11 magnified 1600 x.
  • FIG. 18 is a photograph showing the indirect immunofluorescent staining of induced RAW 264.7 cells with mouse IgG 1 monoclonal antibody 1E8-B8 magnified 1600 x.
  • FIG. 19 is a photograph showing the indirect immunofluorescent staining of induced RAW 264.7 cells with mouse IgG 1 monoclonal antibody 2A12-A4 magnified 1600 x.
  • FIG. 20 is a photograph showing the indirect immunofluorescent staining of induced human monocytes with mouse IgG 1 monoclonal antibody 1E8-B8 magnified 1600 x.
  • FIG. 21 is a photograph showing the indirect immunofluorescent staining of induced human monocytes with mouse IgG 1 monoclonal antibody 2A12-A4 magnified 1600 x.
  • Fig. 22 is a photograph of mouse peritoneal cavity lavage cells 16 hrs. after induction with lipopolysaccharide showing indirect immunofluorescent staining of iNOS with mouse IgG., kappa monoclonal antibodies 5B3-E6 magnified 800 x.
  • Fig. 23 is a photograph of mouse peritoneal cavity lavage cells 16 hrs. after induction with lipopolysaccharide showing indirect immunofluorescent staining of iNOS with mouse IgG., kappa monoclonal antibodies 2D2-B2 magnified 800 x.
  • Fig. 24 is a photograph of mouse buffy coat cells 12 hours after induction with lipopolysaccharide showing indirect immunofluorescent staining of iNOS with mouse IgG 1 kappa monoclonal antibodies 5B3-E6 magnified 800 x.
  • Fig. 25 is a photograph of mouse buffy coat cells 12 hours after induction with lipopolysaccharide showing indirect immunofluorescent staining of iNOS with mouse IgG,, kappa monoclonal antibodies 2D2-B2 magnified 800 x.
  • Fig. 26 is a photograph of human A-172 cells 40 hours after induction with CM/LPS showing indirect immunofluorescent staining of hiNOS with mouse IgG., kappa monoclonal antibodies 5B3-E6 magnified 1600 x.
  • Fig. 27 is a photograph of human A-172 cells 40 hours after induction with CM/LPS showing indirect immunofluorescent staining of hiNOS with mouse IgG,, kappa monoclonal antibodies 2D2-B2 magnified 1600 x.
  • Fig. 28 is a photograph of mouse RAW 264.7 cells 40 hours after induction with CM/LPS showing indirect immunofluorescent staining of iNOS with mouse IgG., kappa monoclonal antibody 2D2-B2 magnified 1600 x.
  • Fig. 29 is a photograph of buffy coat cells obtained from a human septic patient (No. 2) showing indirect immunofluorescent staining of hiNOS with mouse IgG,, kappa monoclonal antibody 2D2-B2 magnified 800 x.
  • Fig. 30 is a graph representing a competitive binding ELISA that measures the quantity of iNOS using mouse IgG., monoclonal antibody 21C10-1D10 in cell lysates obtained from A-172 and RAW 264.7 cells 40 hours after induction with CM/LPS.
  • Fig. 31 is a graph representing a competitive binding ELISA that measures the quantity of hiNOS using mouse IgG, monoclonal antibody 21C10-1D10 in cells lysates obtained from the buffy coat cells of whole blood from three different human septic shock patients.
  • Fig. 32 is a panel of four graphs depicting the increased sensitivity in the competitive binding ELISA for hiNOS using mouse IgC, monoclonal antibody 21C10-1D10 and the 30 amino acid long A3 + A4 peptide, PS-5251, as compared to the 18 amino acid A4 peptide, PS-5104, at four different dilutions of ascites fluid.
  • Fig 33 is a graph representing a competitive binding ELISA using mouse IgG,, monoclonal antibody 21C10- 1D10, the A4 peptide (PS-5104), and the carboxyl terminal truncation peptides (PS-5265 to PS-5268) of Table V which locates the antibodies epitope to the amino terminal region of the A4 locus .
