GB2062644A

GB2062644A - Glucagon fragment and utility hereof

Info

Publication number: GB2062644A
Application number: GB8033248A
Authority: GB
Original assignee: Toyo Jozo KK
Current assignee: Toyo Jozo KK
Priority date: 1979-10-16
Filing date: 1980-10-15
Publication date: 1981-05-28
Also published as: FR2467841B1; NL8005703A; DE3039122A1; GB2062644B; SE8007074L; JPS5657753A; FR2467841A1

Abstract

Known glucagon fragments are not effective in producing antibodies against glucagon but the problem has been overcome using the glucagon fragment (16-29) of formula [I]: H-Ser-Arg-Ala-Gln-Asp-Phe-Val- Gln-Trp-Leu-Met-Asn-Thr-OH which peptide may be conjugated with a protein such as albumin. The conjugate may be employed to prepare antibodies against the peptide of formula [I] by administering the conjugate to a mammal to sensitise the mammal to the peptide. The antibody is also active against glucagon (1-29) and therefore may be employed to determine glucagon (1-29) by contacting a glucagon- containing sample with the said antibody.

Description

SPECIFICATION Glucagon fragment and utility thereof This invention relates to a glucagon fragment, to a conjugated form of the fragment and a protein, to a process for preparation of a specific antibody using the conjugate and tq a method of determining glucagon employing this antibody.

Glucagon is a hormone which acts on carbohydrate metabolism. It is known as a 1-29 peptide of the pancreas of the formula: 123456 H-His-Ser-Gln-Gly-Thr-Phe- 7 8 9 10 11 12 13 Th r-Ser-Asp-Tyr-Ser-Lys-Tyr- 14 15 16 17 18 19 20 Leu -Asp-Ser-Arg-Arg-Ala-Gln- 21 22 23 24 25 26 27 Asp-Phe-Val -Gln -Trp-Leu-Met- 28 29 Asn-Thr-O H [Il] [hereinafter designated as glucagon(1-29)]. The determination of its blood level is very important for clinical assay.

Radioimmunoassay, fluorescent immunoassay, enzyme immunoassay and similar assay methods based on immune reaction have been employed as the major assay methods for trace amounts of glucagon. Therefore an antibody specific to glucagon is required. However, specific antibody can only be obtained incidentially by using a combination of glucagon (1-29) as a hapten and bovine serum albumin (BSA). Moreover reproducibility cannot be expected.A few examples of the production of antibody using a combination of a fragment of glucagon(1-29) as a hapten, for example a glucagon fragment consisting of a peptide of the 1 8th to 29th amino acids in glucagon [hereinafter designated as glucagon( 18-29)], a glucagon fragment consisting of a peptide of the 1 9th to 29th amino acids in glucagon [hereinafter designated as glucagon(19-29), Japanese Patent Publication No.53-99320] or a glucagon fragement consisting of a peptide of the 1 5th to 29th amino acids in glucagon [hereinafter designated as glucagon (15-29), Japanese Patent Publication No. 54-24868], and BSA have been reported.

We have studied methods for obtaining an effective antibody for an assay based on the immune reaction of glucagon (1-29), and have found that the combination of a protein and a glucagon fragment constiting of a peptide of the 1 6th to 29th amino acids in glucagon produces antibody effectively. This antibody reacts advantageously with labelled glucagon (1-29) and various labelled glucagon fragments in immune reaction.

The present invention therefore provides a peptide of the formula: 16 29 H-Ser-Arg-Arg-Ala-Gln-Asp-P he-Val-Gln-Trp-Leu-Met-Asn-Thr-OH [I] [hereinafter designated as glucagon fragment(1 6-29)]. The invention also provides a conjugate of this peptide and a protein. This conjugate can be employed to prepare antibody against the peptide of formula [I] by administering the conjugate to a mammal to sensitize the mammal to the peptide. This antibody is also active against glucagonl--29) itself, and therefore can be employed to determine glucagon(1-29) by contacting a glucagon-containing sample with the antibody.

