JPH0218836B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing a lyophilized product containing stable β-D-galactosidase (hereinafter sometimes abbreviated as "β-Gal."). β-Gal. is an enzyme that hydrolyzes lactose into galactose and glucose, and recently it has also been used as a labeling enzyme in diagnostic reagents, especially in enzyme-linked immunosorbent assays (hereinafter sometimes abbreviated as "EIA"). Its importance has increased. However, β-Gal. is unstable in a solution state both when it is alone (free state) and when it is combined with other components (e.g. immunoactive substances). If the concentration is as low as below, it is extremely unstable, and therefore the quality of the measurement reagent is difficult to maintain and stable results cannot be obtained. Furthermore, it is well known that the enzymatic activity of β-Gal. is significantly reduced even when it is freeze-dried. In view of these drawbacks, the present inventors conducted extensive research with the aim of finding a method for stabilizing β-Gal., and found that a freeze-dried product of an aqueous composition containing β-Gal. and sugar or sugar alcohol was found. It was discovered that the intended purpose could be achieved, and as a result of further research based on this finding, the present invention was completed. The present invention provides 100 pg to 10 μg of β-D-galactosidase or β-D-galactosidase per ml in an aqueous composition containing β-D-galactosidase or its conjugate with an immunoactive substance and sugar or sugar alcohol. , a method for producing a lyophilizate for labeling, which comprises subjecting the aqueous composition containing sugar or sugar alcohol at a concentration of 0.2 to 5 w/v % to lyophilization. The β-D-galactosidase used in the present invention may be of any origin, but it is easy to obtain, and 0-nitrophenyl-β-D-galactopyranoside or 4-methylumbelliferyl-β
β-Gal. derived from Escherichia coli, which has high specific activity against synthetic substrates such as -D-galactopyranoside, is preferably used. β-Gal. may be used alone, or may be in a bound state in which it is bound to an immunologically active substance. Examples of the immunologically active substance include antigens, antibodies, and haptens such as drugs. Antigens include hormones [pancreatic glucagon (hereinafter referred to as "PG"), its fragment peptide, insulin, its fragment peptide, parathyroid hormone, calcitonin, adrenocortical hormone, growth hormone human chorionic gonadotrobin (hereinafter referred to as " (abbreviated as “hCG”).
its fragment peptides, progesterone, thyroid stimulating hormone, etc.], proteins [IgG, IgA,
IgM, IgE, α-fetoprotein, CEA, etc.] and viral antigens (rubella virus HA antigen, Sendai virus HA antigen, etc.). As antibodies, rabbit, rat,
Antibodies (anti-PG antibodies,
anti-hCG antibody, anti-IgG antibody, anti-IgM antibody, anti-IgE antibody, etc.). Examples of haptens include steroids, vitamins, catecholamines, and drugs including antibiotics (penicillin, cephalosporin, β-adrenergic agonist, etc.). The above hCG fragment peptide has the general formula H-R ₁ -Pro-Ser-Asp-Thr-Pro-Ile-
Leu―Pro―Gln―OH [In the formula, R ₁ is represents 1 to 14 partial peptide chains containing Gly at position 14 of the peptide shown by ] Examples include peptides represented by the following. The general formula for the above PG fragment peptide is H―R ₂ ―Phe―Val―Gln―Trp―Leu―R ₃ ―
Asn―Thr―OH [In the formula, R ₂ is 1 to 10 partial peptide chains containing Asp at position 10 of the peptide represented by R ₃ represents Met or Nle, respectively. The fragment peptides of insulin include the general formula H-R ₄ -Gly-Phe-Phe-R ₅ -OH [wherein R ₄ is

1 to 3 partial peptide chains containing Arg at the 3-position shown by [Formula], R ₅ is

1 to 5 partial peptide chains containing Tyr at position 1 of the peptide represented by the formula are each represented. ] Examples include peptides represented by the following. R ₁ in the above hCG fragment peptide
indicated by The 1 to 14 partial peptide chains containing Gly at position 14 of the peptide include, for example, Gly, Pro-
Gly, Leu-Pro-Gly, Arg-Leu-Pro-Gly,
Ser―Arg―Leu―Pro―Gly, Pro―Ser―Arg―
Leu―Pro―Gly, Ser―Pro―Ser―Arg―Leu―
Pro―Gly, Pro―Ser―Pro―Ser―Arg―Leu―
Pro―Gly, Leu―Pro―ser―Pro―Ser―Arg―
Leu-Pro-Gly, Ser-Leu-Pro-Ser-Pro-
Ser―Arg―Leu―Pro―Gly, Pro―Ser―Leu―
Pro―Ser―Pro―Ser―Arg―Leu―Pro―Gly,
Pro―Pro―Ser―Leu―Pro―Ser―Pro―Ser―
Arg―Leu―Pro―Gly, Pro―Pro―Pro―Ser―
Leu―Pro―Ser―Pro―Ser―Arg―Leu―Pro―
Gly, Ala―Pro―Pro―Pro―Ser―Leu―Pro―
Examples include Ser-Pro-Ser-Arg-Leu-Pro-Gly. Indicated by R ₂ in the above PG fragment peptide The 1 to 10 partial peptide chains containing Asp at position 10 of the peptide include, for example, Asp, Gln-Asp,
Ala―Gln―Asp, Arg―Ala―Gln―Asp, Arg―
Arg―Ala―Gln―Asp, Ser―Arg―Arg―Ala―
Gln―Asp, Asp―Ser―Arg―Arg―Ala―Gln―
Asp, Leu―Asp―Ser―Arg―Arg―Ala―Gln―
Asp, Tyr―Leu―Asp―Ser―Arg―Arg―Ala
-Gln-Asp, β-Ala-Tyr-Leu-Asp-Ser
-Arg-Arg-Ala-Gln-Asp, etc. Indicated by R ₄ in the insulin fragment peptide above.

One to three partial peptide chains containing Arg at position 3 of the peptide of [Formula] include, for example, Arg, Glu-Arg, Gly-
Examples include Glu-Arg. Also designated by R ₅

Examples of 1 to 5 partial peptide chains containing Tyr at position 1 of the peptide of [Formula] include Tyr, Tyr-Thr, Tyr-Thr
―Pro, Tyr―Thr―Pro―Lys, Tyr―Thr―Pro
-Lys-Thr etc. The various peptides described above can be produced by known conventional methods of peptide synthesis. Either a solid phase synthesis method or a liquid phase synthesis method may be used, but the liquid phase synthesis method is often advantageous. Examples of such methods of peptide synthesis include “The Peptides”, Volume 1 (1966), Schroder and Lubke, Academic
Press, New York, USA or methods described in "Peptide Synthesis", Izumiya et al., Maruzen Co., Ltd. (1975), such as azide method, chloride method, acid anhydride method, mixed anhydride method, DCC method, activity Ester method, method using Woodward reagent K,
Carbodiimidazole method, redox method, DCC/
Examples include additive methods (eg, HONB, HOBt, HOSu). A covalent bond can be cited as a form of bond between β-Gal. and an immunoactive substance. As for the joining method,
Various known methods may be followed, for example Clinica Chimica Acta.