  • Fig. 34 is a graph depicting the competitive binding ELISA using mouse IgG, monoclonal antibody 21C10- 1D10, the standard A4 peptide (PS-5104) , and amino terminal elongation series (PS-5211 to PS-5216) of Table VII which locates the antibodies epitope to PS-5213, VTQDDLQ.
  • Fig. 35 is a graph depicting the competitive binding ELISA using mouse monoclonal antibody 2D2-B2, the standard F6 peptide (PS-5166) , and peptides from the three mid-region elongation series of Table X which locates the antibody's epitope to PS-5294, VQGILERV.
  • Fig. 36 is a graph depicting the competitive binding ELISA using mouse monoclonal antibody 5B3-E6, the standard F6 peptide (PS-5166) , and peptides from the three mid-region elongation series of Table X which locates the antibody's epitope to PS-5294, VQGILERV.
  • a panel of mouse monoclonal antibodies specific for the inducible form of human NOS (hiNOS) has been developed.
  • the monoclonal antibodies were characterized by a number of different techniques including enzyme-linked immunosorbent assay (ELISA) , western immunoblots, immunoprecipitation of 125 I-hiNOS, and indirect immunofluorescent staining of cells. All the monoclonals were initially detected by ELISA, and all perform well in ELISA based assays. However, in all the other assay formats tested, some of the anti-hiNOS monoclonal antibodies worked well and others did not. Only one monoclonal antibody, 1E8-B8, has been found to perform well in all the assay formats tested.
  • purified human iNOS was employed to immunize mice and to develop a panel of monoclonal antibodies.
  • the monoclonal antibodies could have been developed using protein fragments, fusion peptides and proteins, or peptide analogues of hiNOS to immunize mice and elicit an immune response to regions of hiNOS.
  • Standard techniques were used to produce the hybridomas, clone the cells, and produce the monoclonal antibodies.
  • the hybridomas and clones were screened by ELISA and western immunoblot and used in the production of monoclonal antibodies as culture supernatant and as ascites fluid from mice.
  • the monoclonal antibodies were characterized by standard techniques and were also isotyped.
  • the monoclonal antibodies were then tested for their ability to inhibit the enzymatic activity of hiNOS.
  • 96 overlapping peptides each 18 amino acids long, were synthesized to cover the entire 1153 amino acid length structure of the hiNOS.
  • Each peptide had a six amino acid long overlap with its nearest neighbors, except the carboxyl terminal peptide which had an 11 amino acid overlap with the prior peptide.
  • the peptides were used to sensitize a specific well on microtiter plates, and culture supernatant or ascites fluid from each clone was applied individually to the wells. The presence of bound monoclonal antibody was then determined. Specific regions of the iNOS protein were identified as being bound by the monoclonal antibodies.
  • Fig. 1 represents peptide sequences which represent the specific regions of human iNOS which were determined to bind to some of the monoclonal antibodies of the present invention.
  • P Value represents the probability of dissimilarity. In other words, the smaller the value, the more likely the probability of there being a match.
  • the results of the BLAST calculations for peptide A3 in Table I found complete sequence homology with hiNOS (25-42) . This was expected since this is the region of hiNOS that this peptide was built to mimic.
  • the computer search only found sequence homology with two other proteins.
  • One sequence homology concern mouse iNOS (25-42) with a P Value of less than 0.02.
  • the other sequence homology was rat iNOS (25-42) with a P Value of less than 0.03. No sequence homology was found to any other proteins in the databases with a P Value of less than 0.1.
  • sequence homology with peptide A4 found homology only with human iNOS (37-54) which is the region mimicked by the peptide. No region of any other protein in the databases was determined to match this sequence with a P Value of less than 0.1 (i.e., the probability that there is a difference is greater than 99.9%).
