JPS5835156A - Fluorescent peptide and determination of human beta-interferon using the same - Google Patents

Abstract

NEW MATERIAL:The peptide of formulaI(R is fluorescent dye). EXAMPLE:Fluorescein.isothiocyanate( FITC )-labeled-Met-Ser-Tyr-Asn-Leu-Leu- Gly-Phe-Leu-Gln-Arg-Ser-Ser-OH. USE:Determination of human beta-interferon by fluorescent depolarization. PROCESS:The novel peptide of formula II is labeled with a fluorescent dye[e.g. FITC, tetramethylrhodamine.isothiocyanate (TRITC), etc.]to obtain the fluorescent peptide of formulaI. The labeling reaction proceeds in a proper buffer solution (e.g. a borate buffer solution) under alkaline conditions, preferably at 8-12pH usually at 0-40 deg.C, preferably at 0-20 deg.C, and completes within about several minutes - 48hr.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel fluorescently labeled peptide and a novel human β-interferon 0IIB method using the same.

The fluorescently labeled peptide of the present invention is represented by the following general formula.

R-Met-5er-Tyr-Asn-Leu
-Leu -Gly-Ph@-Leu-Gln-Arg
-8er -ser filOH [In the formula, R represents a fluorescent dye. ] The present inventors discovered the fact that the fluorescently labeled peptide represented by the above general formula (1) specifically immunoreacts with human β-type interferon antibodies and forms an immune complex, and this fluorescently labeled peptide. We discovered the fact that human β-type interferon can be measured by using a peptide in a fluorescence immunoassay. The present invention was completed based on these findings.

In this specification, when amino acids, peptides, protecting groups, active groups, etc. are indicated by abbreviations, IUPAC,
The IUB regulations or common symbols in the field shall be followed, and examples are given below.

Arg! Fulginine Asn H Asparagine Ser; Serine Glni Glutane Met; Medeonine Leu; Leucine Pbs+ s Phenylalanine Tyr i Tyrcine (jIF; Glycine 2; Carbobenzoxy group Su i Succinic acid indo group Tos i P) Luenesulfoel group Boci 8th The fluorescently labeled peptide represented by the general formula (1) of the butoxycarbonyl basic invention can be produced by labeling the peptide represented by the following general formula (21) with a fluorescent dye.

The fluorescent dye used above and combined with the peptide of general formula (2) and incorporated as KR in the fluorescently labeled peptide of general formula (1) of the present invention is selected from conventional fluorescent labeling reagents. Specific examples include fluorescein inthiocyanate (FITC), tetramethylrhodamine inthiocyanate (TRITC), substituted rhodamine inthiocyanate (XRITC), rhodamine B isothiocyanate, dichlorotriazine fluorescein (DTAF), etc. The amount of the fluorescent dye to be used is not particularly limited, but it is usually 1 to 10 times the mole, preferably 2 to 4 times the mole of the peptide of general formula (2).

The labeling reaction of the peptide of the above general formula (2) with a fluorescent dye can be carried out, for example, in an appropriate buffer at alkaline, preferably pH
It will be held from 8 to 12. Buffers that exhibit properties within the above pH range are not particularly limited and may be selected from a wide range, such as borate buffer, bicarbonate buffer, glycine buffer, barbital buffer, Tris buffer, Amediol buffer, Ringer's buffer, etc. can be used. or,
If the peptide and/or fluorescent dye of the general formula () to be subjected to the reaction does not dissolve in the above buffer, for example, methanol,
The above labeling reaction can be carried out by adding a common solvent capable of dissolving the double peptide and fluorescent dye, such as ethanol, acetone, and DMF (dimethylformamide), into the reaction system.

The above labeling reaction is usually carried out at 0°C to 40°C, preferably θ°C to 2°C.
The process proceeds at 0°C and ends in about a few minutes to 48 hours.

In this way, a fluorescently labeled peptide represented by general formula (1) of the present invention is obtained. This can be done using conventional separation means, e.g. extraction,
Single-nI! by partitioning, column chromatography, gel filtration, etc. ! be done.

The peptide represented by the general formula (2) used as a raw material in the labeling reaction is a new compound, and can be produced, for example, by the method shown in Reaction Scheme-1 below.

Reaction scheme-1> A-Leu-Gln-B (31 A-Leu-G17-Phe-Leu-Gln-Arg
-8@r-8er-OH2 OH A-Tyr-A@n-Leu-Leu-Gly-Phe
-Leu-Gln-Arg-5er-8er-OHa#
↓ H-Tyr-Asn-Leu-Leu-Gly-Pb@
-Leu-Gln-Arg-5er-8er-OR(I
sA-Met-8er-Tyr-Asn-Leu-Le
u-Gly-Phc-Leu-Gln-Arg-8er
-8er-OHα9↓ H-Met-8er-Tyr-Asn-Leu-Leu
-Gly-Phe-Leu-Gln-Arg-5er-
8er-OH (21 [in each of the above formulas, A is amine) group protecting group, B an active group of hydroxyl group or carboxyl group, and C a protecting group of guanidino group of arginine, respectively. ] The reaction between peptide (3) and peptide (4) can be carried out in the presence of a solvent. As the solvent, there are various types of 401 which can be used in the usual begutide condensation reaction, such as anhydrous or hydrous dimethylformand, dimethylformand, etc.

Methyl sulfoxide, pyridine, tetraroform, dioxane, dichloromethane, tetrahydro7ran 1 ethyl acetate% N-methylpyrrolidone, hexamethylphosphate triand, or a mixed solvent thereof can be used. The ratio of peptide (3) to peptide (41) is not particularly limited and can be selected as appropriate within a wide range, but it is usually used in an equal to 5 times amount, preferably in an equal to 1.5 times amount. The reaction temperature is generally about -40 to about 60°C, preferably about -20 to about 40°C, and the reaction time is generally from several minutes to 80 hours at 1°C.

Reaction between peptide (6) and amino acid (7), peptide (
The reaction between 9) and peptide (to), the reaction between peptide (to) and peptide 0, and the reaction between peptide (to) and peptide (2) are all based on the above-mentioned peptide (3) and peptide (4).
) can be carried out in the same manner as the reaction with

After the completion of the above condensation reaction, the binding groups of Vepti EQn(DA and C) obtained can be removed by a conventional method. Examples of such a method include a reduction method using metallic sodium in liquid ammonia.

