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WO1992004633A1 - Procede et composition servant a detecter des peptides bioactifs - Google Patents

Procede et composition servant a detecter des peptides bioactifs Download PDF

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Publication number
WO1992004633A1
WO1992004633A1 PCT/US1991/006115 US9106115W WO9204633A1 WO 1992004633 A1 WO1992004633 A1 WO 1992004633A1 US 9106115 W US9106115 W US 9106115W WO 9204633 A1 WO9204633 A1 WO 9204633A1
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WIPO (PCT)
Prior art keywords
terminal
amino acid
antibodies
amidated
peptide
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
PCT/US1991/006115
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English (en)
Inventor
Christopher J. Evans
Karen L. Valentino
Patricia M. Bassett
Tejinder Singh
Donald H. Yamashiro
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Elan Corp PLC
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Neurex Corp
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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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6842—Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • 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
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors

Definitions

  • This invention relates to a method for screening bio ⁇ logical material for the presence of bioactive peptides, and to an antibody composition useful in the method.
  • the peptide detected in the second species has been found to have structural and biolo ⁇ gical functions which are quite similar to the original peptides, this has not always been the case.
  • the immunoreactive material purified from chicken brain had the sequence LPLRF-amide, and the immunoreactive material found in rat brain was attributable to the peptide neur ⁇ peptide Y (NPY) having the sequence: YPSKPDNPGEDAPAEDLARYYSALFHYINLITRQRY- amide.
  • the mammalian immuno ⁇ reactive material has been determined to be gastrin re- leasing peptide (GRP) , a 27 amino acid peptide, which has continuous series of seven amino acids in common with frog bombesin.
  • GRP gastrin re- leasing peptide
  • homologous sear ⁇ ching strategies described above have reduced to some degree the labor required to identify novel peptides.
  • these strategies generally limit discovery to a subset of homologous or associated peptides, and rely on the ability of antibodies to recognize epitopes with high specificity.
  • Tatemoto and Mutt developed a chemical method to detect the presence of C-terminal amino acid amides in isolated peptides (K. Tatemoto et al, Proc Nat Acad Sci USA (1978) 75:4115-19; K. Tatemoto, Proc Nat Acad Sci USA (1982) 7_9_:5485-89) .
  • a tissue extract is subjected to various separation procedures culminating in high performance liquid chromatography (HPLC) in order to isolate a series of chromatography (HPLC) in order to iso ⁇ late a series of chromatographically pure peptides.
  • HPLC high performance liquid chromatography
  • HPLC series of chromatography
  • Each isolated peptide is then subjected to an enzyme hydrolysis procedure followed by dansylation to identify the component amino acids.
  • the presence of a C-terminal amidated amino acid in a particular fraction recommends that fraction for further study.
  • this method is able to detect truly novel (non-homologous), non-precursor) peptides, it requires relatively high amounts of starting material from a deduced source. It is also labor intensive, in that numerous peptide fractions must be processed methodically or at random in order to find a novel amidated peptide.
  • tissue sources can be rapidly screened by immunocytochemical methods to detect novel amidated or pyroglu peptides. These antibodies are also useful for monitoring purification of the peptides from relatively small quantities of material.
  • the rational behind the development of this method is somewhat counter ⁇ intuitive, as described below. It is generally known that small molecules such as peptides are not detectably immunogenic unless couples to a carrier protein.
  • NPY the FMRF-amide immunoreactive material described above which has a C-terminal Y-amide
  • NPY the FMRF-amide immunoreactive material described above which has a C-terminal Y-amide
  • the antiserum was found to recognize a sequence contained in the N terminal region of NPY (J.M. Lundberg et al, J Neurosci (1984) 4.:2376-86) .
  • the method of the invention has utilized amino acid amides, pyroglutamate or short (2-3 amino acid) peptides terminating in C-terminal amide of N-terminal pyroglutamate conjugated to carrier proteins to produce antibodies which recognize potential biologically active peptides. Since the sequences of the conjugated haptens have only 1-3 amino acids, the antibodies produced to them can detect novel peptides which may or may not be highly homologous to currently known peptides. It should be pointed out that, as noted by Barchas et al, (1987) , other researchers have occasionally discovered novel peptides serendipitously using antibodies produced against known homologous peptides, due to cross-reactivity of the specific antisera used. Our approach is different in that it deliberately uses "nonspecific" antibodies, directed toward the N- or C-terminus without restriction by the amino acid sequence further down- or upstream, in order to detect novel bioactive peptides.