  • sequence homology to peptide F6 which is hiNOS (781-798) found sequence homology with human iNOS and with mouse and rat iNOS. No homology was found during this search to any other protein with a P Value of less than 0.1.
  • sequence homology with peptide Gil which is hiNOS (985-1002) , found homology to a number of proteins in Table I. These included mouse and rat iNOS, human nNOS (1256-1273) , human eNOS (1017- 1031) , and bovine eNOS (1019-1033) .
  • the computer search for sequence homology to peptide HI which is hiNOS (1009- 1026) , found homology only with rat and mouse iNOS. No other sequence homology was found with a P Value less than 0.5. It should be noted that a small amount of homology was found with human eNOS and human nNOS, but the P Values are greater than 0.5.
  • sequences from each of the 18-mers to which monoclonal antibodies are found to bind i.e., peptides A3 (PS-5103), A4 (PS-5104), F6 (PS-5166) , GH (PS-5183), and HI (PS-5185) , Table I, were used to design and make a series of epitope mapping peptides for these regions.
  • a series of four truncation peptides from the amino acid terminal end of the 18-mers, as well as a series of four truncations from the carboxyl terminal of each of the 18- mers were fashioned.
  • Fig. 7 represents amino acid sequences showing such truncated peptides which were bound by some of the monoclonal antibodies of the present invention.
  • peptide homologues were designed and synthesized based on the BLAST search. These peptide homologue were used to characterize the specificity of the monoclonal antibodies to proteins other than hiNOS.
  • proteins include hNOS, heNOS, mouse iNOS, and rat iNOS.
  • the panel of monoclonal antibodies of Table III were tested for their ability to detect hiNOS in samples by western immunoblot techniques.
  • cells in culture were induced with a cytokine/LPS mix.
  • the latter technique induced the production of iNOS by the cells which was detectable in western immunoblots by the monoclonal antibodies of the present invention.
  • each of the monoclonal antibodies in the panel of Table III was tested for its ability of immunoprecipitate hiNOS. This was tested by radioimmunoassay (RIA) techniques using 125 I-labeled hiNOS. Ten of the 20 different monoclonal antibodies in the panel were determined to immunoprecipitate hiNOS by this method. Of the ten positives found, monoclonal antibodies 2H11-D11, 5B3-E6, and 21C10-1D10 were found to be the best at immunoprecipitating the radiolabeled protein.
  • RIA radioimmunoassay
  • the ability of the monoclonal antibodies in Table III to recognize and bind to iNOS in fixed cells was also investigated. Induction of iNOS production was examined in three very different types of cultured cells by indirect immunofluorescent staining of the induced cells using the anti-hiNOS monoclonal antibodies as the primary antibody. The three types of induced cultured cells tested were A-172 (a human glioblastoma cell line), RAW 264.7 (a mouse macrophage cell line) , and normal human monocytes isolated from blood.
  • Peptides of defined amino acid sequences were prepared, which mimicked either the amino terminal or the carboxyl terminal of each of the isoforms of human NOS.
  • Each peptide was synthesized by solid phase peptide synthesis utilizing the fmoc protecting strategy.
  • the synthetic peptides were cleaved from the solid support resin, isolated, and purified by standard procedures including preparative HPLC. They were analyzed for purity by analytical HPLC.
  • Each synthetic peptide was conjugated onto a carrier protein, keyhole limpet hemocyanin (KHL) , using either the EDAC or sulfo-MBS chemistries to construct the immunogens for the elicitation of antibodies.
  • KHL keyhole limpet hemocyanin
  • Each peptide/protein conjugate was used as an immunogen in rabbits.
  • the different immunogens were employed to immunize groups of 2-4 rabbits each.
  • the rabbits were immunized, boosted, and bled following a standard protocol developed for the production of anti- peptide antibodies in rabbits.
  • Table II represents a summary of such synthetic peptides.
  • mice were aseptically removed, splenocytes were isolated and were fused with SP2/0 - Ag 14 myeloma cells with polyethylene glycol .