The sulfhydryl group of β-Gal. and the amino group of the immunoactive substance are cross-linked using the method described in Volume 81, Section 1 (1977), that is, m-maleimidobenzoyl-N-hydroxy succinimide. A method for cross-linking the sulfhydryl group of β-Gal. with the sulfhydryl group of an immunoactive substance using 0,0'-phenylene dimaleimide.
Examples include a method of cross-linking the amino group of β-Gal. with the amino group of the immunoactive substance, and a method of creating a peptide bond between the amino group or carboxyl group of β-Gal. and the carboxyl group or amino group of the immunoactive substance using carbodiimide. . Examples of sugars in the present invention include monosaccharides such as pentose arabinose, xylose, ribose, hexose glucose, fructose, etc.
Mannose, galactose, rhamnose, etc.2
Sugars include maltose, cellobiose, trehalose, gentiobiose, lactose, sucrose, etc.
Examples of trisaccharides include raffinose and maltotriose. Examples of sugar alcohols include xylitol, which is a monosaccharide alcohol with 5 carbon atoms,
Sorbitol as a monosaccharide alcohol with 6 carbon atoms,
Examples include mannitol and inositol.
Among them, arabinose, mannitol, inositol, sucrose, and raffinose are preferred, and sucrose is most preferably used. Furthermore, a mixture of sugar or sugar alcohol may be used. Further, the β-Gal.-containing aqueous composition of the present invention may contain albumin in addition to the sugar or sugar alcohol, and may further contain albumin and a magnesium and/or calcium ion generating substance. This prevents a decrease in enzyme activity that occurs during the freeze-drying operation, and also prevents a decrease in immune activity in the case of a conjugate with an immunoactive substance, and makes it possible to improve the shape after freeze-drying. Examples of the albumin include serum albumins such as human serum albumin, horse serum albumin, bovine serum albumin, and sheep serum albumin, and bovine serum albumin is preferably used. The magnesium and/or calcium ion generating substance may be any compound that can generate magnesium and/or calcium ions, but usually includes magnesium salts and/or calcium salts, preferably magnesium chloride and calcium chloride. Can be mentioned. In the composition of the present invention, the content of β-Gal. is usually 100 pg to 1 ml per ml of the aqueous composition.
It is 10 μg (or 3 micro units to 0.3 mm units). The content of saccharides in the aqueous substance is usually 0.2 to 5 W/V%. The content of albumin in the aqueous composition is usually from 0.01 to
5 W/V%, preferably 0.1 to 1 W/V%.
The magnesium or/and calcium ion generating substance is such that the ion concentration in the aqueous composition is
0.0001~0.1M/, preferably 0.0001~
It is 0.001M/. When preparing an aqueous composition, the order of addition of each substance is not particularly limited. The lyophilizate of the aqueous composition can be prepared in a conventional manner, e.g.
freezing the aqueous composition at 30°C to about -50°C;
It is then produced by sublimating ice under reduced pressure at a temperature of about 10°C to about 20°C. After freeze-drying, sealing with nitrogen gas or vacuum sealing is more preferable in order to prevent spoilage and deterioration due to contamination with mold, bacteria, etc. Nitrogen gas sealing is usually performed by sufficiently substituting the freeze-dried material with nitrogen gas and then sealing the container under a nitrogen gas flow. In addition, vacuum seals are usually used to store freeze-dried products under reduced pressure (for example, 10
~0.01mmHg). The lyophilized aqueous composition containing β-Gal. and sugar or sugar alcohol thus obtained is useful as a reagent because the enzymatic activity of β-Gal. is hardly lost. In addition, the lyophilized aqueous composition containing the conjugate of β-Gal. and an immunoactive substance and sugar or sugar alcohol obtained in this way is useful as a diagnostic reagent, and especially β-Gal. Since it is relatively inexpensive and has a high specific activity, it is useful as a measurement reagent for EIA. In such EIA, an antigen bound to an enzyme is added to the antigen in a test solution to be quantified together with a certain amount of antibody, and the competitive binding method is used to competitively bind to the antigen and measure the enzyme activity bound or not bound to the antibody.