  • the composition of the invention includes a plurality of antibodies which are immunoreactive with multiple distinct antigens, which may be (a) different amidated C- terminal amino acid residues, (b) different N-terminal pyroglu/penultimate amino acid residues, or (c) a combina ⁇ tion of (a) and (b) antigens.
  • the anti ⁇ bodies in the composition are contacted, singly or in combinations, with a biological material to be screened, producing in the material, an immunoconjugate complex with a bioactive peptide containing such antigen. The material is then examined for the presence of immunoconjugate.
  • the method may further include isolating bioactive peptide(s) from material found to contain such immunoconjugate(s) .
  • the invention provides an immunogen for producing the above antibody composition.
  • the immuno ⁇ gen has the structure of-formula (a) or (b) , for generating antibodies which are immunoreactive with (a) different amidated C-terminal amino acid residues or (b) distinct N- terminal pyroglu/penultimate amino acid residues, respec- tively:
  • X is X X 2 -X ⁇ , or X 3 -X 2 -X j ; and X b is a direct bond, X 4 , or X 4 -X 5 , where X l t X 2 , ⁇ 3 , ⁇ 4 and X 5 are each independently alanine, cysteine, cystine, aspartic acid, glutamic acid, phenylalanine, glycine, histi ⁇ ine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, argi- nine, serine, threonine, valine, tryptophan, tyrosine, ornithine, desmosine, isodesmosine, or a derivative there ⁇ of; X j is a linking group or a direct bond; and X c is a suitable carrier protein.
  • the linking group X,* has formula: where X f and X c , are as above.
  • peptide refers to molecules composed of a number of amino acids linked covalently by peptide bonds. In general, peptides within this definition will have from 2 to about 60 amino acid resides. However, as only one to three amino acids at the C-terminal of the molecule are recognized in the method of the invention, there is no practical upper limit to the size of the peptide or protein which may be detected. Peptides may also have other post- translational modifications, such as glycosylation, myristylation, acetylation, and covalent modifications of amino acid side chains (e.g., histidine N-Methylation) , formation of pyroglutamate, and the like.
  • post- translational modifications such as glycosylation, myristylation, acetylation, and covalent modifications of amino acid side chains (e.g., histidine N-Methylation) , formation of pyroglutamate, and the like.
  • C-terminal amidated and “amidated” peptide refer to a peptide having a -C0NH 2 group at the carboxy terminus.
  • An amidated amino acid is similarly structured.
  • aminodated blycine refers to the molecule H 2 N- CH 2 -CONH 2 .
  • An amidated dipeptide will have the general structure HjN-CHRj-CONH-CHR ⁇ CON ⁇ , where Rj and R 2 are the side chains of the amino acids.
  • ⁇ is -CH 2 OH and R 2 is -CH 2 .
  • Pyroglu peptide refers to peptides in which the N- terminal amino acid is glutamine, wherein the side chai amide cyclizes with the N-terminal amine group to for pyrrolidone carboxylic acid, also known as pyroglutamate.
  • pyroglu amino acid means pyroglutamate
  • N-terminal pyroglu/penultimate amino aci residues refers to the two N-terminal amino acid residues of a peptide including the penultimate N-terminal residue and the N-terminal pyroglu residue.
  • an “amidated peptide” refers to a dipeptide, tripeptide, or single amino acid wherein the C-terminus is amidated.
  • pyroglu peptide when applied to the haptens and immunogens of the invention refers to a dipeptide or tripeptide wherein the N-terminus is pyroglu, or the amino acid pyroglutamate.
  • i munogen as used herein refers generally to a conjugate comprising a hapten of the invention (amidated or pyroglu amino acid or peptide) linked to a suitable carrier molecule.
  • X a is a direct bond, ⁇ 4 , or X 4 -X 5 , where X., ⁇ 2 , ⁇ 3 , ⁇ 4 , and X 5 are each independently alamine, cysteine, cystine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methio- nine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, tyrosine, ornithine, desmosine, isodesmosine, or a derivative thereof; X,* is a joining or linking group, or a direct bond; and X c is a suitable carrier protein.
  • NH 2 shown in formula I indicates that the molecule is amidated at the C-termi ⁇ nus.
  • the ring structure in formula II is N-terminal pyroglu.
  • “Derivatives" of amino acids include, without limitation, hydroxylated amino acids (e.g., 4- hydroxyproline, hydroxylysine, and the like) , phosphorylat- ed amino acids (e.g., phosphoserine, phosphotyrosine, phosphothreonine, phosphohistidine, and the like) , acetyla- ted amino acids (e.g., N-acetyllysine, acetylserine, and the like), sulfated amino acids (e.g., sulfotyrosine, sulfothreonine, etc.), glycosylated amino acids, myristyla- ted amino acids, methylated amino acids (for example, epsilon-N-methyllsine,
  • suitable carrier refers to a protein or other large molecule which may be used to carry a hapten, and present the hapten appropriately to an animal's immune system for the generation of a specific immune response.