Synthesis of glucagon fragment( 1 6-29) can be carried out as follows: one or more amino acids and/or one or more lower peptides are reacted by condensation in the order of the amino acid sequence of formula [I], and the protective group for the reactive group is released at the final stage of the reaction. A lower peptide can contain up to 6, for example up to 4, amino acid residues. Condensation can be carried out by conventional peptide synthesis with repeated attachment and removal of protective groups and condensation. The protective groups for the synthesis of the starting materials or intermediates are conventional protective groups for peptide synthesis and are easily removable by hydrolysis, acid decomposition, reduction, aminolysis or hydrazinolysis.

For example, a-amino groups may be protected conventionally by a benzyloxycarbonyl group such as benzyloxycarbonyl, p-nitrobenzyloxycarbonyl or p-methoxybenzyloxycarbonyl, or an aliphatic oxycarbonyl group such as t-amyloxycarbonyl or t-butoxycarbonyl.

The carboxyl group can be protected by esterification. The ester group can be formed employing an alkanol such as methanol or ethanol, or an araikanol such as benzylalcohol, p-nitrobenzylalcohol, p methoxybenzylalcohol or dichlorobenzylalcohol.

The hydroxy group in serine and threonine may optionally be protected by esterification or etherification. A benzyloxycarbonyl protecting group may be used in the case of esterification. A benzyl protecting group can be employed in etherification.

The amino group of the guanidino group in arginine can be protected by a tosyl or benzyloxy carbonyl group. However it is not always necessary to protect the guanidino group.

After the end of the final step of condensations, these protective groups are split, preferably by acid decomposition such as by hydrogen fluoride in one step removal. Therefore, a reactive side chain group in serine, arginine, aspartic acid or threonine is preferably protected by a protective group which can be easily removed by hydrogen fluoride.

For example, the hydroxyl group in serine and threonine is preferably protected by a benzyl group; the amino group in the guanidino group in arginine is preferably protected by a tosyl group; and the side chain carboxyl group in aspartic acid is preferably protected by a benzyl ester.

Other a-amino and carboxyl groups are preferably protected by a protective group which can be removed by conditions under which side chain protective groups are not removed. For example, an aamino group is preferably protected by a t-butoxycarbonyl or t-amylcarbonyl group which can be removed by trifluoroacetic acid, or by a benzyloxycarbonyl group which can be removed by catalytic reduction. A carboxyl group is protected by a methyl or ethyl ester which can be removed by dilute aqueous sodium hydroxide. The C-terminal carboxyl group is preferably protected by a benzyl ester which can be removed by anhydrous hydrogen fluoride.

Condensation of amino acid(s) and/or peptide(s) can be carried out as follows. For example, an amino acid or peptide having a protected amino group and an activated terminal carboxyl group is reacted with an amino acid or peptide having a free a-amino group and protected terminal carboxyl group. Alternatively, an amino acid or peptide having an activated amino group and protected terminal carboxyl group is reacted with an amino acid or peptide having a free terminal carboxyl group and a protected amino group.

The carboxyl group can be activated by, for example, conversion to acid azide, acid anhydride, acid imidazolide, isoxazolide or active ester, or reaction with carbodiimide or N,N'-carbonyl-diimidazole.

Preferred condensation reactions are the carbodiimide, azide or active ester methods. In the condensation reaction, racemization should carefully be avoided. Preferred methods are the azide method, active ester method using succinimide ester or p-nitrophenyl ester, Wunsch method [Z.

Natufforsch, 216,426(1966)] or Geiger method [Chem.Ber., 103,788 (1970)1. especially a modified Geiger method using N-ethyl, N'-3-di-methylaminopropyl-carbodiimide as a condensation agent.

Thus the peptide [I] carrying protective groups is obtained. These protective groups are split, preferably by acid decomposition such as a one-step removal by hydrogen fluoride, and finally the product [I] can be obtained. Purification of peptide [I] can be achieved by known column chromatography using a carrier.