  • Hybridomas were screened by ELISA for production of mouse IgG or IgM antibodies to hiNOS. Positive hybrids were expanded and cloned via limiting dilution. The clones were screened by ELISA and western immunoblot techniques.
  • Positive clones were expanded, frozen down in liquid nitrogen for cryopreservation, and used for the production of monoclonal antibodies as culture supernatant, as well as ascites fluid from Balb/C female mice.
  • the monoclonal antibodies produced by the various clones were characterized by a number of different techniques. These include ELISA, western immunoblot, immunoprecipitation of 125 I-hiNOS (I. P.), and indirect immunofluorescent staining of cells (I.F.A.). The monoclonal antibodies were also isotyped. Table III represents these results:
  • the peptides were used to epitope map the panel of monoclonal antibodies by ELISA techniques. Each peptide was used to sensitize a specific well on a series of microtiter plates. The culture supernatant or ascites from each monoclonal antibody was then applied individually to all the wells of a sensitized plate. The wells were then tested for the presence of bound mouse monoclonal antibody.
  • homologs to iNOS found by the BLAST search were employed to characterize the specificity of the iNOS monoclonal antibodies.
  • a series of four truncation peptides from the amino terminal end of each of the 18-mers as well as a series of four truncations from the carboxyl terminal of each of the 18-mers were made.
  • Each series deleted three amino acids in turn from either the carboxyl or amino terminal of the 18-mers. This resulted in two series of truncation peptides for each 18-mers which were successively shorter by three amino acids from each end.
  • Table V and Figs. 7A-7D and Fig. 8 list the truncation peptides and peptide homologs to hiNOS that were built, the latter were from regions of human nNOS, mouse and rat iNOS, and human eNOS, if any sequence homology was found to these regions by the BLAST computer search, hereinbefore discussed:
  • monoclonal antibody 21C10-1D10 would only bind strongly to peptide A-3 (PS-5103) and weakly to the mouse homolog miNOS (25-42) , (PS-5241) . 21C10-1D10 would not bind to any of the truncated peptides nor to the rat homolog riNOS (25-42) , (PS-5242) .
  • monoclonal antibody 21C10-1D10 bound only to the original 18-mer A4 (PS-5104) and to the hiNOS (37-51) peptide sequence (PS-5265) , which is truncated three residues on the carboxyl terminus.
  • monoclonal antibody 24B10-2C7 was found to bind to HI (PS-5185) .
  • This monoclonal did not recognize either the human eNOS or nNOS homologs, PS-5281 and PS-5282, respectively, but it did bind strongly to the first two amino terminal truncation series peptides, PS-5283 and PS-5284.
  • Monoclonal 24B10-2C7 also bound weakly to the next shorter amino terminal truncation peptide, PS-5285.
  • Table III were tested for their ability to detect and/or quantitate hiNOS in samples, illustrated in Fig. 9.
  • the results shows that clones 1E8-B8, 21C10-1D10, 2A1-A4 , and
  • IgG monoclonal antibodies were used as detection antibodies, for example 1E8-B8 and 2D2-B2, shown in Fig.
  • any of the mouse IgG clones could be used as detection antibody; this includes 1E8-B8 (IgG,) ,
  • the panel of monoclonal antibodies of Table III was tested for their ability to detect hiNOS in samples by western immunoblot techniques.
  • Samples were electrophoresed on 7.5% SDS-PAGE gels which separates the proteins by molecular weight.
  • the proteins were transferred onto PVDF membranes, and the membranes were blocked with evaporated goats milk diluted 1:4 with PBS/Tween 20 buffer.
  • the primary anti-hiNOS monoclonal antibodies were bound, and then the membranes were developed using HRP-conjugated goat anti-mouse IgG antibody, shown in Fig. 12.
  • the monoclonal antibodies have also been tested in western blots using cell lysates obtained from cells which have been reported to contain iNOS following induction with cytokine/LPS mix.