  • the amidated or pyroglu dipeptides, tripeptides, and amino acids used as immunogens herein are too small to stimulate an immune response in isolation, and must be conjugated to a larger molecule.
  • the carrier is preferably a moderately large protein, which does not exhibit detrimental activity upon injection.
  • suitable carriers used in the art include bovine serum albumin (BSA) , bovine thyroglobulin (BTG) , keyhole limpet hemocyanin (KLH) , synthetic carriers, and the like.
  • BSA bovine serum albumin
  • BSG bovine thyroglobulin
  • KLH keyhole limpet hemocyanin
  • synthetic carriers and the like. Suitable carriers and methods for conjugating haptens to carriers are described, inter alia, in B.F. Erlanger
  • Coupled reagent or "linking reagent” - is used to indicate those reagents which are useful for forming a covalent bond between an antigen and a carrier, whether any part of the reagent molecule remains in the resulting complex or not. Most commonly, the coupling will comprise a covalent bond between the amidated or pyroglu amino acid or peptide and a free carboxyl or amine group present on the carrier protein. Alternatively, peptides and carriers may be coupled using standard crosslinking reagents, such as formaldehyde, glutaraldehyde, carbodi- imide, disuccinimidylsuberate and the like.
  • any technique which is currently useful for attaching enzy ⁇ matic labels to antibodies or antigens, and which does not chemically alter the amino acid or peptide epitope may be employed.
  • An amidated amino acid or peptide may be co- valently attached to the carrier by a peptide bond, for example by selecting a carrier protein having the desired amino acid(s) at the c-terminal (where the C-terminus is exposed on the surface of the protein) , and amidating the C-terminal amino acid.
  • N-terminal pyroglu immunogens may be generated by selecting a carrier having the desired N-terminal sequence (beginning with Gin) , and cyclizing the terminal Gin, preferably by enzymatic means (W.H. Fischer et al, Proc Nat Acad Sci USA (1987) £4:3628- 32) .
  • the carrier protein may be reco bi- nantly engineered to terminate with a selected amino acid, followed by C-terminal amidation or N-terminal cyclization
  • the modification may involve truncation of the gene, site specific mutagenesis, fusion to an additional nucleotid sequence, or a combination thereof.
  • one ma 5 cleave a carrier protein to expose the selected amino acid, followed by C-terminal amidation or N-terminal cyclization.
  • a 10 sently preferred technique is to couple an amidated or pyroglu amino acid, dipeptide, or tripeptide, particularly a tripeptide, to a carrier protein using a coupling agent such as 3-(3-dimethylaminopropyl_-carbodiimide (EDC) .
  • a coupling agent such as 3-(3-dimethylaminopropyl_-carbodiimide (EDC) .
  • Another presently preferred linker is a polyvalent
  • X f is a joining or linking group, or a direct bond: the free valence is 5 linked to an antigen molecule.
  • This branched linking group may be coupled to the carrier directly, or using an addi ⁇ tional coupling agent such as a short (1-10 amino acid) peptide sequence or polymer of similar length.
  • X e is a direct bond, a coupling agent as described for X,-, or a pep ⁇ tide of 1-10 amino acids.
  • the C-terminal Lys is peptide bonded to Cys, either directly or through 1-5 intervening amino acids, and succinoyl amidated amino acids or pyroglutamic acid are coupled to the free Lys amine groups using the l- hydroxybenxotriazole -dicyclohexylcarbodiimide method.
  • a presently preferred branched linker of the invention has the following structure:
  • the product complex is then conjugated to a carrier, e.g., bovine thyroglobulin (BTG) using the heterobifunctio- nal reagent succinimidyl 4-maleimidomethyl)cyclohexane-l- carboxylate (SMCC) .
  • a carrier e.g., bovine thyroglobulin (BTG)
  • BBG bovine thyroglobulin
  • SMCC succinimidyl 4-maleimidomethylcyclohexane-l- carboxylate
  • antibody and Ab as used herein refer to im unoglobulins and fragments thereof capable of binding specifically to a C-terminal amidated or N-terminal pyro- glu/penultimate amino acid residues. Both polyclonal anti ⁇ sera and monoclonal antibodies (MAbs) are included within this definition, as well as binding fragments such as Fab and F(ab , ) 2 .