Glucagon fragment (1 6-29) of the present invention is bound with a protein to produce a conjugated form for antibody production. Examples of the protein conventionally used are BSA or a modified version of BSA formed by splitting the inner molecular disulfide group by alkaline or sodium laurylsulfate and mercaptoethanol treatment.

An example of a conjugation reagent for glucagon and protein is a polyfunctional conjugation reagent such as glutaraldehyde or 3-(2'-benzothiazolyl-dithio) propionate succinimide ester (Japanese Patent Appln. No. 53-85900, G.B. Specification No. 2029825A). These polyfunctional conjugation reagents are selected for functional groups which participate in the bonding of protein.

The ratio of glucagon fragment (1 6-29) to protein such as BSA is one or more moles of glucagon fragment (16-29) per mole of protein such as BSA, preferably ten moles of glucagon(1 6-29) per mole of BSA. In the binding reaction, for example the necessary amount of glucagon fragment(1 6-29) is added to an aqueous medium of pH 7-8, and a polyfunctional conjugation reagent is added thereto.

Reaction proceeds under cooling or at ambient temperature for 1 4 hours. After purifying by gel filtration, BSA is added and reacted at ambient temperature for 1-4 hours. A conjugated product of glucagon fragment(1 6-29) and BSA is obtained after purification, such as by gel filtration and dialysis, and if necessary is stored after lyophilization.

The conjugated form of glucagon fragment(1 6-29) and protein is administered to mammals such as rabbit, rat, guinea pig or mouse to sensitize. For example, the conjugated substance is suspended in Freund's complete adjuvant and is subcutaneously injected into a guinea pig at a ratio of 30-70 p of glucagon(1 6-29) for 4 7 times in 2 weeks intervals. Antiserum is obtained from the blood of the sensitized animals by a conventional procedure such as blood collection and centrifugation.

The antiserum contains a higher concentration of the specific antibody and can be stored in a freezer, and can be used by aliquot dilution. The specific antibody immunologically binds not only labelled glucagon(1-29) but also to various labelled glucagon fragments such as glucagon fragment(1 6-29), giucagon fragment( 1 7-29) and glucagon fragment( 19-29).

As explained above, the glucagon fragment( 1 6-29) of the present invention is useful for antibody production in a conjugated form with a protein. The thus obtained antibody has excellent specific binding in immune reaction. This immune reaction can be advantageously applied for radio immunoassay, fluorescent immunoassay and enzyme immunoassay of glucagon( 1-29) in vivo.

It has been reported (Japanese Patent Publication No. 55-39702) that the glucagon fragment( 1 5-29) has an activity with a conjugate protein. However, no examples of glucagon fragment (1 6-29) are mentioned. Moreover the glucagon fragment( 1 6-29) of the present invention is more reactive and specific as compared with the fragment( 5-29) due to the N-terminal amino acid serine of glucagon fragment(1 6-29).

The abbreviations in the description, claims and abstract mean: Ser; L-serine Val; L-valine Arg; L-arginine Trp; L-tryptophane Ala; L-alanine Leu; L-leucine Gln; L-glutamine Met; L-methionine Asp; L-aspartic acid Asn; L-asparagine Phe; L-phenylalanine Thr; L-threonine BOC; t-butoxycarbonyl OBzl; benzyl ester AOC; t-amyloxycarbonyl OSU; N-hydroxysuccinimide ester Z; benzyloxycarbonyl ONP; p-nitrophenyl ester Bzl; benzyl TFA; trifluoroacetic acid Tos; tosyl NMM; N-methylmorphine OMe; methyl ester Et2O; diethyl ether THF; tetrahydrofuran DMF; dimethylformamide HOBT; 1 -hydroxybenzotriazole WSC; N-ethyl, N'-3-dimethylaminopropylcarbodiimide The following Examples illustrate the present invention. Percentages are by weight.In the Examples, the following carrier and developing solvent for thin layer chromatography [TLC] are used: |TLC| Carrier: Merck cellulose Art 571 6.