For metallic sodium, the reaction immersion liquid turns permanent blue.8
It is used in such an amount that it remains colored for about 0 seconds to 10 minutes. This reduction is usually carried out at about -40 to -7 et.

Peptide (4) used in the above reaction scheme-I
is produced, for example, by the method shown in reaction line 1 formula-2 below.

Reaction scheme-2>H-5et-OR' tS A-8er-B A-8er-8er-
OR'a seconds (company) H-8er-Bar-ORn ■ A-Arg-5er-8er-OR (21A-Argi
er-5er-OH@41H-Arg-8er-8er
-OH (41 [In each of the above formulas, R1 represents a lower alkyl group. A, B and C are the same as above.] The reaction between the asanoic acid (2) and the heptanoacid (to) is the reaction of the peptide ( The reaction can be carried out in the same manner as the reaction between 3) and peptide (4).

The binding group of A of Peptide Co., Ltd. can be removed by conventional methods. Such methods include, for example, reductive methods (e.g., hydrogenation using palladium or palladium black catalyst, hydrogenation using metallic sodium in liquid ammonia). reduction), acidolysis (
Examples include acidolysis using strong acids such as trifluoroacetic acid, hydrogen fluoride, methanesulfonic acid, and hydrobromic acid. In the case of hydrogenation using a catalyst, it is usually carried out at a temperature of 0 to 40°C under a hydrogen pressure of 1 atmosphere. The amount of catalyst used is usually 100
The reaction time may be about 4 to 1 f, and the reaction is generally completed in about 1 to 48 hours. In addition, in the case of acidolysis, usually 0
~80'Cm degrees, preferably 0~20°C,
Generally, the reaction is completed in about 15 minutes to about 1 hour. The amount of acid to be used is usually 5 to 10 times the amount of the raw material compound. Further, the reduction with metallic sodium in liquid ammonia can be carried out under the same conditions as in 1 above for the deprotection reaction of the peptide αη.

The reaction between Peptide (Inc.) and the amino acid @ can be carried out in the same manner as the reaction between peptide (3) and peptide (4).

The reaction of hydrolyzing peptide 123t to obtain the peptide is carried out using water, methanol,
Normally 20-50℃ in a solvent such as ethanol or dioxane
The process takes about 80 minutes to 8 hours.

As a method for removing protecting group A from peptide (goods),
Examples include methods such as hydrogenation and acidolysis using catalysts such as palladium and palladium black.

These methods outside the prefecture are the same as the nine conditions already mentioned◇
Peptides (6), (9), a2 and t1!9 are produced by removing the protecting group A from peptides (51, (81, αυ and a4), respectively, in the same manner as described above.

Also, the peptide (
2) is produced, for example, by the following reaction scheme-8.

<Reactivity 1 formula-8> H-Leu-OR@A-Asn-B A-Asn-Leu-
OR'@ Koichiichiin H-Asn-Lau-OR@ A-Tyr-Asn-L@u-OR' (!.

↓ A-Tyr-Asn-Leu-OHC11) ↓ A-Tyr-Asn-Lea-B Q3 [In the above, R', A%B and C are the same as above. ] Amino acid (c)
The reaction between peptide (3) and amino acid (2) and the reaction between peptide (2) and amino acid (2) are as follows:
You can increase your batting power in the same way as the reaction.

The removal of the protecting group A from the peptide field is carried out in the same manner as the removal of the protecting group A from the peptide (2) above.
The hydrolysis reaction of can be carried out in the same way as the hydrolysis reaction of Peptedro.

Preferred methods for obtaining peptide a3 from peptide (conversion) include, for example, the mixed acid anhydride method and the azide method. The mixed acid anhydride method is carried out using an alkyl halocarboxylic acid in the presence of a suitable basic compound. Examples of the alkyl halocarboxylic acid include methyl chlorotetraformate, ethyl bromoformate, ethyl chlorotetraformate, etc. ethyl taformate,
Ethyl bromoformate, isobutyl bromoformate, etc. can be used. Examples of basic compounds include triethylamine, triethylamine, pyridine, and dimethylamine IJ.
y, N-methylmorpholine, 1. b-Diazabishifu palm [
4,8,0] nonene-6 (DBN), 1,6-diazabicyclo[6,4,0) kundecene-6 (DBU), violet,
4-Diazabicyclo[2,2,rioctane (DABCO)
) Inorganic bases such as potassium carbonate, sodium carbonate, potassium hydrogen carbonate, and sodium hydrogen carbonate can be used.As the solvent, solvents commonly used in the mixed acid anhydride method, specifically methylene chloride, Halogenated hydrocarbons such as chlorine, chlorine, and Schiff's tetraethane, aromatic hydrocarbons such as benzene, toluene, and xylene, ethers such as diethyl ether, dihydrofuran, and dimethoxyethane, methyl acetate, and ethyl acetate. fathom esters, N, N
- Non-polar polar solvents such as dimethyl formamide, dimethyl sulfoxide, and hexamethyl phosphate trioxide can be used. The reaction is generally carried out at -20 to 100"C, preferably -20 to 60"C for 5 minutes to 10 hours, preferably 5
It takes between minutes and 2 hours.

In the azidation method, the activated carboxyl group, e.g.
The carboxyl group activated with ttf/ttf/tyl alcohol, ethyl alcohol, or benzyl alcohol is reacted with hydrazine hydrate in a suitable solvent such as dioxane, dimethyl formand, dimethyl sulfoxide, or a mixed solvent thereof. It will be done soon. In this reaction, hydrazine hydrate is usually used in an amount of 6 to 20 times the amount of the activated carboxyl group, preferably in an amount of Is to 10 times the amount of activated carboxyl group. The reaction is usually 6G”C
The following steps are preferably carried out at -20 to 80°C.

In this way, a compound (hydrazine derivative) in which the carboxyl group of the terminal amino acid is substituted with hydrazine can be produced. A compound in which the terminal amino acid carboxyl group moiety is substituted with azide can be produced by reacting the hydrazine derivative obtained above with a nitrous acid compound in a suitable solvent in the presence of an acid. Hydrochloric acid is usually used. Examples of the solvent include dioxane, dimethyl formand, dimethyl sulfoxide, and a warm solvent thereof. Examples of the nitrite compound include sodium nitrite, isoamyl nitrite, and nitrosyl chloride, and the amount of the nitrite compound is usually 1 to 2 times the amount of the hydrazine derivative, preferably 1 mole. It is preferable to use 5 times the molar amount.