  • the C-terminal or N-terminal amino acids or acids form the immunoreactive epitope responsible for the Ab specificity. Due to the small size of epitope, a cer ⁇ tain amount of cross-reactivity is inevitable.
  • immunoconjugate refers to a non-covalent complex of antibody and amidated amino acid or peptide or pyroglu amino acid or peptide which forms spontaneously upon contacting the amidated or pyroglu peptide with an Ab specific for an immunogen of the invention.
  • amidated peptides formula I
  • X is the same amino acid as the C-terminal amidated amino acid of the amidated peptide.
  • an immunogen of formula II is employed.
  • Immunoconjugates are detected by means known in the art for immunoassays, typically by reaction with a labeled specific binding partner. For example, where rab ⁇ bit antisera is used as the primary Ab, detection of immu ⁇ noconjugates may be accomplished by treating the reaction products with a labeled goat anti-rabbit IgG.
  • rab ⁇ bit antisera is used as the primary Ab
  • detection of immu ⁇ noconjugates may be accomplished by treating the reaction products with a labeled goat anti-rabbit IgG.
  • label refers to any atom or molecule which can be used to provide a detectable (preferable quantifiable) sig ⁇ nal, and which can be attached to a protein. Labels may provide signals detectable by fluorescence, radioactivity, colorimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, and the like. Suitable labels include fluorophores, chromophores, radioactive atoms (particularly 32 P and 125 I) , electron-dense reagents, enzymes, and ligands having specific binding partners. Enzymes are typically detected by their activity.
  • horseradish peroxidase can be detected by its ability to convert 3,3' ,5,5'-tetramethylbenzidine (TMB) to a blue pigment, quantifiable with a spectrophotometer.
  • TMB 3,3' ,5,5'-tetramethylbenzidine
  • Specific binding partner refers to a protein capable of binding a ligand molecule with high specificity, as for example in the case of an antigen and a monoclonal antibody specific therefor.
  • Other specific binding partners include biotin and avidin or streptavidin, IgG and protein A, and the numerous receptor-ligand couples known "in the art. It should be understood that the above description is not meant to categorize the various labels into distinct clas ⁇ ses, as the same label may serve in several different modes.
  • 15 I may serve as a radioactive label and as an electron-dense reagent.
  • HRP may serve as enzyme or as antigen for an Ab.
  • Abs and avidin also require labels in the practice of this invention: thus, one might label a probe with biotin, and detect its presence with avidin labeled with 125 I, or with an anti- biotin Ab labeled with HRP.
  • Other permutations and pos ⁇ sibilities will be readily apparent to those of ordinary skill in the art, and are considered as equivalents within the scope of the instant invention.
  • animal refers to vertebrates having a functional immune system, i.e., capable of producing appropriate antibodies in response to immuniza ⁇ tion with a coupled peptide carrier complex ("immunocompe- tent").
  • Animals include without limitation mammals, such as rabbits, rats, mice, Guinea pigs, dogs, cats, cattle, goats, horses, minks, man, monkeys, apes, and the like, and birds, such as chickens, turkeys, pigeons, quail, and the like.
  • Presently preferred animals for the generation of antibodies are rabbits and goats, particularly rabbits.
  • Suitable Abs are generated by immunizing an animal having an intact (antibody-producing) immune system with a selec ⁇ ted immunogen of formula I or II.
  • Amidated amino acids, pyroglutamic acid, and amino acid derivatives are commer- cially available (for example, from Sigma Chemical Co., St. Louis, MO) , or may be prepared from amino acids using tech ⁇ niques known in the art.
  • Dipeptides and tripeptides may also be obtained commercially (e.g., from Bachem Bioscienc- es) , or may be prepared from an amino acid or dipeptide and an amidated amino acid.
  • Carrier proteins are also available from commercial source, such as Calbiochem, San Diego, CA.
  • the carrier and immunogen of formula I or II are coupled using conventional techniques, such as described by Erlanger, supra.
  • the resulting immunogen is then used to immunize a suitable animal, preferably a rabbit or goat. Rabbits and goats are preferred due to the volume of serum obtainable, and the availability of labeled anti-rabbit and anti-goat antibodies. Immunization is generally performed by mixing or emulsifying the immunogen in saline, preferably in an adjuvant such as Freund's complete adjuvant, and injecting the mixture or emulsion parenterally (generally subcutane- ously or intramuscularly) . A dose of 50-200 ug/injection is typically sufficient.
  • Immunization is generally boosted 2-6 weeks later with one or more injections of immunogen in saline, preferably using methods known in the art, which for the purposes of this invention is considered equivalent to in vivo immunization.