Alternatively, the Sandermansch method is used, in which the antigen or antibody in the test solution is reacted with the antibody or antigen on the solid phase, and the enzyme activity bound to the solid phase is measured, and various modified methods are also used [Clinica Chimica, Clinica Chimica Acta, Volume 81, Section 1 (1977)]. In the enzyme immunoassay method using the competitive binding method described above, (1) a test sample containing an unknown amount of antigen is
by adding a fixed amount of the enzyme conjugate and a fixed amount of the antibody in insoluble form and measuring the enzyme activity on the antigen-enzyme conjugate in the liquid phase or on the reacted antigen-enzyme conjugate in the solid phase. Method for quantifying antigen in a test solution [FEBS Letters
(2) Add a fixed amount of antigen-enzyme conjugate and a fixed amount of soluble antibody to the antigen-containing test solution to allow competitive binding, and then Add a certain amount of an insoluble second antibody against the antibody [for example, anti-rabbit immunoglobulin G (IgG) antibody when the antibody was prepared using a rabbit], and add it to the liquid or solid phase in the same manner as in (1) above. A method for quantifying antigen in a test solution by measuring oxygen activity (see the above Febus Letters), and (3) adding a fixed amount of antigen-enzyme conjugate and soluble After adding a certain amount of antibody and binding competitively, if the second antibody is an anti-animal IgG antibody, add a certain amount of normal animal serum to form a precipitate, and proceed as in (1). A method for quantifying antigens in a test solution by measuring oxygen activity in a liquid or solid phase [Clinica Chimica Acta]
Vol. 67, p. 283 (1976)]. In either method, when measuring enzyme activity in the liquid phase, it is advantageous to measure the activity in the solid phase, as contaminants present in the test solution may interfere with the measurement of the enzyme activity. be. Furthermore, from the viewpoint of reproducibility and sensitivity, the "double antibody method", which allows measurement using a trace amount of antibody, ie, methods (2) and (3), is more preferably used. For example, when measuring hCG, it is preferable to use an hCG enzyme immunoassay kit comprising the following reagents. The test set for the quantitative method using the insolubilized second antibody mainly consists of (1) peptide-β of anti-hCG antibody added to the reaction system;
- Bind a certain amount of Gal. (preferably about 5
(2) Peptide-β-Gal. (lyophilizate of the present invention)
(3) 0 to 500 IU standard hCG; (4) Buffer used for diluting the reagents and test samples in (1) to (3) above. solution (preferably a phosphate buffer solution with a pH of 6 to 9), (5) a certain amount (an amount that can bind all of the first antibody in (1) above) of an anti-animal IgG-binding carrier (as a carrier) in an insoluble form; Examples include cellulose, cefdex, cephalose, polystyrene spheres, silicon pieces, glass spheres, etc.), (6) a certain amount (preferably 1 to 100 μg) of the enzyme substrate, (7) the second antibody of (5) Buffer (preferably phosphate buffer or carbonate buffer) used to wash the bound carrier and dissolve the enzyme substrate in (6), (8) Buffer used to stop the enzyme reaction (preferably carbonate buffer or diluted caustic soda) aqueous solution). Further, the above kit is preferably used, for example, by the following method. Reagent (1) 10-300μl, reagent (4) 100-500μl and standard
After adding 5 to 100 μl of hCG or test sample, and then adding 10 to 300 μl of reagent (2), react at 0 to 40°C. After 1 to 72 hours of reaction, a certain amount of reagent (5)
Add and stir thoroughly, and further react at 0 to 40°C for 0.5 to 24 hours. After the reaction is completed, the second antibody-bound carrier is washed with reagent (7), and then 10 to 1000 μl of reagent (6) is added to start the enzyme reaction. After reacting at 20-40°C for 0.5-24 hours, 1-5 ml of reagent (8) is added to stop the reaction. Measure the absorbance or fluorescence intensity in the reaction solution to determine the hCG concentration in the test solution. In the above-mentioned immunochemical measurement method using the Sand-Deutsch method, an excess amount of antibody held on a carrier is added to a test solution containing an unknown amount of antigen and reacted (first reaction), and then labeled with a labeling agent. A fixed amount of excess antibody is added and reacted (second reaction). The activity of the labeling agent retained on the carrier or the labeling agent not retained on the carrier is measured. The first reaction and the second reaction may be performed simultaneously or at different times. Generally, in an immunochemical assay using the Sandermansch method, the antibodies used in the first reaction and the second reaction are prepared from the same antiserum. However, in the immunochemical measurement method of the present invention using the Sanderutsch method, which specifically measures antigens, in order to eliminate measurement errors due to cross-reaction with protein hormones that have a chemical structure similar to the antigen, it is necessary to The antibody and the antibody used in the second reaction are two types of antibodies that do not have overlapping antigen-determining sites, and one of the antibodies is an antibody that specifically reacts with the antigen. For example, a quantitative kit used in the immunochemical measurement of hCG using the Sanderutsch method described above mainly consists of (1) an antibody supported on a carrier, and (2) an antibody labeled with β-Gal (of the present invention). (lyophilized product) (3) 0 to 100 IU of standard hCG (4) Buffer used for diluting the reagents (1) to (3) above and the test sample (a buffer solution that can be used to dilute the reagent and test sample) (5) After incubation, the buffer used for washing the carrier (the buffer used for washing the carrier) Any buffer that can be used for washing the carrier may be used, examples of which include phosphate buffer or glycine buffer. (6) Enzyme substrate (preferably 4-methylumbelliferyl) β-D-galactoside or orthonitrophenyl-β-D-galactoside), a buffer used to dissolve the enzyme substrate (preferably a phosphate buffer), and a buffer used to stop the enzyme reaction (preferably a carbonate buffer or glycine buffer solution)
can be mentioned. Moreover, the above (1) and (2) may be mixed in advance. The kit described above is preferably used, for example, in the following manner. Approximately 10 to 200 μl of standard hCG or test solution and reagent (4)
Add and dilute a certain amount of reagent (1), then reagent (2).
After adding about 10 to 300 μl of the solution, react at about 0 to 40°C. After reacting for about 1 to 24 hours, the carrier is washed with reagent (5) and the activity of the labeling agent bound on the carrier is measured. Add about 10 to 1000 μl of substrate solution and incubate at about 20 to 40°C for about 0.5 to 1000 μl.