The reaction is usually carried out at -20 to 0°C, preferably -20 to -10°C.
The reaction is generally completed in 5 to 10 minutes at 1°C.

The peptide of general formula (2) produced as described above is single-purified from the reaction mixture by conventional peptide separation methods such as extraction, partitioning, column chromatography, etc.

The fluorescently labeled peptide represented by the general formula (1) of the present invention has a function as a hapten for human β interferon, and has the feature of forming an immune complex with an antibody for human β interferon. Therefore, the scissors fluorescently labeled peptide is
Utilizing this, human β-type interferon can be measured by fluorescence immunoassay, and the present invention also provides a method for measuring such human β-type interferon.

Conventionally, interfines have been selected using biological measurement methods that are related to their physiological activity [FinLe et al.
r, N, B, in Interferon and In
Terferon Inducers (ed, F
inter.

N, B,) 186-170 (North-Hollan
d.

Amsterdam, 197B) lo i.e.
The interferon titer is determined by directly or indirectly quantifying the degree of cell death caused by the virus using living cells and a living virus that infects them. However, in this biological measurement method, since the material used for measurement is a living organism, it is unavoidable that it is unstable and that there is a large dispersion between measured values. Furthermore, if the test sample is contaminated with eyelid matter having an antiviral effect other than interferon, the quantification of interferon by this method becomes completely meaningless. Furthermore, according to the method,
It is impossible to selectively measure human β interferon. In addition, the method requires several days for measurement, which, together with complicated operations, does not meet the demands of the medical community.

The method of the present invention, unlike the above-mentioned conventional method, allows human β-type interferon to be measured simply, quickly, and at a high rate, and is therefore particularly preferable as it meets the current requirements of the industry.

Furthermore, although the fluorescent light depolarization method has been well known for some time, there has never been any case where this method has been used to treat interferon. This is because it is difficult to produce purified fluorescent 4111wt interferon, and even if it is possible, if a labeling substance with such a large molecular weight is used, the change in the polarization degree (P value) is small and sufficient. This is because it is not possible to obtain sufficient quantitative sensitivity. In contrast, the fluorescently labeled peptide of the present invention is effective as a labeling substance that can quantify β-lid interferon (by increasing the change in the degree of polarization (P value) and with sufficient quantitative sensitivity). With the development of photolabeled peptides, it became possible for the first time to quantify human β-type interferon using the above-mentioned fluorescence resolution method.

The method for quantitatively measuring water chestnut β-gourd interferon according to the present invention will be described in detail below.

The method of the present invention is fundamentally different from conventional fluorescence depolarization methods in its measurement procedure and operation. The method is carried out using human β-type interferon as a standard antigen, an antibody against it, and a fluorescently labeled peptide of the present invention. ζThe standard antigen used here is live (Nati MaC).
) Human β-type interferon itself or the peptide of general formula (2) which is antigenically equivalent to it can be used. When using the peptide of general formula 12), human β-type interferon against antibodies can be used. By determining the cross-reactivity with the peptide of general formula (2), it is possible to easily calculate the interferon titer of an unknown sample. The use of the peptide of the general formula (2) is preferable, especially considering the labor, cost, and operation required to obtain a purified product as a standard antigen. Also, as the interferon antibody used in the method of the present invention, Although all antibodies against human β-type interferon and the peptide of general formula (2) can be used, antibodies specific for human β-type interferon obtained by using the peptide of general formula (2) as a hapten can be used. It is better to use high-quality antibodies. The antibody is produced by using the peptide of general formula (2) as a hapten and reacting it with a carrier in the presence of a hapten-carrier binding reagent to produce a human β-type interferon antigen consisting of a peptide-carrier complex. (411 Application No. 66-47842).

In the production of antigens for producing the above-mentioned antibodies, a wide range of high-molecular natural or synthetic proteins that are commonly used in the production of antigens can be used as carriers, such as horse serum albumin, bovine serum albumin, rabbit serum albumin, etc. , human serum albumin, animal serum albumin such as sheep blood albumin, horse serum globulin, bovine serum globulin,
Animal serum globulins such as rabbit serum guprine, human serum guprine, and sheep serum gupulin; animal teroglobulins such as equine teroglobulin, bovine teroglobulin, rabbit teroglobulin, human thylogp-purin, and sheep teroglobulin; , animal hemoglobulins such as horse hemoglobin, bovine hemoglobulin, rabbit hemoglobulin, human hemoglobulin, and ovine hemoglobulin, animal hemocyanins, and flea white matter extracted from roundworms (Ascaris extract, JP-A-Sho). 158-18434), polylysine, polyglutamic acid, lysine-glutanic acid copolymers, and copolymers containing lysine or ornithine.

As the hapten-carrier binding reagent, those commonly used in the preparation of antigens can be widely used.
Alipid dialdehydes such as 1, malondialdehyde, geltaraldehyde, succinic aldehyde, and azibaldehyde, cross-linking thiol groups with thiol groups,
For example, N, go0-phenylene dimaleimide, N, N
'-m-phenylene simaleimide-based simalimide compounds, which cross-link amino groups and thiol groups, such as metamaleimidobenzoyl-N-hydroxysuccinidoester% 4-(maleicetomethyl)-cyclohexane-1-carboxyl -N-Hydroxysuccinimide ester compound, a reagent used in the usual peptide bond-forming reaction to form an amide bond between an amino group and a carboxyl group, such as N-hydroxysuccinimide ester, etc. , N-dicyclohexylcarpodiindo, N-ethyl-d-dimethylaminocarbodiimide, 1-ethyl-8-cyclohexylcarbodiimide, 1-cyclohexyl-8-(2-
Examples include dehydration condensation agents such as carbodiimides such as Jl ruforinyl-4-ethyl)carbodiimide;
Furthermore, a combination of diazonium arylcarboxylic acids of the p-diazonium phenylacetic acid moiety and a conventional peptide bond forming reaction reagent, such as the above dehydration condensation agent, can also be used.