  • Polyclonal antisera are obtained by bleeding the im ⁇ munized animal into a glass or plastic container, incubat ⁇ ing the blood at 25°C for one hour, followed by incubating at 4°C for 2-18 hours.
  • the serum is recovered by centrifu- gation (e.g., 1,000 x G for 10 minutes). About 20-50 L per bleed may be obtained from rabbits.
  • Monoclonal antibodies are prepared using the method of Kohler and Milstein, Nature (1975) 256:495-96. or a modifi ⁇ cation thereof.
  • a mouse or rat is immunized as described above.
  • the spleen is removed and dissociated into single cells.
  • the spleen cells may be screened (after removal of non-specifically adherent cells) by applying a cell suspension to a plate or well coated with the immunizing antigen.
  • B-cells expressing membrane- bound immunoglobulin specific for the antigen bind to the plate, and are not rinsed away with the rest of the sus ⁇ pension.
  • Resulting B-cells, or all dissociated spleen cells are then induced to fuse with myeloma cells to form hybridomas, and are cultures in a selective medium (e.g., hypoxanthine, aminopterin, thymidine medium, "HAT") .
  • the resulting hybridomas are plated by limiting dilution, and are assayed for the production of antibodies which bind specifically to the immunizing antigen (and which do not bind to unrelated antigens) .
  • the selected MAb-secreting hybridomas are then cultured either in vitro (e.g., in tissue culture bottles or hollow fiber reactors) , or in vivo (as ascites in mice) .
  • antibodies whether polyclonal or monoclonal
  • antibodies may be labeled using conventional technique.
  • the antibody composition of the invention includes a plurality or battery of antibodies enzymes which together are effective in screening biological material for the presence of bioactive peptides having different amidated C- terminal residues and/or different N-terminal pyroglu amino acid residues.
  • the most exhaustive composition will include antibodies capable of reacting with each possible amidated C-terminal L-amino acid or each possible N- terminal pyroglu L-amino acid. Each composition would thus include about 20 different-specificity antibodies.
  • the number of antibodies in a composition used for screening can typically be reduced by taking advantage of cross reactivity among certain antibodies.
  • composition antibody components may be selected for immureactivity with a selected group of amidated amino acid residues only.
  • the distribution of amidated C-terminal residues of known bio ⁇ active peptides, as listed in Table I shows a dominance of Gly, Met, Pro, Phe, Val, Ala, Leu, and Tyr C-terminal amidated residues (each with three or more representative) .
  • a composition of antibodies specific against this group of eight C-terminal residues would be effective to screening a large portion of bioactive peptides with C- terminal amidated ends.
  • composition of antibodies which are specific against N-terminal pyroglu/penultimate residues may be reduced by selecting cross-reacting antibodies, or antibodies specific against a smaller number of pyroglu/- penultimate residues, such as the penultimate residues shown in Table II.
  • the present invention allows one to search for biologically active peptides having amidated C- termini or pyroglu N-termini.
  • the antibodies of the invention employed are specific for C- and N-terminal resi ⁇ dues, as described above, and are thus not limited by sequence variations within the peptide.
  • Using a different antibody in the composition one could next search for all peptides having, for example, amidated glycineserine.
  • the screening procedure includes contacting the test biological material with the sequentially or in combinations with the antibod ⁇ ies in the above antibody composition.
  • the method of the invention com- prises contacting a biological material with the antibodies in the antibody composition, thereby forming an immunogenic complex between one or more of the antibodies and one or more bioactive peptides in the material.
  • the material is then examined for the presence of the immunogenic complex.
  • the sample may be a tissue preparation (e.g., tissue extract, thin section, etc.), serum or blood, a secretion, a cell culture supernatant, or the like.
  • the method of the invention is employed to detect the presence of C-terminal amidated peptides and N-terminal pyroglu peptides in tissue sections and extracts.
  • tissue sections are prepared using methods known in the art for immunohistochemical imagings, and antibodies of the invention are applied and allowed to incubate. After a suitable incubation period, the section is rinsed and examined for formation of immunoconjugates. If the specific antibodies have been labeled with radioac ⁇ tive isotopes, fluorophores, or similar indicators, the sections are generally examined by autoradiography or fluorescent photomicroscopy. Unlabeled Abs are generally detected by using a labeled specific binding partner which binds to the Abs. For example, if the specific Abs used are polyclonal rabbit antibodies, detection may be accom ⁇ plished using FITC-conjugated goat anti-rabbit IgG antibod ⁇ ies.
  • the method of the invention is also employed to detect the presence of C-terminal amidated peptides and N-terminal pyroglu peptides in tissue extracts.