After 24 hours of reaction, the enzyme reaction is stopped and the absorbance in the reaction solution is measured. The present invention will be explained in more detail below with reference to examples and reference examples, but it goes without saying that these do not limit the scope of the present invention. In the following reference examples, thin layer chromatography was performed using a silica gel plate 60F ₂₅₄ manufactured by Merck and the following developing solvent, unless otherwise specified. Rf ¹ : Chloroform: methanol = 95:5 Rf ² : Chloroform: methanol: acetic acid = 9:1:0.5 Rf ³ : Ethyl acetate: pyridine: acetic acid: water = 60:20:6:10 Rf ⁴ : n-butanol: Pyridine: acetic acid: water = 30:20:6:24 Rf ⁵ : ethyl acetate: n-butanol: acetic acid: water = 1:1:1:1 Rf ⁶ : n-butanol: acetic acid: water = 12:3:5 In this specification, when amino acids, peptides, protective groups, active groups, etc. are indicated by abbreviations,
They are IUPAC - IUB Commission on
Abbreviations based on Biological Nomenclture or common abbreviations in the field, examples of which are listed below. Furthermore, when an amino acid or the like can have optical isomers, the L-isomer is indicated unless otherwise specified. Ala: Alanine Pro: Proline Ser: Serine Leu: Leucine Arg: Arginine Gly: Glycine Asp: Aspartic acid Thr: Threonine Ile: Isoleucine Gln: Glutamine Glu: Glutamic acid Z: Benzyloxycarbonyl OBu ^t : t-Butyl ester HONB: N- Hydroxy-5-norbornene-2,3-dicarboximide ONB: N-hydroxy-5-norbornene-
2,3-dicarboximide ester DMF: N,N'-dimethylformamide DCC: N,N'-dicyclohexylcarbodiimide DMSO: Dimethyl sulfoxide THF: Tetrahydrofuran HOBt: 1-hydroxy-benzotriazole OSu: N-hydroxysuccinimide ester ECDI :1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide CMCT:1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide meth-
Para-toluenesulfonate GLA: Glutaraldehyde m-MBHS: Meta-maleimidobenzoyl-
N-hydroxysuccinimide ester p-MCHS: para-maleimidomethylcyclohexane-1-carboxyl-N-hydroxysuccinimide ester reference example 1 hCG-βC-terminal fragment peptide (123-
145) Production: (a) Production of Z―Pro―Gln―OBu ^t : Z―Gln―
12.5 g of ^OBut is dissolved in 500 ml of methanol and catalytically reduced using palladium black as a catalyst. After removing the solvent by filtration and distilling off the solvent, the residue is dissolved in 300 ml of ethyl acetate. In this Z-Pro-OH9.7
Z- synthesized from g, HONB8.4g, and DCC8.8g
Add 200 ml of Pro-ONB ethyl acetate solution and stir for 5 hours. Add the reaction solution to 0.2N hydrochloric acid, 4%
Wash with sodium bicarbonate and water, and dry with anhydrous sodium sulfate. Ethyl acetate is distilled off, and petroleum benzine and a small amount of ether are added to the residue to form crystals, which are then recrystallized from the same solvent system. Yield 13.1g (81.5%), melting point 86-88℃, [α] ²⁶ _D
-64.8゜(c=0.5, methanol), Rf ¹ 0.42,
^Rf2 0.73. Elemental analysis C ₂₂ H ₃₁ C ₆ N ₃ : Calculated value C 60.95;
H 7.21; N 9.69, analysis value: C 61.04; H
7.20; N 9.49. (b) Structure of Z-Leu-Pro-Gln-OBu ^t : Z-
Dissolve 13 g of Pro-Gln-OBu ^t in 500 ml of methanol and reduce in a hydrogen stream using palladium black as a catalyst. The catalyst was filtered off, the solvent was distilled off, the residue was dissolved in 300 ml of ethyl acetate, and Z-Leu-
Add an ethyl acetate solution of Z-Leu-ONB synthesized from 7.9 g of OH, 6.5 g of HONB, and 6.8 g of DCC,
Stir for 5 hours. Add the reaction solution to 0.2N hydrochloric acid, 4
%-Sodium hydrogen carbonate solution, wash with water, dry over anhydrous sodium sulfate, and evaporate the solvent. Add petroleum benzine to the residue and filter it as a powder. Yield 10.6g (66.2%), melting point 74-77℃,
[α] ²³ _D −81.4° (c=0.6, methanol), Rf ¹ 0.38
,
^Rf2 0.66. Elemental analysis C ₂₈ H ₄₂ O ₇ N ₄ : Calculated value: C
61.52; H 7.74; N 10.25. Analysis value: C
61.61; H 7.94; N 9.92. (c) Production of Z―Ile―Leu―Pro―Gln―OBu ^t : Z
-Leu-Pro-Gln-OBu ^t 10.6g in methanol
After catalytic reduction using palladium black as a catalyst, methanol was distilled off and the residue was dissolved in 300 ml of ethyl acetate. Z―Ile― to this
Add 200 ml of an ethyl acetate-dioxane mixed solution (1:1) of Z-Ile-ONB synthesized from 5.1 g of OH, 4.2 g of HONB, and 4.4 g of DCC, and stir for 16 hours. The solvent was distilled off from the reaction solution, and the residue was dissolved in 400 ml of ethyl acetate, washed with 0.2N hydrochloric acid, 4% aqueous sodium bicarbonate, and water, and dried over anhydrous sodium sulfate. The solvent was distilled off, petroleum benzine was added to the residue, and the powder was collected by filtration. Yield 12.1g (94.5%), melting point 78-80℃ (decomposition), [α] ²³ _D -87.0゜ (c = 0.42, methanol),
Rf ¹ 0.23, Rf ² 0.67. Elemental analysis C ₃₄ H ₅₃ O ₈ N ₅ Calculated value: C 61.89;
H 8.10; N 10.62. Analysis value: C 62.35; H
8.31; N 10.16. (d) Production of Z-Pro-Ile-Leu-Pro-Gln-OBu ^t : Dissolve 12 g of Z-Ile-Leu-Pro-Gln-OBu ^t in 500 ml of methanol and use palladium black as a catalyst in a hydrogen stream. After reduction, the catalyst is filtered off and the solvent is distilled off. Dissolve the residue in 100ml of DMF, and add 4.7g of Z-Pro-OH and 3.0g of HOBt.
was added, cooled to 0℃, added 4.3g of DCC, and cooled to 0℃.
Stir for 4 hours at room temperature and 10 hours at room temperature. After the precipitate was filtered off and the solvent was distilled off, the residue was dissolved in 400 ml of ethyl acetate, washed with 0.2N hydrochloric acid, 4% aqueous sodium bicarbonate, and water, and dried over anhydrous sodium sulfate. The solvent is distilled off, ether is added to the residue, the mixture is heated, the supernatant is removed, ether is further added, and the mixture is filtered as a powder. Yield 12.6g (91.5%), melting point 83-87℃ (decomposition), [α] ²³ _D -121.0゜ (c = 0.5, methanol),
Rf ¹ 0.31, Rf ² 0.84. Elemental analysis C ₃₉ H ₆₀ O ₉ N ₆ Calculated value: C 61.88;
H 7.99; N 11.10. Analysis value: C 62.04; H
8.21; N 10.70. (e) Z―Thr―Pro―Ile―Leu―Pro―Gln―
Manufacture of OBu ^t : Z-Pro-Ile-Leu-Pro-Gln
-Dissolve 12.5g of OBu ^t in 500ml of methanol and reduce in a hydrogen stream using palladium black as a catalyst. After filtering off the catalyst and distilling off the solvent, the residue is dissolved in 100 ml of DMF. In this solution, Z-
Add and dissolve Thr-OH4.2g and HOBt2.7g,
Cool to 0°C. Next, 3.7 g of DCC was added and the mixture was stirred at 0°C for 4 hours and at room temperature for 8 hours, and then the precipitate was filtered off. The solvent was distilled off and the residue was dissolved in ethyl acetate.
Extract to 400ml, wash with 0.2N hydrochloric acid, 4% sodium bicarbonate solution, and water, and dry over anhydrous sodium sulfate. The solvent was distilled off, ether was added to the residue, and the powder was collected by filtration. Yield 11.8g (86.8%), melting point 101-105℃,
[α] ²³ _D -124.3° (c=0.58, methanol).