Developer: 1: butanol - pyridine - acetic acid - water (15:10:3:11) 2: upper layer of butanol - pyridine - acetic acid - water (10:3:0.1:11) 3: upper layer of butanol - pyridine - acetic acid - water (5:3:0.1:11) Carrier: Merck silica-gel Art 5721.

Developer: 4: chloroform - methanol - acetic acid (95:5:3) 5: chloroform - methanol - acetic acid (80:25:2) 6: chloroform - ethanol - ethyl acetate (5:2:5) 7: chloroform - methanol - acetic acid (85:15:5) EXAMPLE 1 Production of H-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln~Trp-Leu-Asn-Thr-OH [glucagon fragment( 16-29)]: Anisole (6 ml), ethanedithiol (0.94 ml, 10 mM) and skatole (131 mg, 1 mM) were added to 2.5 g (1mM of BOC-Ser(Bzl)-Arg(Tos)-Arg(Tos)-Ala-Gln-Asp(OBzl)-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr(Bzl)-OBzl.

The mixture was added to anhydrous hydrogen fluoride (HF, 20 ml), stirred at 0 C for one hour, and HF was rapidly distilled off in vacuo. Et2O (100 ml) was added to the residue to separate the precipitate. The precipitate was dissolved in 50-80% acetic acid (100 ml) and centrifuged. The supernatant was freeze dried to obtain a powder (1.22 g). The powder was dissolved in 8 M aqueous urea (150 ml) and adjusted to pH 9 by adding aqueous ammonia. The solution was charged onto a column of carboxymethyl cellulose (4 x 20 cm) equilibrated with 8 M aqueous urea. After 30 minutes, the column was washed completely with water to remove urea, and eluted by the continuous concentration gradient procedure using water (1 lit.) adjusted with pH 4.5 by acetic acid to aqueous 0.2 M ammonium acetate (pH 4.5, 1 lit.).Thereafter the column was eluted with aqueous 0.5 M ammonium acetate (pH 4.5, 500 ml), and finally eluted with aqueous 0.5 M ammonium acetate (pH 4.5) containing 8 M urea to fractionate the active fractions II (Nos. 80--1 10 tubes), IlI(Nos. 120-1 70 tubes), IV(Nos.

171-230 tubes) and V(nos. 231-290 tubes) in 7 ml fractions per tube, and these active fractions were freeze dried.

The dried powder prepared from active fraction V was dissolved in 50% acetic acid (50 ml).

Thioglycolic acid (2.5 ml) was added to the solution which was allowed to stand at 450C for 21 hours.

Urea (24 g) was dissolved in the solution. The insoluble part was removed by filtration. The filtrate was charged onto a column (4.2 x 104 cm) of Sephadex LH-20 ("Sephadex" is a Registered Trade Mark) and eluted with 50% acetic acid. The fractions from tubes nos. 62-79, 7 ml per fraction, were collected and freeze dried to obtain a powder (150 mg). TLC: Rf1 = 0.50, Rf2 = 0.34.

The lyophilized powder obtained from active fraction IV was dissolved in 50% acetic acid (20 ml).

Thioglycolic acid (1 ml) was added and the solution was allowed to stand at 450C for 21 hours. Urea (9.6 g) was dissolved in this solution and insoluble material was removed by filtration. The filtrate was charged onto a column (3.3 x 120 cm) of "Sephadex" LH-20 and eluted with 50% acetic acid. The active fractions, corresponding to tubes nos. 32-37, 7 ml per fraction, were collected and freeze dried to obtain a powder 10 mg).

TLCRfi=0.50 Amino acid analysis: Asp 2.03 (2), Thr 0.96 (1), Ser 0.87 (1), Glu 2.07 (2), Ala 1.02(1),Val0.91 (1), Met 0.95 (1), leu 1, Phe 0.95 (1), Arg2.19(2),Trp 1.04 (1).