The human β-lid interferon antigen is produced by reacting the above-mentioned batten and carrier in the presence of a hapten-cuff binding reagent. The above reaction taste, aqueous solution or pH
7 to 100 in a normal buffer, preferably a pH 8 to 90 buffer, at 0 to 40°C, preferably around room temperature, and about 1
The reaction is completed in ~24 hours, preferably 8 to 6 hours.
As a typical lli liquid used in the above, the following can be exemplified.

0.2N sodium hydroxide - 0.2M boric acid - o, g
M potassium chloride buffer, 04M sodium carbonate-0,2M boric acid-0,2M potassium chloride buffer, 0,05M sodium tetraborate-0,2M boric acid-0
, 05M sodium chloride mild solution, 0.1M potassium dihydrogen phosphate-0.05M Shihoku-Shun sodium buffer In the above, the proportions of Hageten, hapten-carrier binding reagent and carrier can be determined as appropriate, but usually hapten It is preferable to use the carrier in an amount of 2 to 6 times the weight, preferably 8 to 6 times the weight, and 5 to 10 times the mole of the hapten-carrier binding reagent. Through the above reaction, the carrier and habten are bound together through the mediation of the cyhutagten-carrier binding reagent, and the human β-inter724 antigen consisting of a nine-pegtedone-carrier complex is obtained. The antigen obtained after completion of the reaction can be easily isolated and purified according to conventional methods, such as dialysis, gel filtration, or fractional precipitation.

The antigen thus obtained usually has an average of 6 to 20 moles of peptide bound to 14 moles of protein, and in both cases it is possible to produce antibodies with high reproducibility and high specificity against human β interferon. However,
In particular, the bond ratio of the peptide to the above protein is l:8.
~ISO ones are preferable because they have higher specificity and can produce antibodies with high titer and high sensitivity.

In order to produce antibodies using the antigen obtained above, a method can be adopted in which the antigen is administered to a mammal according to a conventional method and the antibodies produced in vivo are collected.

Although it does not have eyes, it is usually best to use a demon guinea pig. For antibody production, a predetermined amount of the antigen obtained above is diluted with physiological saline to an appropriate concentration, and Freund's supplementary solution (Complet@Freund@Jdjuvan) is added.
t) to prepare a suspension, which may be administered to a mammal. For example, rabbits may be immunized by injecting the above suspension intrafundially (antigen amount: 0.5 to 15 lIIPZ times) and then administering it every two weeks for 2 to 10 months, preferably 4 to 6 months. . Antibodies are collected after the final administration of the above suspension, when a large amount of antibodies are produced, usually 1 to 2 weeks after the final administration, blood is collected from the immunized animal, and the blood is centrifuged and the serum is separated. It is done by collecting.

The antibody thus obtained has particularly excellent y% isomerism of human β-heavy interferon, as shown in the test examples below.

In the method of the present invention, first, a standard antigen is diluted with an appropriate diluent to prepare a dilution series. t-1 as a diluent,
41 is not limited, and various types commonly used in this type of measurement method can be used. Specifically, for example, boric acid buffer, Tris-HCl buffer, phosphate buffer, acetate buffer,
Citrate phosphate buffer, glycine buffer pH 6-1
Examples include a buffer solution having a pH of 0°C, preferably 7 to 811°C. Also, bovine serum albumin (BSA) to prevent adsorption.
The usual additives of sodium azide, EDTA, sodium chloride A$ as preservatives may be added to the diluent.
Next, a certain amount of the above-mentioned antibody and a certain amount of the fluorescently labeled peptide of general formula (1) were added to the dilution series of this standard antigen, and the mixture was prepared as a slurry and left at 0 to 87°C for about 30 minutes to 48 hours. The linear fluorescence polarization intensity (Iya) and the horizontal fluorescence polarization intensity (IHB) are measured by conventional means.
It can be easily kept constant using soxJ (manufactured by IllμNion Giken Co., Ltd.).

In addition, in the same manner as the above sample, an identical reference sample was prepared except that the fluorescently labeled peptide of the general formula (11) was not included, and the 1 direct fluorescence polarization intensity (IVR) and the horizontal fluorescence polarization intensity (IHR) were similarly prepared. ) is measured. Thus, the degree of polarization (P value) for the dilution series of the standard antigen is measured.
Obtain the standard curve using the following formula.

Using a sample containing an unknown degree of human β-type interferon instead of the standard antigen, the P value can be calculated in the same manner, and the human β-type interferon in the sample can be quantified using the standard curve obtained above. .

When raw β-heavy interferon is used as the standard antigen, the interferen titer of the sample can be determined immediately using the standard curve.

In addition, when the peptide of general formula (2) is used as the standard antigen, it is sufficient to first determine the cross-reactivity with human β-type interferon using raw human β-type interferon as the standard antigen and convert it into the interferon titer. .

Reference examples and examples will be given below to explain the present invention in more detail, but the present invention is not limited thereto.

The Rf value in the reference example was measured by thin layer chromatography on silica gel using the following mixed solvent.

Rf'...1-butanol-acetic acid two (4! 6) R
f''-...l-butanol-pyridine-acetic acid-water (16
SlO: 8! 12) Synthesis of peptide> Reference example I Z-8et-8et-OMe Op synthesis Z -Ser -NHNH! 6.06 F to 60
ml of dimethylformamide and 6.68 ml of isoamyl nitride after cooling to -16°C in 6N-HCI/dioxane. , 68 wIll and stir for 6 minutes.

6.604 of triethylamine is then added to neutralize. This solution was mixed with H-8er ・OMe -HCl 8.
804 in dimethylformamide containing 2.80 μL of triethylamine at 4°C.
Stir for 0 hours. Dimethylformamide was distilled off, and the resulting residue was extracted with ethyl acetate, washed with water, and then dried over Glauber's salt. After distilling off the ethyl acetate, add ether to crystallize, and re-precipitate with ethyl acetate-ether.Z-8er-8
Obtain er-OMe IL76 F.

R11 value: 0.64, RfI value: 0.77 Elemental analysis value (
CHN Calculated value (%) 52.94 6.92 8.28
Actual value (chi) 52.67 6.89 &f3
6 Reference Example 2 Z-Arg(Tos)-8er-5er-OMe
Manufacture of Z-8er-8er-OMe $! , 6 F to 6
0 ml methanol and 7.84 feed (1 (D lN)
-Dissolved in HC6 and added 6001f1 value: o,os Z-Arg(Tos)-OH&89F to 40-tetrahydrofuran and 0.764ON-methyl phosphorine, and after cooling to -16°C 0.97g4 Add 200 mg of imputirucproformei) and stir vigorously for 30 seconds.