  • Cell lines A-172 and RAW 264.7 were purchased from American Type Culture Collection of Rockville, Maryland (ATCC) , were expanded, and cells were harvested before and after induction with a cytokine/LPS mix, Figs. 13 and 14.
  • Such cytotoxic mix is described in the Geller et al., article, hereinbefore noted, as a cytokine/LPS mixture.
  • the cell pellets were thoroughly washed after harvesting with PBS to remove extraneous proteins.
  • the cells were lysed by two freeze-thaw cycles and sonification.
  • the cell lysates were diluted 1:2 with SDS-PAGE sample buffer and boiled for ten minutes. The samples were electrophoresed on 7.5% gels as described above. The uninduced cells did not contain iNOS whereas, after induction with the cytokine/LPS mix, a band at 130kd was present. This shows that the cytokine/LPS mix had induced iNOS and that the monoclonal antibodies of Table III can detect iNOS in unknown samples in the western blot format.
  • the ability of the various monoclonal antibodies to bind to iNOS in cells that have been induced to produce iNOS was examined in three different cell types, A-172 a human glioblastoma cell line, RAW 264.7 a mouse macrophage cell line, and normal human monocytes.
  • the cells were cultured for two (2) days in normal medium and then induced to produce iNOS by treatment for 40 hours with a cytokine/LPS mixture (CM) . Following the treatment, the cells were processed in one of two ways, either for lysis or for immunostaining.
  • the cells that were to be lysed were detached from the culture flask, washed five (5) times, and frozen in a small volume of PBS to lyse.
  • FIG. 15-17 illustrate the indirect immunofluorescent staining pattern observed on induced A-172 cells with three (3) different mouse anti- hiNOS monoclonal antibodies, 1E8-B8, 2A12-A4, and 2H11-D11 of Table IV, respectively.
  • Figures 18 and 19 illustrate the indirect immunofluorescent staining observed on the fixed RAW 264.7 cells with anti-hiNOS monoclonal antibodies, 1E8-B8 and 2A12-A4 of Table IV, respectively.
  • the monocytes were isolated from normal human blood by density gradient centrifugation using Optiprep obtained from Accurate Chemical and Scientific Corp,
  • the enzymatic activity of hiNOS was determined by measuring the amount of nitrite produced in the presence of the substrates and co-factors.
  • Example 2 The procedure of Example 2 was followed by the de novo preparation of ascites fluid from the cryopreserved cells of Example 1. This procedure was followed approximately 12 months following the initial preparation of Example 2.
  • the competitive binding ELISA in Table VI is based upon the competition for antibody binding between a synthetic peptide which is coated onto microtighter ELISA plates and iNOS in standards or unknowns which are in solution. It may be apparent a number of monoclonal antibodies of the present invention were found to bind to specific linear synthetic peptide analogues of protein.
  • the assay can be placed in any of a number of formats acceptable by clinical lab technicians.
  • the competitive binding ELISA as depicted in Table VI has a minimum sensitivity of 20 fmole and takes three hours to complete. It is theorized that the addition of pre-incubation will further increase the sensitivity by a factor of two to three and provide an ELISA with a minimum sensitivity in the 6-10 fmole range.
  • Cells were washed for one minute each with three changes of phosphate buffer saline (PBS) followed by a quick rinse in water. The water was drained well. Cells grown on glass were fixed in 100 percent acetone. Cells grown on plastic were fixed in 80 percent acetone and 20 percent water. Fixing took place for 10 to 15 minutes at room temperature, samples are air dried and stored at 20 degrees centigrade.
  • Monoclonal antibodies were applied as cultured supernatants which were diluted 1:10/1:20/1:40 with PBS. Also, monoclonal antibodies were applied as ascites fluid and were diluted 1:500/1:2,500/1:12,500.
