Internet for Pharmaceutical and Biotech Communities
| Newsletter | Advertising |
 
 
 

  

Pharm/Biotech
Resources

Outsourcing Guide

Cont. Education

Software/Reports

Training Courses

Web Seminars

Jobs

Buyer's Guide

Home Page

Pharm Patents /
Licensing

Pharm News

Federal Register

Pharm Stocks

FDA Links

FDA Warning Letters

FDA Doc/cGMP

Pharm/Biotech Events

Consultants

Advertiser Info

Newsletter Subscription

Web Links

Suggestions

Site Map
 

 
   



 

Title:  Antibody to GALP and uses thereof
United States Patent: 
7,173,112
Issued: 
February 6, 2007

Inventors: 
Matsumoto; Hirokazu (Ibaraki, JP), Horikoshi; Yasuko (Ibaraki, JP), Kitada; Chieko (Osaka, JP), Ohtaki; Tetsuya (Ibaraki, JP)
Assignee: 
Takeda Pharmaceutical Company Limited (Osaka, JP)
Appl. No.: 
10/490,917
Filed: 
September 25, 2002
PCT Filed: 
September 25, 2002
PCT No.: 
PCT/JP02/09840
371(c)(1),(2),(4) Date: 
March 26, 2004
PCT Pub. No.: 
WO03/027150
PCT Pub. Date: 
April 03, 2003


 

Pharm/Biotech Jobs


Abstract

The present invention intends to provide a novel monoclonal antibody having a binding specificity to GALP or its derivative, which is useful in developing therapeutic agents, preventive agents or diagnostic agent for diseases associated with GALP or its derivative, and a method of quantifying GALP using the antibody. More specifically, the present invention provides an antibody specifically reacting with a partial peptide in the C-terminal region of a polypeptide having the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, or its derivative, and a method of quantifying GALP or its derivative.

DISCLOSURE OF THE INVENTION

The present invention intends to provide an antibody (preferably a monoclonal antibody), which can specifically quantify GALP or its derivative with high sensitivity, a method of detecting/quantifying GALP or its derivative using the antibody, and a diagnostic product using the same.

The present inventors have made extensive investigations to solve the foregoing problems and as a result, developed an immunoassay method, which involves using [Cys.sup.43] rat GALP (43-560) as an immunogen, preparing a plurality of monoclonal antibodies, and using them in combination, and by which GALP or its derivative can be specifically detected with high sensitivity. That is, using the complex of keyhole limpet hemocyanin (hereinafter referred to as KLH) and [Cys.sup.43] rat GALP (43-60) as an immunogen, monoclonal antibodies (e.g., GR-1Ca) capable of recognizing the C-terminal partial peptide of GALP or its derivative were obtained. These antibodies showed an extremely high affinity to GALP in the competitive immunoassay using peroxidase (HRP)-labeled [Cys.sup.43] rat (43-60). Furthermore, it has also been found that a sandwich-immunoassay for GALP with extremely high sensitivity is provided by using this antibody in combination with the GR2-1Na antibody specifically reacting with a partial peptide in the N-terminal region of GALP or its derivative, which was already developed (Published Japanese Patent Application KOKAI No. 2000-157273). The present invention enables to assay GALP in a simple manner with high sensitivity, and greatly serves in clarifying the physiological functions of GALP or its derivative by determining changes in GALP in body fluids such as blood, cerebral fluid, urine, etc.

The present invention provides an antibody (preferably a monoclonal antibody), which specifically reacting with a partial peptide in the C-terminal region of GALP or its derivative; a hybridoma cell producing the monoclonal antibody; a method of producing the antibody and the hybridoma; a method of immunological assay for GALP or its derivative by the sandwich method, etc., in combination with the antibody (GR2-1Na) specifically reacting with a partial peptide in the N-terminal region of GALP or its derivative; and the like.