Stir for 2 minutes at 40° C. and 16 minutes at room temperature, respectively. After distilling off the dihydratone and dimethylformamide, the mixture was extracted with ethyl acetate, washed three times with IN-citric acid, then five times with water, and dried over sodium sulfate. The solvent was distilled off, reprecipitation was performed with methanol-ethyl acetate and ether, and Z-
Arg(Tos)-8er-8er-OM@L66
0H11 value customer 0.61%RfK@s o that gets t,
7 B elemental analysis value (as CI8 H8B '601°S) CI N Calculated value (1G) 61,68 6.88 119
1 Actual value (91) 61.62 6.82 12.7
8 Reference Example 8 Production of Z-Arg(Tos)-8er-8er-OH Z-Arg(Tos)-8er-8@r-OMe L
Dissolve 6y in methanol Is Omg and 10 ml of water, add 8.06 ml of 1N-NaOH, and add 4.0 ml of 1N-NaOH at room temperature.
Stir for b minutes, distill off the tanol, and then extract with n-butanol. After washing with water, n-butanol and water were distilled off, ether was added to the residue to crystallize it, reprecipitation was performed with methanol-ethyl acetate, and Z-Arg(Toe)-8@r-8er
-OH2,6Of is obtained.

R11 value: 0.28, RfI: 0.57 Elemental analysis value (as C, □-6N601°S, 1/I H, 0)
CHN Calculated value C%) 50.22 6.77 1. OL! Value in the blank column (%) 50J4 Is, 62 11!0 Reference example 4 Production of H-Arg(Tos)-5er-8*r-OH Z
-Arg(Tos)-8et-8er-OR1,85f
methanol 50- and! 0-”Acetic acid 1011’41?
H-Arg(Tos-)-8er-8er-
Get OH.

RZI value! 0.084 Reference Example 6 Z-Asn-Leu-OEt Dflli manufactured Z-Asn
-OR3,82jl to nato2 human 07j/604, dimethylformamide 104 and N-methylmorpholine 9
．． ．． 044 and isobutyl chloroformate l!
, 64d, H-Leu-OKt-HC68,911' and 40- in dimethylformamide containing 2.80-trietheramine, the reaction is carried out according to the method of Reference Example 2. After distilling off the solvent, add 1 molar citric acid aqueous solution,
The precipitate is collected, washed with water, dried under reduced pressure, and recrystallized from ethyl acetate to obtain Z-Asn-L@u-OEL 6.1 f.

R/! Value: 0.71, Rf”full! 0.80 Elemental analysis value (C!oH29N, as 06) CI
N Calculated value (1G) 68.93 7.17 10.81 Actual value (*) 58.99 7.28 10.26 Reference example 6 Production of Z-Tyr-AIn-Leu-01Ct Z-As
n-Leu-Oft B, 01 f with methanol 6
04 and IN-HCJ? , 4d and subjected to catalytic reduction at room temperature and normal pressure in the presence of palladium black 50G-
-OR/' value to obtain -AIn-Leu-OEt-HCI:
0.26 H-Asn-L@u-OEt-HCg obtained above was dissolved in dimethylformamide 40. nl and triethyl 7 electrons, dissolve OIs sJK, then Z-Tyr-ONH8
Add 8,85y and leave at room temperature for 20 hours. After distilling off dimethylformamide, add 1 molar citric acid aqueous solution,
The precipitate was collected, washed with water, and reprecipitated with methanol-ethyl acetate to give Z-Tyr-Asn-Leu-OEt 8.5
Get 8 f.

RfI value! 0.7B, RZI value: 0.82 elemental analysis value (
C! I H88N4 o, as) (HN Calculated value (56) 61.04 6.71 9
．． 82 Actual value (%) 60.84 6.70
9.89 Reference Example 7 Production of Z-Tyr-Asn-Leu-NHNHg Z-T
yr-Asn-Leu-OEt'i! , 78F to methanol 801f+4! Dissolved in hydrazine hydrate 1
．． 214 was added and left overnight, the resulting crystalline P was removed and washed with a small amount of methanol to form Z-Tyr-Asn-Leu.
-NHNHIB, obtain toy.

RfI value: 016, RfI value! 0.75 elemental analysis value (C
! ? H86NS Of) CHN Calculated value (%) 58.26 g,! 52 1
6.10 Actual value (%) 67.91 g, 66
16.12 Reference Example 8 (a) Boc-Leu-Gln-Arg (To@)
Preparation of -8er-8er-OH Boc-Leu-Gln-OR0,84f is carried out according to the method of Reference Example 2 using Tetrahydrof. After distilling off Tetrahydro7ran and dimethylformamide, the residue is extracted with butanol. After washing the butanol layer with 2-acetic acid, the butanol was distilled off and reprecipitated with methanol-ethyl acetate to obtain Boc-Leu-Gl n-Arg(Tos)-8.
er-5er-OR1,64t is obtained.

RfI value! 0.18, RfI value: O, lS7 elemental analysis value (C86H5? NI OIs S, Hl 0) CHN calculated value (ch) 48.77 6.90 14.
62 Actual column value (1G) 48.76 6.61
14.78(b) H-Leu-Gln-Arg(To
s) -8er-8er-OH production Boc-L@u-Gin-Arg(Tom)-8@r-
8er-OR1, 50f t-10ml of trifluoroacetic acid was left at room temperature for 915 minutes to remove Boc, and crystallized by adding dry bee ether to form H-Leu-Gln-Arg (
Tos)-8et-8et-OB is obtained. RfI value! 0.04 Reference Example 9 (a) Z-Phe-Leu-Gln-Arg(To
s) -8er-8er-OH Production Ii-Leu-Gln-Arg (T
os)-8er-8et-OR was dissolved in 80 ml of dimethylformamide and 0.24 d of triethylamine, then Z-Phe-ONH8O,? 7 f was added and the mixture was allowed to stand at room temperature for 1.0 hours. After distilling off the dimethylformamide, the remaining liquid was extracted with butanol and washed with 2-acetic acid. The butanol layer was diluted, ether was added to crystallize it, the obtained crystalline V was removed, and the crystals were washed with hot ethanol and Z
-Phe-Leu-Gln'-Arg(Tos)-
8er-8@r-OH1,40F is obtained.