  • the supernatants or ascites fluid were added directly to fixed cells at room temperature and incubated for one hour at 37 degrees centigrade, or two hours at room temperature. Each sample was then washed four times in PBS for one minute each and drained. FITC conjugated goat anti-mouse secondary antibodies were then added with Hyclone at 1:120 dilution in PBS. The samples were incubated 30 minutes at 37 degrees centigrade or 45 minutes at room temperature. Again, they were washed three times in PBS for one minute each and then rinsed in water to remove salts. After draining, a cover slip was mounted using glycerine based mounting medium which contains DABCO to reduce fading. The immunofluorescent staining was observed using an epi- fluorescent microscope equipped with excitation and emission wave length set for FITC.
  • Figs. 22-29 represent the indirect immunofluorescent staining of samples using the reagents of Example 9.
  • the assay was validated using A-172 cell lysates
  • the ABC complex was diluted with and equal volume of PBS and 0.1 percent Tween 20. 2% NHS and 0.1 percent crystalline BSA were added to the ABC mixture. 100 microliters per well of the pre-incubated ABC solution were added at room temperature and allowed to rest for 30 minutes. The samples were washed eight times. A color reaction was executed in 10X phosphate citrate buffer at pH 5.0 with 0.5 mg/ml of OPD and 0.08% hydrogen peroxide for 55 minutes at 37° C.
  • the ELISA was "tweaked" with a longer synthetic peptide to coat the plates and effect ABC amplification. A fifty to sixty fold increase was achieved in sensitivity. The ELISA was determined to take three hours to complete. It is believed that the addition of a pre-incubation step will further increase the sensitivity by a factor of two to three and will result in an ELISA with a minimum sensitivity in the 6-10 fmole range.
  • Figs.30 and 31 represent the ELISA assay of this Example for quantifying iNOS and hiNOS.
  • Fig. 32 shows the increased sensitivity of the assay of this Example using the A3 + A4 peptide instead of the A4 peptide.
  • the assay was validated using A-172 cell lysates
  • hiNOS glioblastoma cell line
  • enzyme enzyme, except under specific culture conditions.
  • Western Blotting Western Blotting
  • polyclonal antibody based RIA polyclonal antibody based RIA
  • monoclonal antibody and peptide based competitive binding ELISA show that no hiNOS was present in these controls. Also it was determined that hiNOS was induced by a mixture of cytokines and LPS.
  • 21C10-1D10 was achieved at the amino terminal of the A4 locus of hiNOS Table VII represents these results.
  • 21C10-1D10 EPITOPE VTQDDLQ
  • Fig. 33 represents the "2nd step" competitive binding ELISA using mouse IgC, monoclonal antibody 21C10- 1D10, the A4 peptide (PS-5104) , and the carboxyl terminal truncation peptides (PS-5265 to PS-5268) of Table V of Example 4.
  • Table VIII represents epitope mapping of peptides PS-5261 to PS-5269 which highlighted the PS-5265 to PS-5268 peptides mapped in Fig. 33.
  • the antibodies epitope was determined to be PS-5213 VTQDDLQ.
  • Table IX represents these results and lists the peptides used, displays the results obtained in the competitive binding ELISA using mouse monoclonal antibodies 2D2-B2 and 5B3-E6 with the series of peptides that map the mid-region of the F6 locus. Table IX also identifies both of these antibodies' epitope as the sequence VQGILERV (hiNOS 784-791) .

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Abstract

On a développé un ensemble d'anticorps monoclonaux susceptibles de reconnaître, et de se lier à, l'enzyme monoxyde d'azote synthétase, inductible, d'origine humaine (iNOS ou iNOS de type II). Ces anticorps monoclonaux peuvent servir aux analyses visant à déceler la présence de l'enzyme iNOS d'origine humaine et/ou à le quantifier.