The lyophilizates obtained from active fractions Ill and II were treated and subjected to column chromatography by the same procedure as for active fraction IV to obtain a powfer (130 mg) (TLC: Rf, = 0.50) from the fractions of tubes nos. 31-36 for active fraction Ill and a powder (50 mg) (TLC: Rf1= 0.50) from the fractions of tubes nos. 39-44 for active fraction II.

EXAMPLE 2 Conjugated form of glucagon fragment(1 6-29) and BSA or modified BSA: (1) Conjugated form of glucagon fragment( 16-29) and BSA: Glucagon fragment(1 6-29) (15 mg) was dissolved in 0.0001 N sodium hydroxide and adjusted to pH 8.0 to prepare a solution (20 ml). Aqueous 25% glutaraldehyde (1.5 ml) was added to the solution and reacted at room temperature for 2 hours. The reaction mixture was passed through a column (5 x 50 cm) of "Sephadex" G-1 5 and the fraction passing through was collected [a solution containing about 11 mg of derivative wherein glutaraldehyde has reacted with N-terminal of glucagon fragment( 6-29)].

BSA (42 mg, a product of Sigma Co.) was added thereto and reacted at room temperature for 2 hours. The reaction mixture was dialysed in distilled water. The dialysate was lyophilized to obtain a conjugated form of glucagon fragment(1 6-29) and BSA [bound molar ratio of BSA:glucagon fragment( 16-29) = about 1:10]. Yield: 48 mg.

(2) Conjugated form of glucagon fragment(1 6-29) and modified BSA: Glucagon fragment(1 6-29) (15 mg) was dissolved in 0.0004 N sodium hydroxide and adjusted to pH 8.0 to prepare a solution (20 ml). 0. 1'% Dimethylformamide solution of 3-(2'-benzothiazolyldithio) propionate succinimide ester (1.575 ml) was added to the solution and reacted at 50C for one hour.The reaction mixture was passed through a column (5 x 50 cm) of "Sephadex" G-1 5 packed with acetate buffer (pH 5) containing dimethylformamide to collect the fraction passing through [a solution containing a derivative (11.5 mg) wherein 3-(2'-benzothiazolyl-dithio) propionyl group was introduced in N-terminal of glucagon fragment (1 6-29)]. The solution was adjusted to pH 7.0 and the modified BSA (45 mg), previously treated overnight with sodium lauryl sulfate and mercaptoethanol at room temperature, was added thereto and reacted at room temperature for one hour. The reaction mixture was dialysed in distilled water and lyophilized to obtain a conjugated form of glucagon fragment (16-29) and modified BSA [bound ratio: modified BSA: glucagon fragment(1 6-29) = about 1:10].

Yield: 52 mg.

The above glucagon fragment(1 6-29) was replaced by glucagon(1 7-29), glucagon(1 9-29) and glucagon( 1-29) (product of Stigma Co.), and treated in the same way to obtain a conjugated form of BSA or modified BSA and glucagon( 17-29), glucagon( 19-29) or glucagon( 1-29).

EXAMPLE 3 I. Antibody production using conjugated form of glucagon fragment(1 6-29) and BSA: (1) The conjugated form of glucagon fragment( 1 6-29) and BSA (1 mg) [248y as glucagon (16-29)] was dissolved in 1.0 ml of 10 mM phosphate buffer (pH 7.2) (containing 0.15 M NaCI).

Freund's complete adjuvant (1.0 ml) was added and mixed thoroughly to obtain an emulsion.

The emulsion (0.5 ml) was injected into the skin of a toe and several subcutaneous parts of the back of a guinea pig. The same amount of emulsion was subcutaneously injected six times with 2 weeks intervals. After 10 days from the final injection, whole blood was collected and allowed to stand for 60 minutes for coagulation. Antiglucagon fragement(1 6-29) serum was obtained by centrifugation at 3000 r.p.m. for 10 minutes.