  • a fresh or frozen tissue is extracted using an acidic solution or acidic organic solvent mixture in conjunction with rapid homogenization of the tissue (e.g., using a blender).
  • the mixture is then further purified by centrifugation and/or further extraction with organic solvent, followed by chromatography. Chromatographic fractions are reduced in volume and redissolved in a suitable buffer for binding to a support, such as an ELISA plate. Following a suitable incubation time, the plate is rinsed, then incubated with antibodies of the present invention.
  • the plate is rinsed, and exa ⁇ mined for formation of immunoconjugates with the antibodies in the composition. Generally, this is done by adding a labeled specific binding partner which binds to the Abs.
  • the label is generally an enzyme, such as horseradish per- oxidase.
  • a suitable enzyme substrate such as a solution containing tetramethylbenzidine and hydrogen peroxide is added, and the optical absorbance determined.
  • the Abs of the invention are also useful during the purification and characterization of potential new amidated or pyroglu peptides. Once a positive reaction has indicat ⁇ ed the presence of a possibly new peptide, the tissue is extracted and the extract fractionated using chromatograph ⁇ ic techniques. The Abs of the invention are used to iden- tify fractions containing the candidate peptide. Once highly pure samples of the peptide have been prepared, antibodies specific for the candidate peptide may be pre ⁇ pared by standard methods, if desired. The peptide may be microsequenced when sufficient material has been collected and purified.
  • Example 1 The examples presented below are provided as a further guide to the practitioner of ordinary skill in the art, and are not to be construed as limiting the invention in any way.
  • Example 1
  • Serum was prepared from the blood by allowing the blood to clot for several hours, removing the clot, and centrifuging the remainder at 1,000 x g for 15 min at room temperature. Serum was aliquoted and stored at -20°C. Serum titers were tested by the ELISA method described below.
  • Polystyrene 96-2311 plates (Falcon 3072, Beckton Dickinson, Oxnard, CA) were coated with goat serum albumin
  • GSA GSA in 0.1 M bicarbonate, pH 9.0 (2 mg GSA/100 L buffer) by adding 0.1 mL of this solution per well. Plates were sealed with Parafilm and stored (1 day - 2 mos) at 4°C.
  • Antigen was coupled to the GSA using a glutaral- dehyde coupling technique.
  • the GSA coated wells were emptied of GSA/bicarbonate solution, then rinsed with 0.1 bicarbonate solution, pH 9.0.
  • a 1-2% (v/v) solution of glutaraldehyde in bicarbonate solution was freshly pre ⁇ pared, and 0.1 mL added to each well and incubated for 15 min at room temperature. The solution was then "flicked" out of the wells, without rinsing.
  • a stock solution of 1.0 mg/mL mastoparan (L-NH 2 C-terminus) was prepared by dissolv ⁇ ing the lyophilized peptide in a volume of acetonitrile equal to 10% of the final desired volume, then diluting to volume with 0.1 M bicarbonate buffer, pH 9.0. Dilutions of the peptide solution were then made so that the peptide was distributed to various wells in the following amounts/well in 0.1 mL: 0 ug, 0.001 ug, 0.01 ug, 1 ug, 10 ug. Plates containing the peptide solutions were then incubated at 37°c for 1 hour.
  • Rabbit antisera were diluted in BLOTTO. Dilu ⁇ tions tested ranged from 1/5000 - 1/50,000. The desired dilution(s) of antisera (0.1 mL) were added to each well and the plate incubated overnight at 4°C. The solution was then removed from the plate by flicking, and the wells washed thoroughly with PBS/0.05% Tween-20 twice, then three times with PBS.
  • Horseradish peroxidase (HRP) conjugated goat anti-rabbit antiserum (affinity purified, TAGO, Berkeley, CA) was diluted 1/3000 in BLOTTO. 0.1 mL of this solution was added to each well. This solution was left on the plate for 1 hour at room temperature. The plate was then washed twice with PBS/Tween-20 and three times with PBS, as above.
  • Color reagent (substrate) was prepared just prior to addition to the plate.
  • the reagent consists of 30 mL 0.1 M citrate buffer, pH 5, 0.03 mL 30% hydrogen peroxide solution, 0.3 mL of a 1% (w/v) solution of tetramethylben- zidine (TMB) in dimethylsulfoxide.
  • TMB tetramethylben- zidine
  • peptides are coupled directly to COBIND 96-well plates. It is important that these plates and any materials that are coupled to them are protected from any nucleophile (e.g., amine, ammonia). Plates are therefore covered tightly during all incubation steps.