Rf ¹ 0.20, Rf ² 0.68. Elemental analysis C ₄₃ H ₆₇ O ₁₁ N ₇ Calculated value: C
60.19; H 7.87; N 11.43. Analysis value: C
59.54; H 7.91; N 11.19 (f) Z―Asp(OBu ^t )―Thr―Pro―Ile―Leu―
Manufacture of Pro-Gln-OBu ^t : Z-Thr-Pro-Ile
-Leu-Pro-Gln-OBu ^t 11.8g in methanol
Dissolve in 500 ml, reduce in a hydrogen stream using palladium black as a catalyst, filter off the catalyst, distill off the solvent, and dissolve the residue in 100 ml of DMF. Add 4.5 g of Z-Asp(OBu ^t )-OH and 2.3 g of HOBt to this, dissolve, and cool to 0°C. Add 3.2 g of DCC and stir at 0°C for 4 hours and at room temperature for 10 hours, filter off the precipitate, and distill off the solvent. Add 150 ml of ethyl acetate to the residue, filter the precipitated gel, crystallize from ethyl acetate and ether, and filter with ether. Yield 12.15g (85.9%), melting point 94-96℃ (decomposition), [α] ²¹ _D -109.1゜ (c = 0.59, methanol),
Rf ¹ 0.13, Rf ² 0.47. Elemental analysis C ₅₁ H ₈₀ O ₁₄ N ₈・H ₂ O Calculated value: C
58.49; H 7.89; N 10.70. Analysis value: C
58.60; H 8.07; N 10.71. (g) Z―Ser―Asp(OBu ^t )―Thr―Pro―Ile―
Manufacturing of Leu-Pro-Gln-OBu ^t : Z-Asp
(OBu ^t )―Thr―Pro―Ile―Leu―Pro―Gln―
12 g of ^OBut is dissolved in 500 ml of methanol and reduced in a hydrogen stream using palladium black as a catalyst.
After filtering off the catalyst and distilling off the solvent, the residue was collected together with 2.93 g of Z-Ser-OH and 2.0 g of HOBt.
Dissolve in 100 ml of DMF and cool to 0°C. to this
Add 2.8 g of DCC and stir at 0°C for 4 hours and at room temperature for 12 hours, filter off the precipitate, and distill off the solvent.
Dissolve the residue in 500 ml of ethyl acetate, wash with 0.2N hydrochloric acid, 4% sodium bicarbonate, and water.
After drying over anhydrous sodium sulfate, the solvent is distilled off. Add ethyl acetate and ether to the residue and filter it as a powder. Yield 11.7g (90.0%), melting point 111-115℃
(decomposition), [α] ²¹ _D -112.3° (c=0.63, methanol), Rf ¹ 0.06, Rf ² 0.31. Elemental analysis C ₅₄ H ₈₅ O ₁₆ N ₉ H ₂ O Calculated value: C
57.17; H 7.73; N 11.11. Analysis value: C
57.34; H 7.77; N 11.14. (h) Z―Pro―Ser―Asp (OBu ^t )―Thr―Pro―
Manufacture of Ile-Leu-Pro-Gln-OBu ^t : Z-Ser
-Asp(OBu ^t )-Thr-Pro-Ile-Leu-Pro-
Dissolve 11.6g of Gln-OBu ^t in 500ml of methanol,
It is reduced in a stream of steam using palladium black as a catalyst. After filtering off the catalyst and distilling off the solvent, the residue was poured into DMF100 with 4.3g of Z-Pro-ONB.
ml and stir at room temperature for 16 hours. The solvent was distilled off and the residue was extracted into 500ml of ethyl acetate.
0.2N-hydrochloric acid, 4%-sodium bicarbonate water,
After washing with water and drying over anhydrous sodium sulfate, the solvent is distilled off. Add ether to the residue and filter it as a powder. Yield 11.1g (88.1%), melting point 117-120℃,
[α] ²¹ _D -119.2° (c=0.61, methanol),
^Rf2 0.45. Elemental analysis C ₅₉ H ₉₂ O ₁₇ N ₁₀ H ₂ O Calculated value: C
57.54; H 7.69; N 11.38. Analysis value: C
57.44; H 7.69; N 11.38. (i) Z―Gly―Pro―Ser―Asp (OBu ^t )―Thr―
Production of Pro-Ile-Leu-Pro-Gln-OBu ^t : Z
-Pro-Ser-Asp(OBu ^t )-Thr-Pro-Ile-
Leu-Pro-Gln-OBu ^t 10g and methanol 500ml
The solution is reduced in a hydrogen stream using palladium black as a catalyst, and after the catalyst is filtered off, the solvent is distilled off. Residue with Z-Gly-ONB4g
Dissolve in 80ml of DMF and stir at room temperature for 12 hours.
The solvent was distilled off, and the residue was dissolved in 500 ml of ethyl acetate, washed with 0.2N hydrochloric acid, 4% aqueous sodium bicarbonate, and water, dried over anhydrous sodium sulfate, and the solvent was distilled off. Dissolve the residue in 180 ml of methanol, add 4.5 ml of hydrazine hydrate, and stir at room temperature for 16 ml.
After stirring for some time, the solvent is distilled off. The residue was dissolved in 500 ml of ethyl acetate, washed with water, dried over anhydrous sodium sulfate, and the solvent was distilled off. Add ether to the residue and filter it as a powder. Yield 7.35g (70.2%), melting point 131-135℃
(decomposition), [α] ²² _D -119.4° (c = 0.35, methanol), Rf ² 0.27. Elemental analysis C ₆₁ H ₉₅ O ₁₈ N ₁₁ H ₂ O Calculated value: C
56.86; H 7.59; N 11.96. Analysis value: C
56.65; H 7.68; N 12.02. (j) Manufacturing of Z-Leu-Pro-OBu ^t : Z-Pro-
20.2 g of ^OBut is dissolved in 800 ml of methanol and reduced in a hydrogen stream using palladium black as a catalyst.
The catalyst was filtered off, the solvent was distilled off, the residue was dissolved in 300 ml of ethyl acetate, 26.3 g of Z-Leu-OSu was added, and the mixture was stirred for 16 hours. Add the reaction solution to 0.2N hydrochloric acid,
Wash with 4% sodium bicarbonate water and water, dry over anhydrous sodium sulfate, and evaporate the solvent to obtain an oil. Yield 27.6g (100%), Rf ¹ 0.71, Rf ² 0.78. (K) Production of Z-Arg(NO ₂ )-Leu-Pro-OBu ^t : Add 13.8 g of Z-Leu-Pro-OBu ^t to methanol.
Dissolve in 500ml and use palladium black as a catalyst.