Thus, the present invention relates to the following features: (1) An antibody specifically reacting with a partial peptide in the C-terminal region of a polypeptide having the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, or its derivative; (2) The antibody according to (1), wherein the partial peptide in the C-terminal region is a peptide having a 44 53 amino acid sequence in the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; (3) The antibody according to (1), wherein the partial peptide in the C-terminal region is a peptide having a 40 60, 41 60, 42 60, 43 60, 44 60, 45 60, 46 60, 47 60, 48 60, 49 60, 50 60, 44 54, 45 54, 46 54, 47 54, 48 54, 49 54 or 50 54 amino acid sequence in the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; (4) The antibody according to (1), which is labeled; (5) The antibody according to (1), which is a monoclonal antibody; (6) The monoclonal antibody according to (5), which is producible from a hybridoma cell shown by GR-1C (FERM BP-7682) and shown by GR-1Ca; (7) A hybridoma cell according to (5), which produces the monoclonal antibody of (5); (8) The hybridoma cell according to (7), which is shown by GR-1C (FERM BP-7682); (9) A method of manufacturing the monoclonal antibody of (5), which comprises culturing the hybridoma cell of (7) in vivo or in vitro and collecting the monoclonal antibody of (5) from the body fluid or culture; (10) A pharmaceutical comprising the antibody according to (1); (11) A diagnostic product comprising the antibody according to (1); (12) A method of quantifying a polypeptide having the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, or its derivative, which comprises using the antibody according to (1); (13) A method of quantifying a polypeptide having the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, or its derivative in a sample fluid, which comprises using the antibody according to (1) and an antibody specifically reacting with a partial peptide in the N-terminal region of a polypeptide having the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, or its derivative; (14) A method of quantifying a polypeptide having the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, or its derivative in a sample fluid, which comprises (1) reacting (i) the antibody according to (1) immobilized on a carrier with (ii) a labeled form of antibody specifically reacting with a partial peptide in the N-terminal region of a polypeptide having the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, or its derivative and (iii) a sample fluid, or (2) reacting (i) an antibody specifically reacting with a partial peptide in the N-terminal region of a polypeptide having the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, or its derivative, which is immobilized on a carrier, with (ii) a labeled form of the antibody according to (1) and (iii) a sample fluid, and then determining the activity of a marker on the immobilization carrier; (15) The quantifying method according to (14), which comprises (1) reacting (i) the monoclonal antibody according to (6) immobilized on a carrier with (ii) a labeled form of monoclonal antibody shown by GR2-1Na, which is producible from a hybridoma cell shown by GR2-1N (FERM BP-6682) and (iii) a sample fluid, or (2) reacting (i) a monoclonal antibody shown by GR2-1N, which is producible from hybridoma cell shown by GR2-1N (FERM BP-6682), which is immobilized on a carrier with (ii) a labeled form of the antibody according to (6) and (iii) a sample fluid, and then determining the activity of a marker on the immobilization carrier; (16) A method of quantifying a polypeptide having the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, or its derivative in a sample fluid, which comprises competitively reacting the antibody according to (1), a sample fluid and a labeled form of the polypeptide having the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, or its derivative, and determining a ratio of the labeled polypeptide having the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 or its derivative, bound to the antibody; (17) A method of diagnosing a disease associated with a polypeptide having the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, or its derivative, which comprises using the antibody according to (1); and, (18) A method of diagnosing obesity, sterility, collagen disease or rheumatic disease, which comprises using the antibody according to (1); and so on.

BEST MODE FOR CARRYING OUT THE INVENTION

Throughout the specification, the proteins (polypeptides) are represented in accordance with the conventional way of describing peptides, that is, the N-terminus (amino terminus) at the left hand and the C-terminus (carboxyl terminus) at the right hand. In the proteins used in the present invention including the polypeptide containing the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, the C-terminus may be in any form of a carboxyl group, a carboxylate, an amide or an ester.

The polypeptide having the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 (hereinafter sometimes referred to as GALP) includes a rat, human or porcine polypeptide, etc. consisting of 60 amino acid residues, and the like (hereinafter sometimes referred to as the peptide of the present invention).

The derivatives of GALP used in the present invention include, for example, those having the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, wherein a part of amino acid residues are replaced by a replaceable group(s), the amino acid residues are in part deleted, the amino acid residues are in part added/inserted, etc.