  • nucleophile e.g., amine, ammonia
  • Peptide (alpha-MSH) was diluted to an appropriate concentration (0.001-10 ug/100 uL) in 0.1 M NaHC0 3 or 0.1 M borate buffer pH 9.0. 100 uL of the alpha-MSH solution was added to each well and incubated at 37°C overnight, tightly covered with Parafilm or Falcon pressure sensitive film.
  • Anti-valine-amide antisera were prepared as described in parts A and B above, substituting V-NH 2 for L- NH 2 .
  • the antisera were diluted in BLOTTO and mixed, 100 uL added per well, and incubated at 4°C overnight. The solution was then flicked out of the wells, and the wells washed thoroughly (2X PBS/Tween, then 3X PBS) . Between each wash step, plates were blotted by inverting on paper towels.
  • Horseradish peroxidase conjugated goat anti- rabbit antiserum was diluted 1/3000 in BLOTTO, and 100 uL were added to each well. The plates were incubated with this solution for 1 hour at room temperature. The wells were then washed twice with PBS/Tween, then three times with PBS. Between each wash step, plates were blotted by inverting on paper towels.
  • the tetra ethylbenzidine color reagent was pre ⁇ pared fresh (5-10 min before use in 0.1 M citrate buffer, pH 5.0) by adding 0.3 mL stock TMB solution (10 g/L in DMSO) to 30 mL citrate buffer and 0.03 mL 30% H 2 0 2 . A 0.1 mL aliquot of this reagent was added to each well and incubated at room temperature for 30 minutes prior to reading the absorbance at 650 nm. Positive results were obtained.
  • Rats Male Sprague-Dawly, 150-250 g were injec- ted with 0.05 mg colchi ⁇ ine (Sigma) under ether anesthesia in 0.025 mL water intraventricularly 48 hours prior to sacrifice. All rats were then anesthetized with Nembutal (pentobarbital sodium, Abbot Laboratories, North Chicago, IL) then perfused through the aorta with Saline followed by fixative (15% picric acid, 4% paraformaldehyde, in 0.1 M phosphate buffer, pH 7.4) .
  • Nembutal pentobarbital sodium, Abbot Laboratories, North Chicago, IL
  • the brain, heart, pituitary, adrenal, pancreas, thymus, spleen, testes, stomach, small intestine, lung, ovary, uterus, placenta, salivary gland, and spinal cord were removed and stored in the same fixative overnight.
  • Tissues were immersed in 10% sucrose in 0.1 M phosphate buffer overnight, frozen with Freeze-it (Chemtronics, Hauppauge, NY) and sectioned at 12 microns on a Reichert Histostat cryostat. Sections were mounted on gelatin/chro alum- coated slides and stored at -80°C.
  • Example 2 (Assay for V-NH 2 Peptides) The procedures described in Example 1 above were repeated, substituting V-NH 2 for L-NH 2 . Three rabbits were immunized with V-NH 2 -thyroglobulin conjugate. Of these, rabbits produced antibodies detect ⁇ able in the ELISA assay at dilutions of 1/20,000. These antisera labeled areas of the brain and pituitary in a pattern consistent with the distribution of two known V-NH 2 peptides, metorphamide and alpha-MSH. No additional staining outside the known distribution of these peptides was observed. Thus, the method of producing the antibodies to single amino acid amides was again validated, although no novel peptides were discovered in the immunocytochemical staining patterns with the antiserum.
  • Example 3 Pyroglutamyl immunogens having branched linkers were prepared by a method similar to synthesis of the "MAP" multiple antigenic peptide system described by J.P. Tarn, Proc Nat Acad Sci USA (1988) 85:5409-13. In this case, pyroglutamic acids were attached to a branched Lys struc ⁇ ture using the 1-hydroxybenzotriazoledicyclohexylcarbodi- imide method. The branched tetravalent structure which resulted was then coupled to bovine thyroglobulin (BTG) .
  • BBG bovine thyroglobulin
  • pGly is pyroglutamyl
  • EC is an ethyl- carbamoyl protecting group.
  • the Arg residues were added to insure solubility, and the Gly residue was added for con ⁇ venience of synthesis.
  • the carrier protein was prepared by mixing a solution of bovine thyroglobulin (81 mg) in 0.1 M phosphate buffered saline (pH 7.0, 4.0 mL) with a solution of SMCC (31 mg) in 1.0 mL DMF, stirring for 30 min at 24°C, and gel filtering on a 1.5 x 40 cm column of Sephadex G-25 in 0.1 M phos ⁇ phate-buffered saline (pH 5.9). All fractions correspond- ing to material detected at 280 nm near the void volume were pooled and stored at 4°C. Exposure time for SMCC-BTG to room temperature was 40 min.