Reduction is carried out in a stream of hydrogen, the catalyst is filtered off and the solvent is distilled off. Residue and Z―Arg―(NO ₂ )―
Dissolve 11.7g of OH and 6.7g of HOBt in 200ml of DMF,
Cool to 0°C. Add 7.5g of DCC to this and bring it to 0°C.
Stir for 4 hours at room temperature and 12 hours at room temperature, filter off the precipitate, and distill off the solvent. 600% of the residue in ethyl acetate
ml, washed with 0.2-hydrochloric acid, 4% aqueous sodium bicarbonate, and water, dried over anhydrous sodium sulfate, and the solvent was distilled off. The residue is left to stand, and the crystals precipitated are filtered with ether and then recrystallized from methanol. Yield 12.4g (60.6%), melting point 170-172℃,
[α] ²⁶ _D -67.8° (c=0.48, methanol),
^Rf3 0.77. Elemental analysis C ₂₉ H ₄₅ O ₈ N ₇ Calculated value: C 56.20;
H 7.32; N 15.82, analysis value: C 55.79; H
7.16; N 15.85. (l) Production of Z―Ser―Arg―Leu―Pro―OBu ^t :
12.3 g of Z-Arg(NO ₂ )-Leu-Pro-OBu ^t was dissolved in 500 ml of methanol, 6.6 ml of 6N-hydrochloric acid was added, and then reduced in a hydrogen stream using palladium black as a catalyst, and the catalyst was filtered off. Afterwards, the solvent is distilled off. Residue with 2.8ml of triethylamine
Dissolve in 100ml of DMF and filter off the precipitated triethylamine hydrochloride. Add Z―Ser― to the filtrate.
Add 4.8g of OH and 5.4g of HONB and cool to 0℃.
Add 4.95 g of DCC and stir at 0°C for 4 hours and at room temperature for 16 hours. After filtering off the precipitate and distilling off the solvent, the residue was extracted into 500 ml of ethyl acetate and thoroughly washed with water saturated with sodium chloride. Dry over anhydrous sodium sulfate and evaporate the solvent to obtain a candy-like residue. ^Rf3 0.53. (m) Production of Z―Pro―Ser―Arg―Leu―Pro―OBu ^t : Z―Ser―Arg―Leu―Pro―OBu ^t 10.5g
was dissolved in 500 ml of methanol with 3 ml of 6N hydrochloric acid, and reduced in a hydrogen stream using palladium black as a catalyst. The catalyst was filtered off, the solvent was distilled off, the residue was dissolved in 100 ml of DMF, and triethylamine was added.
Add 2.52ml to neutralize. After filtering off the precipitated triethylamine hydrochloride, Z-Pro-
Add 8.4 g of ONB, stir at room temperature for 16 hours, distill off the solvent, add 300 ml of ethyl acetate and 300 ml of salt-saturated water to the residue, and mix well. After standing still, the oily substance that precipitates in the water layer is collected, and ether is added to it to form a powder. Next, this powder is dissolved in methanol, insoluble matter is filtered off, the solvent is distilled off, ether is added again, and the powder is collected by filtration. Yield 8.45g (70.8%), melting point 115-120℃
(decomposition), [α] ²² _D -84.7° (c = 0.53, methanol), Rf ³ 0.41. Elemental analysis C ₃₇ H ₅₈ O ₉ N ₈・HCl・H ₂ O Calculated value: C 54.63; H 7.56; N 13.78; Cl
4.36. Analysis value: C 54.50; H 7.70; N
14.11; Cl 4.21. (n) Z―Ser―Pro―Ser―Arg―Leu―Pro―
Manufacture of OBu ^t : Z-Pro-Ser-Arg-Leu-Pro
-Dissolve 3.8g of OBu ^t in 200ml of methanol and reduce in a hydrogen stream using palladium black as a catalyst.
After filtering off the catalyst, distill off the solvent and remove the residue.
Dissolve in 30ml DMF. In this solution, Z-Ser-
DMF solution (10ml) of Z-Ser-ONB synthesized from 1.37g of OH, 1.24g of HONB and 1.30g of DCC
Add and stir at room temperature for 16 hours. After filtering off the precipitate, the solvent was distilled off, and ethyl acetate was added to the residue.
ml and 1 ml of acetonitrile to dissolve it, then add ether to precipitate it, turn it into a powder, and collect it by filtration. This powder was dissolved in 2 ml of a 1:1 mixture of Rf ³ solvent and ethyl acetate, and a silica gel column (5.6 x 9.0 cm) was packed with the same solvent.
and develop with the same solvent. A portion of 365 ml to 746 ml is collected, the solvent is distilled off, and ether is added to the residue, which is filtered as a powder. Yield 2.12g (50.2%), melting point 130-135℃
(decomposition), [α] ²² _D -83.5° (c = 0.38, methanol), Rf ³ 0.34. Elemental analysis C ₄₀ H ₆₃ O ₁₁ N ₉・HCl・H ₂ O Calculated value: C 53.35; H 7.39; N 14.00; Cl
3.94. Analysis value: C 53.67; H 7.45; N
13.72; Cl 3.52. (o) Production of Z―Ser―Leu―Pro―OBu ^t : Z―
Dissolve 13.8 g of Leu-Pro-OBu ^t in 700 ml of methanol and reduce in a hydrogen stream using palladium black as a catalyst. The catalyst was filtered off and the solvent was distilled off, and the residue was dissolved in 200 ml of acetonitrile along with 7.9 g of Z-Ser-OH and 8.9 g of HONB, and cooled to 0°C. Next, add 7.5 g of DCC and stir at 0°C for 4 hours and at room temperature for 16 hours. The precipitate was filtered off, the solvent was distilled off, and the residue was extracted into 500 ml of ethyl acetate.
Wash with 0.2-hydrochloric acid, 4% aqueous sodium bicarbonate, and water, dry over anhydrous sodium sulfate, and evaporate the solvent to obtain 17 g of an oil. This was dissolved in 15 ml of a mixed solvent of chloroform and methanol (200:3), applied to a silica gel column (5.4 x 20 cm) packed with the same solvent, and developed with the same solvent.
A portion of 1300-2100 ml was collected and the solvent was distilled off to give an oil. Yield 12.2g (73.1%), Rf ¹ 0.38, Rf ² 0.69. (p) Z―Ser―Leu―Pro―Ser―Pro―Ser―Arg
-Manufacture of Leu-Pro-OBu ^t : Z-Ser-Pro-
Ser―Arg―Leu―Pro―OBu ^t 1.0g with N-hydrochloric acid
Dissolve together with 1.2 ml in 60 ml of methanol, reduce in a hydrogen stream using palladium black as a catalyst, filter off the catalyst, distill off the solvent, and remove the residue.