Examples of the derivatives of polypeptide having the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 are (i) those wherein at least 1 or 2 (preferably approximately 1 to 10, more preferably several (1 to 5) and most preferably 1, 2 or 3) amino acids are deleted of the amino acid sequence described above; (ii) those wherein at least 1 or 2 (preferably approximately 1 to 20 amino acids, more preferably approximately 1 to 10 amino acids, much more preferably several (1 to 5) and most preferably 1, 2 or 3) amino acids are added to the amino acid sequence described above; (iii) those wherein at least 1 or 2 (preferably approximately 1 to 20 amino acids, more preferably approximately 1 to 10 amino acids, much more preferably several (1 to 5) and most preferably 1, 2 or 3) amino acids are inserted into the amino acid sequence described above, or (iv) those wherein at least 1 or 2 (preferably approximately 1 to 10, more preferably several (1 to 5), and most preferably 1, 2 or 3) amino acids in the amino acid sequence described above are replaced with other amino acids.

As the partial peptide in the N-terminal region or the partial peptide in the C-terminal region of GALP or its derivatives used in the present invention, there are peptides having the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, wherein a part of the amino acid residues are deleted, those wherein a part of the amino acid residues are replaced with replaceable groups (e.g., Cys, hydroxyl group, etc.), or those wherein a part of the amino acid residues are deleted and a part of he amino acid residues are replaced with replaceable groups (e.g., Cys, hydroxyl group, etc.), and the like.

Examples of partial peptides in the C-terminal region of GALP or its derivatives include GALP or its derivatives, of which approximately 42 to 54 residues are deleted in the N-terminal region of GALP or its derivatives.

More specifically, the partial peptides in the C-terminal region are the following polypeptides in the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO:2 or SEQ ID NO:3;

(i) polypeptide having the 40 60 amino acid sequence,

(ii) polypeptide having the 41 60 amino acid sequence,

(iii) polypeptide having the 42 60 amino acid sequence,

(iv) polypeptide having the 43 60 amino acid sequence,

(v) polypeptide having the 44 60 amino acid sequence,

(vi) polypeptide having the 45 60 amino acid sequence,

(vii) polypeptide having the 46 60 amino acid sequence,

(viii) polypeptide having the 47 60 amino acid sequence,

(ix) polypeptide having the 48 60 amino acid sequence,

(x) polypeptide having the 49 60 amino acid sequence,

(xi) polypeptide having the 50 60 amino acid sequence,

(xii) polypeptide having the 44 54 amino acid sequence,

(xiii) polypeptide having the 45 54 amino acid sequence,

(xiv) polypeptide having the 46 54 amino acid sequence,

(xv) polypeptide having the 47 54 amino acid sequence,

(xvi) polypeptide having the 48 54 amino acid sequence,

(xvii) polypeptide having the 49 54 amino acid sequence,

(xviii) polypeptide having the 50 54 amino acid sequence, and

(xix) those wherein a part of amino acid residues (e.g., 1 residue) are replaced with replaceable groups in these polypeptides; and so on.

Examples of the partial peptides in the N-terminal region of GALP or its derivatives are those deleted of approximately 40 to 50 residues in the N-terminal region of GALP or its derivatives.

The partial peptides in the N-terminal region include the following polypeptides in the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3;

(i) polypeptide having the 1 4 amino acid sequence,

(ii) polypeptide having the 1 5 amino acid sequence,

(iii) polypeptide having the 1 6 amino acid sequence,

(iv) polypeptide having the 1 7 amino acid sequence,

(v) polypeptide having the 1 8 amino acid sequence,

(vi) polypeptide having the 1 9 amino acid sequence, and

(vii) those wherein a part of amino acid residues (e.g., 1 residue) in these polypeptides are replaced with replaceable groups; and so on.

The antibodies of the present invention, which specifically react with the partial peptides in the C-terminal region of GALP or its derivatives, may be any antibody so long as they specifically react with partial peptides in the C-terminal region of GALP or its derivatives (preferably partial peptides in the C-terminal region of the peptide represented by SEQ ID NO:2). These antibodies include antibodies specifically reacting with the following polypeptides in the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO:2 or SEQ ID NO:3: (i) a polypeptide having the 40 60 amino acid sequence, (ii) a polypeptide having the 41 60 amino acid sequence, (iii) polypeptide having the 42 60 amino acid sequence, (iv) polypeptide having the 43 60 amino acid sequence, (v) polypeptide having the 44 60 amino acid sequence, (vi) polypeptide having the 45 60 amino acid sequence, (vii) polypeptide having the 46 60 amino acid sequence, (viii) polypeptide having the 47 60 amino acid sequence, (ix) polypeptide having the 48 60 amino acid sequence, (x) polypeptide having the 49 60 amino acid sequence, (xi) polypeptide having the 50 60 amino acid sequence, (xii) polypeptide having the 44 54 amino acid sequence, (xiii) polypeptide having the 45 54 amino acid sequence, (xiv) polypeptide having the 46 54 amino acid sequence, (xv) polypeptide having the 47 54 amino acid sequence, (xvi) polypeptide having the 48 54 amino acid sequence, (xvii) polypeptide having the 49 54 amino acid sequence, (xviii) polypeptide having the 50 54 amino acid sequence, and (xix) polypeptide wherein a part of amino acid residues (e.g., 1 residue) in these polypeptides are replaced with replaceable groups; etc.