R11 value! 0.22, RIK value t 0.69 Elemental analysis value (as C47N14 N1゜o14s, n, o) C
HN Calculated value (%) 54,12 6.28 18.
48 Actual value C%') 54.41! 6.88
18.84 Reference Example 10 (a) Z-Leu-Gly-Phe-Leu-Gi
n-Arg(Tos)-8et-8et-OR(2>
Preparation Z-Leu-Gly-NHNHI O,89F was dissolved in dimethylform 7tYrs@l and 6N-HCII/dioxane 0.68.1 and isoamyl nitrite 0.16 ml.

Triethylamine 0.49 gI4 and Reference Example 9(a)
Z-Phe-eu-Gln-Arg(Tos) obtained in
H-Phe-Leu-Gln-Ar obtained by debinarizing -8er-8er-OR in the same manner as in Reference Example 8(b)
A reaction was carried out in the same manner as in Reference Example 1 using a dimethylformamide 104 solution of g(Tos)-8*r-8vr-OH1,0Or, and then an equivalent amount of KZ-Leu-GlyN was added and the reaction was carried out at 4°C for 20 hours. Do the following. After extraction with butanol and washing with 2-acetic acid, butanol and acetic acid are distilled off, and ether is added to crystallize. Wash the crystals with hot methanol to obtain %Z-Leu-Gly-Phe-L.
eu-Gln-Arg(Tos)-8er-8er-O
Obtain HO, 66f.

R11 value: 0.19, RITi value: o, 6° elemental analysis value (as C66N78 N1! 016 S' H! O) CHN calculated value (%) 64.44 6.64 18.8
15 fresh measurements (96) 54.74 166 18
．． 98 Reference Example 11 (a) H-Tyr-Asn-Leu-Leu-Gl
y-Phe-Leu-Gln-Arg(Tos)-
Production of 8er-8er-OH Z-Leu-Gly-P
he-Leu-Gin-Arg(Tos) -8er-
8er-OH60G- in methanol 60 ml and to
-emto-1ice understanding parajiu A! Catalytic reduction was carried out at normal temperature and pressure in the presence of H-Leu-Gl
y-Phe-Leu-Gln-Arg(Tos)-8
Obtain er-8er-OH. R11 value: o, t-5 Z-Tyr-Asn-Leu-NHNH! 420mW
was dissolved in dimethylformamide ml and 6N-HCI/dioxane 0, B7vmllK, and using 0.1 On of isoamylnitite and 0.814 of triethylamine, an azide compound was produced according to Method 10 on the reference side, and this solution was dissolved in H- Leu-Gly-Phe-Leu-Gl
5 keys of n-Arg(Tos)-8er-8er-OR? /? 9/II) Add to a mixed solution of 104 dimethylformamide and stir at 4°C for 20 hours. Dimethylformamide was distilled off, the residue was extracted with butanol, washed with 2-acetic acid, the butanol was distilled off, ether was added to the residue for crystallization, and the resulting Z-Tyr-Asn-Leu-Leu- Gly-Ph
e-Leu-Gln-Arg(Tos)-8er-8
er-OH was dissolved in methanol 80J and 10-acetic acid 10-, and after catalytic reduction in the presence of palladium at room temperature and normal pressure, it was gel-filtered with Sephadex G-25 (BX120am, eluent 60-acetic acid). TeH-Tyr-Asn-
Leu-Leu-Gly-Phe-Leu-Gln-A
rg(Tos)-8@r-8er-OH660@p is obtained.

RfI value! 0.0 M, HIN value $ 0.68 Elemental analysis value (C,,) I, 8N1.0. , S , 2 H,
0) CH, N Calculated value UG) 6B, ff18 6.91 1
&08 Yi side value (ts), 58.10 6.48
1484 Reference example 1! (a) Z-Met-5er-Tyr-Asn-Le
u-Leu-Gly-Phe-Leu-Gl n-Ar
Production of g(To@)-8er-5er-OH Z -M et -Ser -N HN Ht
eye] - to di) lfk ho to muamide &-1 and 6N-H
Cl/dioxane 0.214, the azide compound was produced according to the method of Reference Example 1 using 0.065 gI&ll of isoazurnitrite and 7 vml of triethylamine, and then this solution was dissolved in H-Tyr- As
n-Leu-Leu-Gly-Phe-Leu-Gln
-Arg(Tos)-3ar-8er-OH80GN,
Dimethylformamide 6#! 4 and hexmethylphosphoric acid triad b- were added to a mixed container f#, and stirred at 4°C for 24 hours. Furthermore, Z −M e t −S e r
Add 818 -NHN Hg and stir at 4°C for 72 hours. Purification was carried out in the same manner as in Reference Example 11(a),
Z-Met-8er-Tyr-Asn-Leu-Leu
-Gly-Phe-Leu-Gl n-Arg(To
s) Obtain -5er-8er-OR◎(b) H-M
et-8er-Tyr-A@u-Leu-Leu-Gl
y-Phe-Leu-Gl n-Arg-8er-8e
Production of r-OR Z-M@t-8e obtained in above (a)
r-Tyr-Ass-Leu-Leu-Gly-Ph
e-Leu-Gln-Arg(Tos)-8er-8e
r-OR is debinarized in the same manner as in Reference Example 8(b) O H-M@t-8@r-Tyr-Asm- obtained above
Lau-Leu-Gly-Phe-Lau-Gln-A
Dissolve rg(Tos)-8et-8ar-OR in liquid ammonium chloride 6g, add a small piece of metallic sodium, and when the reaction solution turns blue, dry ammonium chloride 1f
is added, and then the annineane is distilled off. The residual solution was dissolved in 60-acetic acid, and Sephadex G-25 (8X 12 G
ms.

Eluent 60-acetic acid), and further LH-L! O(2X 85
The eluent was purified with 0.001 N-HCN) and H-
Me t-8er-Tyr-Asn-Leu-Leu-
Gly-Phe-Leu-Gln-Arg-8er-5
er-OHl 19 *f is obtained.