Dissolve in 20ml DMF. Z-Ser-Leu-Pro-
Dissolve 700 mg of OBu ^t in 7 ml of trifluoroacetic acid,
After 50 minutes, the solvent was distilled off, the residue was washed with a mixed solvent of ether and petroleum ether (1:2), the washing liquid was removed, and the remaining oil was evaporated under reduced pressure under a vacuum pump until it became a powder. After drying, immediately add and dissolve in the above DMF solution together with 407 ml of HONB, and cool to 0°C. 466 mg of PCC was added thereto, stirred at 0°C for 6 hours and at room temperature for 16 hours, the precipitate was filtered off, and the solvent was distilled off. residue
³³³
A portion of ~572 ml was collected, the solvent was distilled off, ether was added to the residue, and the powder was collected by filtration. Yield 450mg (33.8%), melting point 110-120℃ (decomposition), [α] ²⁴ _D -106.6゜ (c = 0.31, methanol),
^Rf5 0.71. (q) Manufacture of Z-Pro-Pro-OBu ^t : Z-Pro-
Dissolve 10.1 g of OBu ^t in 500 ml of methanol, reduce with palladium black as a catalyst in a hydrogen stream, filter off the catalyst, distill off the solvent, and remove the residue with Z-Pro-
OH8.47g, HOBt5.4g and 300 ethyl acetate
ml and cooled to 0°C. Next, DCC7.5g
The mixture was stirred at 0°C for 4 hours and at room temperature for 12 hours, and the precipitate was filtered off. The filtrate is washed with 0.2-hydrochloric acid, 4% aqueous sodium bicarbonate, and water, dried over anhydrous sodium sulfate, and the solvent is distilled off. Filter the residue crystals with ether. Yield 9.85g (74.1%), melting point 94-96℃,
[α] ²² _D -116.9° (c=0.54, methanol),
Rf ¹ 0.58, Rf ² 0.72. Elemental analysis C ₂₂ H ₃₀ O ₅ N ₂ Calculated value: C 65.65;
H 7.51; N 6.96. Analysis value: C 65.42; H
7, 38; N 7.20. (r) Manufacturing of Z―Pro―Pro―Pro―OBu ^t : Z―
Dissolve 9 g of Pro-Pro-OBu ^t in 300 ml of methanol, reduce in a hydrogen stream using palladium black as a catalyst, filter off the catalyst, distill off the solvent, and dissolve the residue into 5.6 g of Z-Pro-OH, Dissolve in 250 ml of ethyl acetate along with 3.63 g of HOBt and cool to 0°C. Next, 5.1 g of DCC was added and stirred at 0°C for 6 hours and at room temperature for 16 hours, and the precipitate was filtered off. filtrate
0.2N-hydrochloric acid, 4%-sodium bicarbonate water,
Wash with water, dry over anhydrous sodium sulfate, and evaporate the solvent. The precipitated crystals are filtered with ether. Yield 9.6g (85.9%), melting point 155-157℃,
[α] ²² _D -176.0° (c=0.55, methanol),
Rf ¹ 0.40, Rf ² 0.69. Elemental analysis C ₂₇ H ₃₇ O ₆ N ₃・1/2H ₂ O Calculated value:
C 63.76; H 7.53; N 8.26. Analysis value: C
63.77; H 7.53; N 8.62. (s) Manufacture of Z―Ala―Pro―Pro―Pro―OBu ^t :
Z-Pro-Pro-Pro-OBu ^t 5g in methanol
Dissolved in 200 ml, reduced in a hydrogen stream using palladium black as a catalyst, filtered off the catalyst, distilled off the solvent, and the residue was 2.23 g of Z-Ala-OH,
Dissolved in 20ml of DMF with 1.62g of HOBt at 0°C.
Cool to Add 2.27g of DCC to this solution and
Stir at ℃ for 4 hours and at room temperature for 12 hours, and filter off the precipitate. The solvent was distilled off and the residue was diluted with 3 ml of 2%
Silica gel column (3.7 x 10.5 cm) dissolved in methanol/chloroform and packed with the same solvent.
and develop with the same solvent. Collect 170-380ml portions, distill off the solvent, and remove 4.05g (71.0ml) of oily substance.
%). Rf ¹ 0.33, Rf ² 0.67. (t) Z―Ala―Pro―Pro―Pro―Ser―Leu―Pro
―Ser―Pro―Ser―Arg―Leu―Pro―OBu ^t production: Z―Ser―Leu―Pro―Ser―Pro―Ser―
Arg-Leu-Pro-OBu ^t 400mg to N-hydrochloric acid 0.8ml
The mixture was dissolved in 80 ml of methanol and reduced in a hydrogen stream using palladium black as a catalyst. The catalyst is filtered off, the solvent is distilled off, the residue is dissolved in 10 ml of DMF, neutralized with 0.11 ml of triethylamine, and the precipitated triethylamine hydrochloride is filtered off.
Dissolve 233 mg of Z-Ala-Pro-Pro-Pro-OBu ^t in 2 ml of trifluoroacetic acid. After 50 minutes, the solvent is distilled off, ether is added to the residue, the powder is collected by filtration, and dried. This, along with 122 mg of HONB, was added to the above DMF solution to dissolve, and the mixture was cooled to 0°C. Add 140 mg of DCC to this solution, stir at 0°C for 6 hours and at room temperature for 12 hours, filter off the precipitate, and distill off the solvent. Ether is added to the residue, the powder is collected by filtration, and purified by reprecipitation from acetonitrile and ethyl acetate. Yield 430mg (82.1%), melting point 152-155℃ (decomposition), [α] ²⁴ _D -153.6゜ (c = 0.45, methanol),
Rf ³ 0.10, Rf ⁵ 0.54. Elemental analysis _{C72H112O19N16}・HCl・_H2O Calculated values: C 55.42; _H 7.43; _N 14.37; _Cl2.27 .