As the antibody of the present invention, which specifically reacts with the partial peptides in the C-terminal region of GALP or its derivative, a monoclonal antibody is preferred. More specifically, the antibody of the present invention, which specifically reacts with the partial peptide in the C-terminal region of GALP or its derivatives, includes an antibody specifically reacting with [Cys.sup.43] rat GALP (43-60), etc. [Cys.sup.43] rat GALP (43-60) is a 43 60 amino acid sequence in the amino acid sequence represented by SEQ ID NO: 1 and means a polypeptide wherein Cys is replaced for the 43rd residue in this amino acid sequence. More preferred antibodies are antibodies which specifically react with the partial peptides in the C-terminal region of GALP or its derivatives but do not react with the partial peptides in the N-terminal region.

An example of the antibody of the present invention, which specifically reacts with the partial peptides in the C-terminal region of GALP or its derivatives, includes the monoclonal antibody shown by GR-1Ca producible from the hybridoma shown by GR-1C (FERM BP-7682).

As such, the antibody of the present invention, which specifically reacts with the partial peptides in the C-terminal region of GALP or its derivatives, can react with GALP or its derivatives by recognizing a particular amino acid sequence in the C-terminal region of GALP or its derivatives.

The antibodies of the present invention, which specifically react with the partial peptides in the N-terminal region of GALP or its derivatives, may be any antibody so long as they specifically react with partial peptides in the N-terminal region of GALP or its derivatives. These antibodies include antibodies specifically reacting with the following polypeptides in the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3: (i) polypeptide having the 1 4 amino acid sequence, (ii) polypeptide having the 1 5 amino acid sequence, (iii) polypeptide having the 1 6 amino acid sequence, (iv) polypeptide having the 1 7 amino acid sequence, (v) polypeptide having the 1 8 amino acid sequence, (vi) polypeptide having the 1 9 amino acid sequence, and (vii) a polypeptide wherein a part of amino acid residues (e.g., 1 residue) in these polypeptides are replaced with replaceable groups. Preferred antibodies specifically reacting with the partial peptides in the N-terminal region of GALP or its derivatives are monoclonal antibodies.

More specifically, the antibodies include those specifically reacting with rat GALP (1 9) (polypeptide having the 1 9 amino acid sequence in the amino acid sequence represented by SEQ ID NO:1). Among them, more preferred are such antibodies that specifically react with the partial peptides in the N-terminal region but do not react with the partial peptides in the C-terminal region. An example of the antibodies specifically reacting with the partial peptides in the N-terminal region includes monoclonal antibody shown by GR2-1Na producible from the hybridoma cell shown by GR2-1N (FERM BP-6682) (Published Japanese Patent Application KOKAI No. 2000-157273).

As such, the antibody, which specifically reacts with the partial peptides in the N-terminal region of GALP or its derivatives, can react with GALP or its derivatives by recognizing a particular amino acid sequence in the N-terminal region of GALP or its derivatives.

Hereinafter, explanation is given to methods of preparing antigens of the antibodies specifically reacting with the partial peptides in the C-terminal region of GALP or its derivatives (hereinafter sometimes referred to as the antibody of the present invention) and methods of manufacturing the antibodies.

(1) Preparation of Antigen

To prepare the antibody of the present invention, any antigen such as GALP or its derivatives, synthetic peptides having 1 or 2 more antigenic determinants, which are the same as in GALP, etc., may be used (hereinafter these antigens are sometimes collectively referred to as the GALP antigen).