R11 value! 0.01. R/” value: 0.45 (1) go
! -15,7'(C"0.254.0.0OIN-H
C4? ) Elemental analysis values (C,, B2°, N,, 0..

S, 5 H, 0, as CMsCOOH) CHN Calculated value (11) 49.7F17.26 Seal 6.
18 Actual value (1G) 49.8OL92 14.9
1 ζ acid analysis value Atp! 0.96, Ser: 11.16, Glnt
l, OB.

MetgO, 92, GIF: 1.05, Leu: t
o g, Tyr: 1.00% Pha: 0.98
, Arg: 0.96 <Production of antigen> Reference Example 1B Peptides (hereinafter abbreviated as "peptide A") 8- and B5A26- obtained in Reference Example 12(b) above were dissolved in ammonium acetate buffer (0.1 mol , pH 7,0)24. A solution of KO, 0.11 ml of a 1 molar gel taraldehyde solution is added and stirred for 6 hours at room temperature.Then the reaction mixture is stirred for 48 hours at 4 °C in water 1. Dialyze at g'. Change the water 6 times during dialysis. Thereafter, the ** containing the peptide-protein complex was lyophilized to produce human β-heavy interferon antigen (hereinafter abbreviated as "antigen!") 81 @
get f. The peptide was bound to 1 mole of antigen IaBSA at an average of 9:E. Incidentally, the bonding rate below this level was measured.

In other words, the antigen obtained above! Furthermore, Sephadex G-
60 (eluate: physiological saline, detection: OD280gsm,
Gel filtration was carried out at an effluent rate of 8-(volume/hour, fractional amount: iJ each), and the fraction of BSAK-bound peptide A [
1] and the other product (dimer of peptide A) 7-lactone [11] were separated, and the fraction (13) was dialyzed against 0.6-saline solution at 4°C for 24 hours and then lyophilized to form a white powder. A peptide A-BSA complex (hereinafter abbreviated as "antigen!'") is obtained.This complex has an average of 9 moles of peptide A bound to 5 moles of B5Al. If the presence of BSA and peptide A was not recognized, a calibration curve of the standard concentration of the dimer of peptide A was created, and from the standard calibration curve, the presence of peptide A of the above 7-lactone
The amount of the dimer was determined, and this was subtracted from the amount of peptide A used as the starting material. The value calculated was calculated assuming that all of the values were bound to BSA.

<Production of antibodies> Reference example 14 Reference example! Antigen I 215 voice f obtained in 3.1.6J
Freund's auxiliary solution lJgn after dissolving in physiological saline
The suspension prepared by adding 1.0 liters of
Administer the same amount subcutaneously at 0 (immediately) and then administer the same amount 6 times every 2 weeks. After that, administer the same amount 8 times every month thereafter at the same amount as the first dose. Seven days after the final administration, blood is collected from the test animal and centrifuged to collect antiserum to obtain human β-o interferon antibody (hereinafter abbreviated as "antibody summer").

Production of 0-labeled peptide Peptide A is labeled by a method using chlorane ζT.

That is, 6 μt of the above peptide in 20 A of 0.2 molar phosphate buffer (pH 7.4)! ni[”'I] Na(MEN
) Add 1 mic 4 Chierly's o, 2 J+Nyl phosphate buffer, then add 20 pg of Flora's 78.6 mP/ml O, G phosphate buffer. Leave for 20 seconds at room temperature and add 60μ1A of sodium metadisulfate in 04M phosphate buffer at 4 mf/all.
-250 column (1,0X8Gam) (eluent 0.1$ BSA and O-0.1 containing 10% NaN5)
4 lutris-hydrochloric acid buffer, pH 8.6). 17 Japanese horsetail rattan is purified by ion exchange chromatography! J
Obtain the above peptide labeled with ○○ Measurement of titer The titer of the antibody obtained above is fixed as follows.

That is, antibodies were dissolved in physiological saline at 10% and 10% 1O11, respectively.
0'%1G'・・・・・・Double dilution (initial),
To 100 μl of each of these, 1.1! Li-labeled peptide (the labeled peptide obtained above was diluted to about 10,000 cpmK) 0.1 ml and 0.1 molar phosphate buffer (1) H-7,4) [0,191BSA
, 0.154=7 with sodium chloride and 0.01%
Contains N a Nm] Q, 2J was added and incubated at 4°C for 24 hours, and the generated antibody and! The conjugate with the labeled antigen was separated by the dextran-activated charcoal method and centrifugation method (4°C
, 16 minutes at 80 G Orpm) to separate from the unreacted (unbound) I'' labeled peptide, count the radiation, and calculate the binding rate of the antibody to the !1!6 labeled peptide at each dilution concentration. Measure (H).The vertical axis shows the antibody ■
The binding rate with the 125-labeled peptide (-) and the dilution factor (initial concentration) of the antibody are plotted on the horizontal axis, and the binding rate is plotted at each concentration. Binding rate is 50- and antibody 1
Human β-type interferon specificity test for 52,0000 antibodies Test samples include various concentrations of human β-interferon (manufactured by Tokyo Metropolitan Clinical Research Institute, specific activity: 8 x 10'U/vp + f protein), peptide A, and human A-lid interferon (Hayashibara). Made by the Institute, Lymph.

blastold Interferon, Lot
, No. 800928). Also, as a standard diluent, 0.111BSA・0,1 Is% #NaC
A 0,1 molar sodium phosphate buffer (pH 7,4) containing 1 and O,OI IsONmN5 * is used.

In each test tube, add 0.2 J of standard diluent, 0 and 1 sample sample.
m11 Reference Example 1 Antibody IO, In obtained from FD (titer: 52,000) and! 1! Add 0.14 S-labeled peptide (the labeled peptide obtained above was diluted to approximately 10,000 cpm) and incubate at 4°C for 48 hours.
p serum) was added at 1 mA', then coated with dextrin, 0.6 mA' of activated carbon suspension was added, left at 4°C for 80 minutes, then heated at 4°C, 8000 rpm.
Centrifuge for 80 minutes under conditions of
``Bound body with secret peptide and unreacted (does not bind) 1
The 116g recognition peptide was separated, its radiation was counted, and the binding rate (Bo), which corresponds to the titer of the nine antibodies used, was calculated as 100.
-, the percentage of the conjugate (6) between the antibody and the labeled peptide at 11t6 degree and dilution rate of each test sample is determined. The results obtained are shown in FIG. The vertical axis in FIG. 1 is the bond - (B/B.