(2) The conjugated form of glucagon( 1 7-29) and BSA (1 mg), conjugated form of glucagon(19-29) and BSA (1 mg), and conjugated form of glucagon(1-29) and BSA (2.5 mg) obtained in Example 2 were used and treated by the same procedures in Example 3 (1). Antiglucagon (17-29) serum, anti-glucagon (19-29) serum and anti-glucagon(1-29) serum were obtained.

II. Immune reactivity of antiserum: (1) Assay method of immune reactivity: The above antisera were diluted to 200, 400, 800, 3200, 6400, 12800, 25600 and 51200 times with 10 mM phosphate buffer (pH 7.4) (containing 0.25% BSA, 1 mM MgCl2, 0.1% NaN3, 3mM EDTA and 0.15 M NaCI). Each 100 ,ul thereof were reacted with 100 l of a phosphate buffer solution (the same composition as above) of labelled glucagon(1-29) or labelled glucagon fragment [label: p- galactosidase, glucagon fragment: glucagon(1 6-29), glucagon(1 7-29), glucagon(1 9-29)] at 50C for 16 hours. Guinea pig IgG (4y) and anti-guinea pig IgG rabbit serum were added thereto and incubated for one hour at room temperature.A precipitate was collected by centrifugation at 3000 r.p.m. for 10 minutes, and was added to 200 1 of 10 mM phosphate buffer (pH 6.7) (containing 0.1% BSA, 1 mM Mg Cl2, 0.1% NaN3 and 0.15 M NaCI) containing 5 mg/ml of O-nitrophenyl-/3-D-galacto- pyranoside and incubated at 370C for 45 minutes to assay ss-galactosidase activity at 420 nm by colorimetery. Combined ratio to labelled glucagon and labelled glucagon fragment in each dilution of antisera was determined by immune reaction.

(a) Labelled glucagon(1-29): p-Galactosidase labelled glucagon(1-29) [40 pg as glucagon(1-29)] obtained by the method mentioned below was used.

(b) Labelled glucagon fragments: /3-Galactosidase labelled glucagon fragment(1 6-29), p-galactosidase labelled glucagon(1 7-29) and ss-galactosidase labelled glucagon(19-29) prepared by the method below were each used in an amount of about 20 pg.

(2) Result and discussion on immune reactivity: The ratios of combination in immune reactivity calculated by the amount of precipitate for each antisera are shown in the table below for the cases of the 800, 6400 and 51200 times dilutions.

Combination ratios for the other dilutions show resemblance to these results.

3-phenoxybenzyl (+ In the table: +++: combination ratio > 75% ++: combination ratio 50-75% +: combination ratio 25-50% -: combination ratio 0-25% As shown in the table, although glucagon fragment(1 6-29) of the present invention resembles glucagon( 1 7-29) and glucagon( 1 9-29) in its structure, the immune reactivity of antibody in the antisera obtained by injection of each fragment is noticeably different. Antibody produced by glucagon fragment( 1 6-29) of the present invention showed sensitive and advantageous immune reactivity against not only glucagon (16-29) but also glucagon(1 7-29), glucagon(1 9-29), and glucagon (1-29).

These results show that C-terminal specific antibody (antibody titer) can be quantitatively determined by using the glucagon fragment of the present invention.

III. Preparation of labelled glucagon(1-29) and labelled glucagon fragments: (1) Labelled glucagon(1-29): Aqueous 100 mM EDTA (10 ,uI) and 0.1% 3-(2'-benzothiazolyl-dithio) propionate succinimide ester in dimethylformamide (264.4 1) were added to glucagon (1-29) (1 mg) dissolved in 50 mM phosphate buffer (pH 8.0) (0.4 ml) and reacted at 50C for one hour. The reaction mixture was passed through a "Sephadex" G-1 5 column (1.5 x 40 cm) for gel filtration.