GALP or its derivatives can be (a) prepared from mammalian tissue or cells of human, monkey, rat, mouse, swine, etc. by publicly known methods or with modifications, (b) chemically synthesized by publicly known peptide synthesis methods using a peptide synthesizer, etc., or (c) manufactured by culturing a transformant bearing a DNA encoding GALP or its derivatives.

(a) Where the GALP antigen is prepared from the mammalian tissues or cells, the tissues or cells are homogenized, then extracted with an acid, an alcohol, etc., and the extract is purified and isolated by a combination of salting-out, dialysis, gel filtration, chromatography techniques such as reverse phase chromatography, ion exchange chromatography, affinity chromatography and the like. Thus, the GALP antigen can be prepared.

(b) Synthetic peptides used when the GALP antigen is chemically synthesized are, for example, a peptide having the same structure as that of the GALP antigen purified from natural one, a peptide containing 1 or 2 more amino acid sequences, which are the same amino acid sequences consisting of at least 3, preferably at least 6 amino acids in an optional region of the amino acid sequence of GALP, etc.

(c) Where the GALP or its derivatives are manufactured using the DNA-bearing transformants, the DNA can be produced in accordance with publicly known cloning techniques (e.g., the method described in Molecular Cloning (2nd ed., J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989), etc.). The cloning techniques include (1) a method in which transformants containing DNAs encoding the GALP or its derivatives are obtained from cDNA library by hybridization using DNA probes or DNA primers designed based on the amino acid sequence of GALP or its derivatives, or (2) a method in which transformants containing DNAs encoding the GALP or its derivatives are obtained by PCR using DNA primers designed based on the amino acid sequence of GALP or its derivatives, etc.

Peptides used as the GALP antigen can be prepared (1) by peptide synthesis methods publicly known, or (2) by cleaving peptides having the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO:2 or SEQ ID NO:3 with an appropriate peptidase.

For the methods for peptide synthesis, for example, any of solid phase synthesis and liquid phase syntheses may be used. That is, the partial peptides or amino acids that can construct the peptide are condensed with the remaining part. Where the product contains protecting groups, these protecting groups are removed to give the desired peptide. By the condensation or removal of the protecting groups, objective peptides can be prepared. Publicly known methods for condensation and removal of the protecting groups are methods described in (i) or (ii) below.

(i) M. Bodanszky & M. A. Ondetti: Peptide Synthesis, Interscience Publishers, New York (1966)

(ii) Schroeder & Luebke: The Peptide, Academic Press, New York (1965)

After the reaction, the product may be purified and isolated by a combination of conventional purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, etc. to give the peptide. When the peptide obtained by the above methods is in a free form, the peptide can be converted into an appropriate salt by a publicly known method; when the peptide is obtained in a salt form, it can be converted into a free form by a publicly known method.

Amides of the peptide may be obtained using commercially available resins for peptide synthesis, which are suitable for formation of the amides. Examples of such resins include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, 4-hydroxymethylmehtylphenyl acetamidomethyl resin, polyacrylamide resin, 4-(2',4'-dimethoxyphenylhydroxymethyl)phenoxy resin, 4-(2',4'-dimethoxyphenyl-Fmoc-aminoethyl) phenoxy resin, etc. Using these resins, amino acids in which .alpha.-amino groups and functional groups on the side chains are appropriately protected are condensed on the resin in the order of the sequence of the objective peptide according to various condensation methods publicly known in the art. At the end of the reaction, the peptide is cut out from the resin and at the same time, the protecting groups are removed to obtain the objective peptide. Alternatively, the objective peptide may also be obtained by protecting the peptide in part with chlorotrityl resin, oxime resin, 4-hydroxybenzoic acid type resin, etc., and removing the protective groups from the taken out peptide in a conventional manner.