PCT/US1997/006500 1997-04-07 1997-04-11 PROCEDE D'ANALYSE IMMUNOLOGIQUE UTILISANT UN ANTICORPS MONOCLONAL REAGISSANT A L'ENZYME iNOS D'ORIGINE HUMAINE WO1998045710A1 (fr)

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Application Number Priority Date Filing Date Title
PCT/US1997/006500 WO1998045710A1 (fr) 1997-04-11 1997-04-11 PROCEDE D'ANALYSE IMMUNOLOGIQUE UTILISANT UN ANTICORPS MONOCLONAL REAGISSANT A L'ENZYME iNOS D'ORIGINE HUMAINE
CA002285408A CA2285408A1 (fr) 1997-04-07 1997-04-11 Procede d'analyse immunologique utilisant un anticorps monoclonal reactif
DE69739091T DE69739091D1 (de) 1997-04-07 1997-04-11 Immunoassay verfahren das einen zu menschlichem inos reaktiven monoklonalen antikörper verwendet
AU27350/97A AU2735097A (en) 1997-04-11 1997-04-11 Immunoassay method employing monoclonal antibody reactive to human inos
EP97921260A EP1012605B1 (fr) 1997-04-07 1997-04-11 PROCEDE D'ANALYSE IMMUNOLOGIQUE UTILISANT UN ANTICORPS MONOCLONAL REAGISSANT A L'ENZYME iNOS D'ORIGINE HUMAINE
JP54272298A JP4117911B2 (ja) 1997-04-11 1997-04-11 ヒトｉＮＯＳに反応するモノクロナール抗体を用いる免疫学的検定法
BR9714657-9A BR9714657A (pt) 1997-04-07 1997-04-11 Processo para imunoensaio empregando reagente de anticorpos monoclonais a inos humano

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Cited By (2)

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WO2003012026A1 (fr) * 2000-07-31 2003-02-13 Robert Webber Dispositif et procede permettant de detecter l'apparition et la presence de conditions de sepsie
CN103946707A (zh) * 2010-09-22 2014-07-23 罗伯特·韦伯 脓毒症血液生物标志物系统

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US20050281826A1 (en) * 2004-05-19 2005-12-22 Research & Diagnostic Antibodies Llc Therapeutic agent for iNOS generating illness cross-references to related applications
WO2017069113A1 (fr) * 2015-10-20 2017-04-27 国立大学法人 東京医科歯科大学 Composition stimulant la croissance des cheveux et son utilisation

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WO1996039858A1 (fr) * 1995-06-07 1996-12-19 Merck & Co., Inc. Anticorps monospecifiques et leurs procedes d'utilisation

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WO1996039858A1 (fr) * 1995-06-07 1996-12-19 Merck & Co., Inc. Anticorps monospecifiques et leurs procedes d'utilisation

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KASHEM A, ET AL.: "EXPRESSION OF INDUCIBLE-NOS IN HUMAN GLOMERULONEPHRITIS: THE POSSIBLE SOURCE IN INFILTRATING MONOCYTES/MACROPHAGES", KIDNEY INTERNATIONAL, NATURE PUBLISHING GROUP, LONDON, GB, vol. 50, no. 02, 1 August 1996 (1996-08-01), LONDON, GB, pages 392 - 399, XP008004803, ISSN: 0085-2538, DOI: 10.1038/ki.1996.328 *
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003012026A1 (fr) * 2000-07-31 2003-02-13 Robert Webber Dispositif et procede permettant de detecter l'apparition et la presence de conditions de sepsie
JP2005524380A (ja) * 2000-07-31 2005-08-18 ロバート ウェーバー 敗血症状態の発症および存在を決定するための装置及び方法
AU2001280912B2 (en) * 2000-07-31 2006-06-22 Robert Webber Apparatus and method for determining the onset and presence of sepsis conditions
AU2001280912B8 (en) * 2000-07-31 2006-11-23 Robert Webber Apparatus and method for determining the onset and presence of sepsis conditions
CN103946707A (zh) * 2010-09-22 2014-07-23 罗伯特·韦伯 脓毒症血液生物标志物系统

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