TABLE

labelled compounds idrf gpt sddsu gluca ss-galactosidase ss-galactosidase ss-galactosidase ss-galactosidase gon and labelled labelled labelled labelled giucagon glucagon (18-29) glucagon (17-29) glucagon (19-29) glucagon (1-29) fragments antisera for immunity dilution x800 ++ ++ +++ +++ glucagon (16-29) x6400 ++ ++ +++ ++ x51200 + + + + x800 - - + + glucagon (16-29) x6400 - + + + x51200 - - - x800 - - - glucagon (16-29) x6400 - - - x51200 - - - x800 + + ++ ++ glucagon (16-29) x6400 + + + ++ x51200 - - - + The fraction passing through was collected to obtain a solution containing a derivative in which a 3-(2'benzothiazolyl-dithio) propionyl group had been introduced onto the amino group of glucagon( 1-29).

,B-Galactosidase (a product of Boehringer Mannheim GmbH) (2.78 mg) was added to 50 mM phosphate buffer (pH 8.0) (2 ml) containing the derivative (25 y9) of 3-(2'-benzothiazolyldithio)propionyl glucagon(1-29), and reacted at room temperature for one hour. The reaction mixture was passed through a column (1.5 x 90 cm) of "Sephadex" G-1 50 for gel-filtration and the fraction passing through was collected to obtain /3-galactosidase labelled glucagon(1-29) in which the amino group of the glucagon(1-29) had combined with the thiol group in p-galactosidase.

(2) Labelled glucagon fragments: Aqueous 100 mM EDTA (10 ,ul) and 0.1% 3-(2'-benzothiazolyl-dithio) propionate succinimide ester in dimethylformamide (625 ,ul) were added to glucagon fragment(1 6-29) (0.5 mg) dissolved in 50 mM phosphate buffer (pH 8.0) (0.4 ml) and reacted at 50C for one hour. The reaction mixture was passed through a "Sephadex" G-1 5 column (1.5 x 40 cm) for gel-filtration. The fraction passing through was collected to obtain a solution containing a derivative in which the 3-(2'-benzothiazolyldithio) propionyl group had been introduced onto the N-terminal amino group of glucagon fragments(1 6-29).

p-Galactosidase (6.19 mg) was added to 50 mM phosphate buffer (pH 8.0) (2 ml) containing the 3-(2'-benzothiazolyl-dithio) propionyl glucagon fragment(1 6-29) (25 g), and reacted at room temperature for one hour. The reaction mixture was passed through a column (1.5 x 90 cm) of "Sephadex" G-1 50 for gel-filtration. The fraction passing through was collected to obtain a galactosidase labelled glucagon fragment(1 6-29) in which the N-terminal amino group of glucagon fragment( 16-29) has combined with the thiol group in ,l3-galactosidase.

The glucagon fragment( 16-29) was replaced by glucagon( 1 7-29) and glucagon( 19-29) and treated by the same procedure in the above to produce a ,B-galactosidase labelled glucagon( 17-29) and p-galactosidase labelled glucagon(1 9-29).

Claims

1. A peptide of the formula [I]: H-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Va l-Gln-Trp-Leu-Met-Asn-Thr-OH [I] in which Ala, Arg, Asn, Asp, Gln, Leu, Met, Phe, Ser, Thr, Trp and Val are as hereinbefore defined.

2. A conjugate of a protein and a peptide of formula [I] as defined in claim 1.

3. A conjugate according to claim 2 wherein the protein is albumin or a modified form thereof.

4. A conjugate according to claim 2 substantially as hereinbefore described in Example 2(1) or 2(2).

5. A process for the preparation of antibody against a peptide of formula [I] as defined in claim 1, which process comprises administering a conjugate as claimed in any one of claims 2 to 5 to a mammal to sensitize said mammal to said peptide.

6. A process according to claim 5 substantially as hereinbefore described in Example 31(1).

7. A method of determining glucagon which is not practised on the human or animal body, which method comprises contacting a glucagon-containing sample with antibody which has been prepared by a process as claimed in claim 5 or 6.