For condensation of the protected amino acids described above, a variety of activation reagents for peptide synthesis may be used, and carbodimides are particularly preferable. Examples of such carbodiimides include DCC, N,N'-diisopropylcarbodiimide, N-ethyl-N'-(3-dimethylaminoprolyl)carbodiimide, etc. For activation by these reagents, the protected amino acids in combination with a racemization inhibitor (e.g., HOBt, HOOBt) are added directly to the resin, or the amino acids previously protected in the form of symmetric acid anhydrides, HOBt esters or HOOBt esters are activated, followed by adding the thus activated protected amino acids to the resin. Solvents suitable for use to activate the protected amino acids or condense with the resin may be appropriately chosen from solvents known to be usable for peptide condensation reactions. Examples of such solvents are acid amides such as N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, etc.; halogenated hydrocarbons such as methylene chloride, chloroform, etc.; alcohols such as trifluoroethanol, etc.; sulfoxides such as dimethylsulfoxide, etc.; tertiary amines such as pyridine, etc.; ethers such as dioxane, tetrahydrofuran, etc.; nitriles such as acetonitrile, propionitrile, etc.; esters such as methyl acetate, ethyl acetate, etc.; and appropriate mixtures of these solvents. The reaction temperature is appropriately chosen from the range known to be applicable to peptide bond-forming reactions and is usually selected in the range of approximately -20.degree. C. to 50.degree. C. The activated amino acid derivatives are used generally in an excess of about 1.5 to about 4 times. The condensation is examined by a test using the ninhydrin reaction; when the condensation is insufficient, the condensation can be completed by repeating the condensation reaction without removal of the protecting groups. When the condensation is yet insufficient even after repeating the reaction, unreacted amino acids are acetylated with acetic anhydride or acetylimidazole to cancel adverse effects on the subsequent reactions.

Examples of the protecting groups used to protect the amino groups of the starting compounds include Z, Boc, t-pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, trifluoroacetyl, phthaloyl, formyl, 2-nitrophenylsulphenyl, diphenylphosphinothioyl, Fmoc, etc. Examples of the protecting groups of a carboxyl group include, in addition to a C.sub.1-6 alkyl group, a C.sub.3-8 cycloalkyl group and a C.sub.7-14 aralkyl group, 2-adamantyl, 4-nitrobenzyl, 4-methoxybenzyl, 4-chlorobenzyl, phenacyl and benzyloxycarbonyl hydrazide, t-butoxycarbonyl hydrazide, trityl hydrazide and the like.

The hydroxyl group of serine and threonine can be protected through, for example, its esterification or etherification. Examples of the groups appropriately used for the esterification include a lower (C.sub.1-6) alkanoyl group, such as acetyl group, etc.; an aroyl group such as benzoyl group, etc., and a group derived from carbonic acid such as benzyloxycarbonyl group, ethoxycarbonyl group, etc. Examples of a group suitable for the etherification include benzyl group, tetrahydropyranyl group, t-butyl group, etc.

Examples of groups for protecting the phenolic hydroxyl group of tyrosine include Bzl, ClBzl, 2-nitrobenzyl, Br-Z, t-butyl, etc.

Examples of groups used to protect the imidazole moiety of histidine include Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, Bom, Bum, Boc, Trt, Fmoc, etc.

Examples of the activated carboxyl groups in the starting compounds include the corresponding acid anhydrides, azides, activated esters [esters with alcohols (e.g., pentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol, cyanomethyl alcohol, p-nitrophenol, HONB, N-hydroxysuccimide, N-hydroxyphthalimide, HOBt)]. As the activated amino acids, in which the amino groups are activated in the starting material, the corresponding phosphoric amides are employed.

To eliminate (split off) the protecting groups, there are used catalytic reduction under hydrogen gas flow in the presence of a catalyst such as Pd-black, Pd-carbon, etc.; an acid treatment with anhydrous hydrofluoric acid, methanesulfonic acid, trifluoromethane-sulfonic acid or trifluoroacetic acid, or a mixture solution of these acids; a treatment with a base such as diisopropylethylamine, triethylamine, piperidine, piperazine, etc.; and reduction with sodium in liquid ammonia; or the like. The elimination of the protecting groups by the acid treatment described above is carried out generally at a temperature of approximately -20.degree. C. to 40.degree. C. In the acid treatment, it is efficient to add a cation scavenger such as anisole, phenol, thioanisole, m-cresol, p-cresol, dimethylsulfide, 1,4-butanedithiol, 1,2-ethanedithiol, etc. Furthermore, 2,4-dinitrophenyl group known as the protecting group for the imidazole of histidine is removed by a treatment with thiophenol. Formyl group used as the protecting group of the indole of tryptophan is eliminated by the aforesaid acid treatment in the presence of 1,2-ethanedithiol, 1,4-butanedithiol, etc. as well as by a treatment with an alkali such as a dilute sodium hydroxide solution, dilute ammonia, etc.