  • the peptide-linker complex (22 mg) was dissolved in water (0.90 mL) and mixed with 1 N NaOH (0.10 mL) to remove the EC group from cysteine. After 45 sec at 24°C, 0.4 M KH 2 P0 4 (0.35 mL) was added to neutralize the solution. The resulting solution was then mixed with one half of the pooled SMCC-BTG fractions, and allowed to react for 3.5 hr at 24°C to form the complete immunogen. The solution was then gel filtered through a 1.5 x 40 cm column of Sephadex G-25 in 25 mM NH 4 HC0 3 .

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Abstract

L'invention décrit un procédé et une composition d'anticorps servant à filtrer un matériau biologique pour détecter la présence de peptides bioactifs. La composition d'anticorps comprend une pluralité d'anticorps qui sont immunoréactifs avec (a) différents résidus amino-acides aminés à terminaison en C, (b) différents résidus amino-acides pyroglu à terminaison en N, et (c) une combinaison d'antigènes des groupes (a) et (b). Dans le procédé, on fait réagir la composition d'anticorps avec le matériau à filtrer et on examine celui-ci pour détecter la présence d'immunoconjugués.
PCT/US1991/006115 1990-09-05 1991-08-27 Procede et composition servant a detecter des peptides bioactifs Ceased WO1992004633A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU736374B2 (en) * 1994-11-08 2001-07-26 Diacrin, Inc. Porcine striatal cells and their use in treatment of neurological deficits due to neurodegenerative disease

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0232717A2 (fr) * 1986-01-10 1987-08-19 Shiosaka Sadao Anticorps spécifique contre une substance de faible poids moléculaire et son procédé de préparation utilisant des particules métalliques colloidales comme support
WO1990010709A2 (fr) * 1989-03-14 1990-09-20 Board Of Regents Of The University Of Nebraska Anticorps monoclonaux pour cations metalliques sur petites molecules

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0232717A2 (fr) * 1986-01-10 1987-08-19 Shiosaka Sadao Anticorps spécifique contre une substance de faible poids moléculaire et son procédé de préparation utilisant des particules métalliques colloidales comme support
WO1990010709A2 (fr) * 1989-03-14 1990-09-20 Board Of Regents Of The University Of Nebraska Anticorps monoclonaux pour cations metalliques sur petites molecules

Non-Patent Citations (8)

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Title
JOURNAL OF IMMUNOLOGY. vol. 138, no. 10, 15 May 1987, BALTIMORE US pages 3332 - 3338; P. MOTTE ET AL: 'Monoclonal antibodies distinguish synthetic peptides that differ in one chemical group' see abstract see page 3338, left column, line 53 - line 55 *
NEUROCHEMISTRY INTERNATIONAL vol. 14, no. 4, 1989, GREAT BRITAIN pages 505 - 510; A. VARRO: 'Identification and characterization of a novel CCK-like immunoreactive peptide in pig CNS' see abstract see page 505, right column, line 12 - line 26 see page 506, left column, line 10 - line 21 *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA. vol. 75, September 1978, WASHINGTON US pages 4115 - 4119; K. TATEMOTO ET AL.: 'Chemical determination of polypeptide hormones' cited in the application see the whole document *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA. vol. 79, September 1982, WASHINGTON US pages 5485 - 5489; K.TATEMOTO: 'Neuropeptide Y: Complete amino acid sequence of the brain peptide' cited in the application see abstract see page 5485, left column, line 1 - line 23 *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA. vol. 83, December 1986, WASHINGTON US pages 9817 - 9821; GRIMMELIKHUIJZEN C.J.P. ET AL.: 'Isolation of <Glu-Gly-Arg-Phe-NH2 (Antho-RFamide), a neuropeptide from sea anemones' *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA. vol. 84, June 1987, WASHINGTON US pages 3628 - 3632; W.H.FISHER ET AL.: 'Identification of a mammalian glutaminyl cyclase converting glutaminyl into pyroglutaminyl peptides' see page 3628, left column, line 1 - line 18 *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA. vol. 85, August 1988, WASHINGTON US pages 5409 - 5413; J.P.TAM: 'Synthetic peptide vaccine design: Synthesis and properties of a high density multiple antigenic peptide system' cited in the application see abstract; figure 1 *
see the whole document esp. the abstract and page 9817, left column, lines 25-28 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU736374B2 (en) * 1994-11-08 2001-07-26 Diacrin, Inc. Porcine striatal cells and their use in treatment of neurological deficits due to neurodegenerative disease

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