X100), and the horizontal axis shows the concentration of the test sample (peptide A, human β-type interferon, and human 4m interferen). In addition, in the figure, curve (a) represents peptide A, curve (b) represents human β-lid interferp, and curve (a) represents peptide A.
C) shows human α-interferon. Figure 1 shows that antibody 1 shows clearly distinct curves in terms of reactivity to human β-type interferon and reactivity to human β-type interferon. It can be seen that this is a highly specific antibody with no cross-over.

Execution side car Peptide A I 111F and FITClmF 6 o - O of methanol,! ! M sodium bicarbonate buffer (p
H=9.6). After reacting at room temperature for 2 hours, the mixture was separated using a thin layer of silica gel and a four-layer gradient (elution 1 [:bumenol:acetic acid:water=4!136]). Suboc) collection J6
/) to obtain 0.8 my of FITCIIIIII peptide.

Rf'*-0,16, Rfn value-0,52 elemental analysis value (
CsC55HoyNno S!・'Hz 0-CsH
(as sCOOH) CHN Calculated value (*) 6160 6.42 12.96 Analysis value (91) fs2.65 6.22 1174
II Theory Example 2 Nine peptide AI dissolved in 0.06 mg of 10”H hydrochloric acid
1112 to 1!10v/v96 methanol O, RM
Dissolve in sodium bicarbonate buffer (pH 9.5). T
RI TClmPi-OJM-Sodium Bicarbonate (pH
9,5) and mix with the above solution. After reacting at room temperature for 2 hours, the mixture was separated by silica gel thin layer chromatography (eluent: butanol-acetic acid:water = 4:1:5).

The spots are collected and eluted with methanol to obtain TRITC-labeled peptides o and steel p.

Rf'*mouth 0.12, RZII value - 0,42 elemental analysis value (C,, II, □? N130!4 S! "H!
0・CB.

(as C0OH) CHN Calculated value (CH) 58, Is2 6.69 18.84 Analytical value (-) 68.20 6.6 18.80 Example 8 (a) Preparation of fluorescently labeled peptide solution In Example 1 The obtained nine FITC-labeled peptides were added at 0.9 w/v *N a
Dissolved in 0.1M sodium phosphate buffer (pH = 7) containing cl and 0.0g%%BSA to a concentration of 8X
Prepare 10-9 MIC. This will be referred to as "rA solution" hereinafter.

(b) Blank of fluorescent am peptide solution Prepare a solution having the same composition as the above solution A except that it does not contain the fluorescently labeled peptide. This will be referred to as "CB liquid" hereinafter.

(C) Preparation of antibody solution Antibody 1 obtained in Reference Example 14 is diluted 800 times with physiological saline. This will be referred to as "liquid C" hereinafter.

(d) Diluent OH H, BO29, 89y/l, Na, B, 0. -10H!
04.82 y/1%NaCl 9 f/1%NaN@
Prepare a pH 7.8 dilution containing 0.006 w/, $ and BSA O,01 W/v-. This will be referred to as "ED liquid" hereinafter.

(e) Preparation of standard dilution series ■ Peptide A, 0.9 w//% NaC4 and 0;
Dissolved in OIM-sodium phosphate buffer (pH-6,6) containing 06 W/V% BSA to 6μF!
/nl Dilute this with the above solution D to make the concentration of peptide A 600.260°121s, 62.
6,81.26.15.6.7.8.8.9.1.9B
.

Prepare a dilution series expressed as 0.925, 0.468, 0 nf/me.

■ Using human β-type interferon (manufactured by Masai Clinical Research Institute, Tokyo), incubate 2 × 0 11 in the same manner as in ■ above.
XIG, 0.6X10. 0.26X10
.

0.126X10. 0.068XlO, 0,081X
10.

Prepare a dilution series of o, otaxtoll, OU/, J0 (f) Measurement of polarization degree P value (e) - Each of the dilution series prepared in 〇 o, g@4'KA
Liquid j, 064. Clll0.1 vrl and D liquid 0.
664 to create a sample of peptide A.

A reference sample of peptide A is prepared in exactly the same manner except that 6 mg of solution 0 and o are used instead of solution A.

From the dilution series prepared in (e)-〇 in the same manner as above,
Prepare a human β-type interferon sample and a reference sample.

Furthermore, 0.06 ml of solution A and 0.95 J of solution D were mixed to prepare - antibody blank sample, solution B o, osmg and D.
A blank reference sample of the antibody was prepared by mixing 0.96+J of the solution.

After each of these samples and each reference sample was incubated at 4° C. for 12 hours, the fluorescence polarization component was measured using a fluorescence polarization intensity measuring device. For each dilution series and blank, the polarization degree P value was calculated using the following formula.

(In the formula, summer VB is the vertical fluorescence polarization intensity of the sample, IH8
is the horizontal fluorescence polarization intensity of the sample, IVR is the vertical fluorescence polarization intensity of the reference sample, and %IHR is the horizontal fluorescence polarization intensity of the reference sample. ) The results are shown in Figures 2 and 8. From both figures, the concentration equivalent to half the change in P value is 1 nf/
About Tubes human β-type interferon 2. I
X 10' U/Tube, and the ratio of both is 2t
oU/pg was obtained as the crossover rate9.

Regarding samples with unknown concentrations of human β-type interferon,
In the same way, the P value was measured and the intellectual ability could be measured as shown in Figure 2 or Figure 8.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the specificity of the nine human β-type interferon antibody obtained in Reference Example 14, and FIGS. It is a graph showing the degree of polarization P value determined by the fluorescence depolarization method. (above), No. 1 52 Fig. 2 97°ke)-A Fig. 3 Human β1 = 2! ? −7, []2

Claims (1)

[Claims] ■ General formula % Formula % E In the formula, R represents a fluorescent dye. ] Fluorescently labeled peptide represented by ■ General formula % Formula % [In the formula, R represents a fluorescent dye. ] A method for measuring human β-type interferon, which is characterized by measuring human β-type interferon by a fluorescence depolarization method using a fluorescently labeled peptide represented by the following.