Protection of the functional groups that should not be involved in the reaction of the starting materials, protecting groups, elimination of the protecting groups and activation of the functional groups involved in the reaction may be appropriately selected from publicly known groups and publicly known means.

In another method for obtaining the amides of the peptide, for example, the .alpha.-carboxyl group of the carboxy terminal amino acid is first protected by amidation; the peptide chain is then extended to a desired length toward the amino group side. Thereafter, a peptide in which only the protecting group of the N-terminal .alpha.-amino group in the peptide chain has been eliminated from the peptide and a peptide (or amino acids) in which only the protecting group of the C-terminal carboxyl group has been eliminated are prepared. The two peptides are condensed in a mixture of the solvents described above. The details of the condensation reaction are the same as described above. After the protected peptide obtained by the condensation is purified, all the protecting groups are eliminated by the method described above to give the desired crude peptide. This crude peptide is purified by various known purification means. Lyophilization of the major fraction gives the amide of the desired peptide.

To prepare the esterified peptide, for example, the .alpha.-carboxyl group of the carboxy terminal amino acid is condensed with a desired alcohol to prepare the amino acid ester, which is followed by procedure similar to the preparation of the amidated peptide above to give the ester form of the desired peptide.

The GALP antigen may be provided for direct immunization in its immobilized form. The GALP antigen may also be bound or adsorbed to an appropriate carrier and the complex produced may be provided for immunization. A mixing ratio of the carrier to the GALP antigen (hapten) may be in any ratio of any type, as long as the antibody can be efficiently produced to the GALP antigen. A high molecular carrier conventionally used to produce an antibody to a hapten may be used in a weight ratio of 0.1 to 100 based on 1 of hapten. As such a high molecular carrier, there are used a naturally occurring high molecular carrier and a synthetic high molecular carrier. Examples of the naturally occurring high molecular carrier used are serum albumin from mammals such as bovine, rabbit, human, etc., thyroglobulins from mammals such as bovine, rabbit, etc., hemoglobins from mammals such as bovine, rabbit, human, sheep, etc or KHL hemocyanin.

As the synthetic high molecular carrier, there may be used, for example, a variety of latexes including polymers or copolymers, etc., such as polyamino acids, polystyrenes, polyacryls, polyvinyls, polypropylenes, etc.

For coupling of the hapten and the carrier, a variety of condensing agents can be used. Examples of the condensing agents, which are advantageously employed, are diazonium compounds such as bis-diazotized benzidine through crosslinking of tyrosine, histidine or tryptophan; dialdehyde compounds such as glutaraldehyde, etc. through crosslinking of amino groups therebetween; diisocyanate compounds such as toluene-2,4-diisocyanate, etc.; dimaleimide compounds such as N,N'-o-phenylenedimaleimide, etc. by crosslinking of thiols therebetween; maleimide activated ester compounds by crosslinking of an amino group with a thiol group; carbodiimide compounds by crosslinking of an amino group with a carboxyl group; etc. In the crosslinking of amino groups with each other, one amino group is reacted with an activated ester reagent (e.g., SPDP, etc.) having dithiopyridyl and then reduced to introduce the thiol group, whereas another amino group is introduced with a maleimide group using a maleimide activated ester reagent, and the two groups may be reacted with each other.

(2) Preparation of Monoclonal Antibody

The GALP antigen is administered to warm-blooded animal either solely or together with carriers or diluents to the site where the production of antibody is possible by administration routes such as intraperitoneally, intravenously, subcutaneously, etc. In order to potentiate the antibody productivity upon the administration, complete Freund's adjuvants or incomplete Freund's adjuvants may be administered. The administration is usually carried out once in every 2 to 6 weeks and approximately 2 to 10 times in total. Examples of the warm-blooded animal are monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats, chicken, etc. with mice being preferred for the preparation of monoclonal antibodies.

In the preparation of monoclonal antibodies, from warm-blooded animals, e.g., mice, immunized with the GALP antigen, the animal wherein the antibody titer is noted is selected, then the spleen or lymph node is collected after 2 to 5 days from the final immunization and antibody-producing cells contained therein are fused with myeloma cells to give anti-GALP monoclonal antibody-producing hybridomas. Measurement of the anti-GALP antibody titer in antisera may be made, for example, by reacting a labeled form of GALP, which will be described later, with the antiserum followed by assaying the binding activity of a marker bound to the antibody. The fusion may be operated, for example, by the known Kohler and Milstein method [Nature, 256, 495 (1975)]. Examples of fusion accelerators are polyethylene glycol (PEG), Sendai virus, etc., of which PEG is preferably employed. Examples of the myeloma cells are NS-1, P3U1, SP2/0, AP-1, etc. In particular, P3U1 or the like is preferably employed. A preferred ratio in count of the antibody-producing cells (spleen cells) to the myeloma cells used is within a range of approximately 1:1 to 20:1. When PEG (preferably, PEG 1000 to PEG 6000) is added in a concentration of approximately 10 to 80% followed by incubation generally at 20 to 40.degree. C., preferably at 30 to 37.degree. C. generally for 1 to 10 minutes, an efficient cell fusion can be carried out.

Various methods can be used for screening of the anti-GALP antibody-producing hybridomas. Examples of such methods include a method which comprises adding the hybridoma supernatant to a solid phase (e.g., microplate) adsorbed with GALP or its derivatives, or partial peptides thereof directly or together with a carrier, then adding an anti-immunoglobulin antibody (when mouse cells are used for the cell fusion, anti-mouse immunoglobulin antibody is used) labeled with a radioactive substance, an enzyme or the like, or Protein A and detecting the anti-GALP monoclonal antibody bound to the solid phase; a method which comprises adding the hybridoma supernatant to a solid phase adsorbed with an anti-immunoglobulin antibody or Protein A, adding GALP labeled with a radioactive substance, an enzyme, etc. and detecting the GALP monoclonal antibodies bound to the solid phase; etc. Screening and plating of the anti-GALP monoclonal antibodies can be performed generally in a medium for animal cells (e.g., RPMI 1640) containing 10 20% fetal calf serum and supplemented with HAT (hypoxanthine, aminopterin and thymidine). The antibody titer in the hybridomas culture supernatant can be assayed as in the assay for the antibody titer in the antisera described above.

Separation and purification of the anti-GALP monoclonal antibody can be carried out by methods applied to conventional separation and purification of immunoglobulins, as in the conventional methods for separation and purification of polyclonal antibodies (e.g., salting-out, alcohol precipitation, isoelectric point precipitation, electrophoresis, adsorption and desorption with ion exchangers (e.g., DEAE), ultracentrifugation, gel filtration, or a specific purification method which involves collecting only an antibody with an activated adsorbent such as a GALP antigen-binding solid phase, Protein A, Protein G, etc. and dissociating the binding to obtain the antibody and the like).

As described above, the antibody of the present invention can be produced by culturing hybridoma cells in a warm-blooded animal in vivo or in vitro and collecting the antibody from the body fluids or culture.

The antibody specifically reacting with the partial peptides in the N-terminal region of GALP or its derivatives can be produced in a similar manner to the methods described above; alternatively, the antibody can also be produced by publicly known methods, e.g., by the method described in Published Japanese Patent Application KOKAI No. 2000-157273.

The antibody of the present invention can quantify efficiently human GALP, rat GALP and porcine GALP or derivatives thereof.

Hereinafter, applications of the antibody of the present invention including the method of quantifying GALP or its derivatives (immunoassay), etc. are described in detail.
 


Claim 1 of 8 Claims

1. An isolated antibody specifically reacting with the partial peptide in the C-terminal region consisting of the 44.sup.th 53.sup.rd amino acid sequence in the amino acid sequence represented by SEQ ID NO:2.

 

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

 

 

     
[ Outsourcing Guide ] [ Cont. Education ] [ Software/Reports ] [ Training Courses ]
[ Web Seminars ] [ Jobs ] [ Consultants ] [ Buyer's Guide ] [ Advertiser Info ]

[ Home ] [ Pharm Patents / Licensing ] [ Pharm News ] [ Federal Register ]
[ Pharm Stocks ] [ FDA Links ] [ FDA Warning Letters ] [ FDA Doc/cGMP ]
[ Pharm/Biotech Events ] [ Newsletter Subscription ] [ Web Links ] [ Suggestions ]
[ Site Map ]