Analysis value: C 56.21; H 7.54; N 14.04; Cl
2.30 (u) Z―Ala―Pro―Pro―Pro―Ser―Leu―Pro
―Ser―Pro―Ser―Arg―Leu―Pro―Gly―
Pro―Ser―Asp(OBu ^t )―Thr―Pro―Ile―
Manufacture of Leu-Pro-Gln-OBu ^t : Z-Gly-Pro
―Ser―Asp(OBu ^t )―Thr―Pro―Ile―Leu―
363 mg of Pro-Gln-OBu ^t is dissolved in 30 ml of methanol and reduced in a hydrogen stream using palladium black as a catalyst. The catalyst is filtered off, the solvent is distilled off and the residue is dissolved in 10 ml of DMF. Z-Ala-Pro-
Pro―Pro―Ser―Leu―Pro―Ser―Pro―Ser
-Dissolve 430 mg of Arg-Leu-Pro-OBu ^t in 3 ml of trifluoroacetic acid and leave at room temperature for 45 minutes. The solvent was distilled off, ether was added to the residue, the powder was collected by filtration, and after drying, along with 103 mg of HONB,
Add to DMF solution to dissolve and cool to 0°C.
Next, 118 mg of DCC was added and the mixture was heated at 0℃ for 6 hours and at room temperature.
Stir for 40 hours. The precipitate is filtered off, the solvent is distilled off, ether is added to the residue, the powder is collected by filtration, and further purified by reprecipitation from DMF and ether. Yield 700mg (95.5%), melting point 120-125℃ (decomposition), [α] ²³ _D 127.7゜ (c = 0.12, methanol),
Rf ⁵ 0.62, Rf ⁶ 0.44. Elemental analysis C ₁₂₁ H ₁₉₁ O ₃₄ N ₂₇・HCl・2H ₂ O Calculated value: C 53.12; H 7.80; N 14.94; Cl
1.40. Analysis value: C 53.39; H 7.62; N
15.10; Cl 1.54. (v) H-Ala-Pro-Pro-Pro-Ser-Leu-Pro
―Ser―Pro―Ser―Arg―Leu―Pro―Gly―
Pro―Ser―Asp―Thr―Pro―Ile―Leu―Pro
-Production of Gln-OH [hCG-β less than C fragment peptide (123-145)]: Z-Ala-Pro-
Pro―Pro―Ser―Leu―Pro―Ser―Pro―Ser
―Arg―Leu―Pro―Gly―Pro―Ser―Asp
(OBu ^t )―Thr―Pro―Ile―Leu―Pro―Gln―
580 mg of ^OBut is dissolved in 50 ml of methanol together with 0.45 ml of N-hydrochloric acid, and reduced in a hydrogen stream using palladium black as a catalyst. The catalyst was filtered off, the solvent was distilled off, 1 ml of water was added to the residue, the mixture was distilled off again, and the residue was dissolved in 10 ml of 90% trifluoroacetic acid water and left at room temperature for 60 minutes. Distill the solvent, add 10ml of water to the residue, dissolve it, and add Amberlite IRA.
- Apply to a 410 (acetic acid type) resin column (1 x 5 cm) and collect the eluate. Wash the resin thoroughly with 50 ml of water, and combine the washings with the previous eluate and freeze-dry to obtain 360 mg of white powder. Next, this was applied to a Sephadex LH-20 column (2×
83cm) and develop with the same solvent. 70~100ml
The fractions are collected and lyophilized, and further subjected to rechromatography on the same column, and the fractions containing the target product are collected and lyophilized to obtain a white powder. Yield 185 mg (35.2%), [α] ²³ _D -194.5° (c=
0.13, 0.1N-acetic acid), Rf ⁵ 0.06, Rf ⁴ (cellulose) 0.78. Amino acid analysis value (theoretical value) Arg1.00(1),
Asp1.00(1), Thr1.00(1), Ser3.74(4), Glu1.03
(1), Pro9.59(9), Gly0.98(1), Ala0.94(1),
Ile0.95(1), Leu2.97(3), average recovery rate 79.3%. Reference example 2 hCG-β C-terminal fragment peptide (130
-145) Manufacturing: Z-Gly-Pro-Ser-Asp
Dissolve 2.16 g of (OBu ^t )-Thr-Pro-Gln-OBu ^t in 100 ml of methanol, use palladium black as a catalyst,
Reduce in a hydrogen stream. The catalyst is filtered off, the solvent is distilled off and the residue is dissolved in 25 ml of DMF. Add Z―Ser―Pro―Ser―Arg―Leu―Pro― to this solution.
Z-Ser-Pro- obtained by TFA treatment of 1.5g of OBu ^t
Add Ser-Arg-Leu-Pro-OH and 1.22 g of HONB and cool to 0°C. Next, add 701 mg of DCC and
The mixture was stirred for 6 hours at room temperature and for 40 hours at room temperature, the precipitate was filtered off, and the solvent was distilled off. Ethyl acetate and ether are added to the residue to form a precipitate, which is collected by filtration. This was dissolved in 5 ml of Rf ³ solvent, applied to a silica gel column (5.7 x 9 cm) packed with the same solvent, and developed with the same solvent. Collect portions of 534-914 ml, distill off the solvent, add ether to the residue, and filter. Yield 1.1g (33.9%), Rf ³ 0.20. Next, 70 mg of this protected peptide was dissolved in 10 ml of methanol and reduced in a hydrogen stream using palladium black as a catalyst.
The catalyst was filtered off and the solvent was distilled off. 90% residue
Dissolve in 1 ml of TFA, evaporate the solvent after 60 minutes, add 3 ml of water to the residue, and dissolve. This aqueous solution was applied to an Amberlite IRA-410 (acetic acid type) column (1 x 1
cm) and lyophilize. Next, add this to N-acetic acid
Dissolve in 0.5 ml, apply to a Sephadex LH-20 column packed with N-acetic acid, and develop with the same solvent. Object [H-Ser-Pro-Ser-Arg-Leu-
Pro―Gly―Pro―Ser―Asp―Thr―Pro―Ile―
The fraction containing Leu-Pro-Gln-OH (hCG-β C-terminal fragment peptide (130-145)) is collected and freeze-dried to obtain a white powder. Yield 22 mg (36.1%), [α] ²³ _D -159.3゜(c=
0.15, 0.1N-acetic acid), Rf ⁵ 0.15. Amino acid analysis (theoretical value) Arg1.01(1), Asp1.01
(1), Thr0.98(1), Ser2.57(3), Glu0.96(1), Pro4.85
(5), Gly1.00(1), Ile0.96(1), Leu2.04(2), average recovery rate 81.1%. Reference example 3 hCG-βC-terminal fragment peptide (130-
145) and β-Gal.: 1.7 mg of the hCG-β C-terminal fragment peptide (130-145) obtained in Reference Example 2 was mixed with 0.5 ml of 0.05 M phosphate buffer (PH7.0) and DMSO0. Dissolve in .5ml,
This solution contains 50 μl of THF containing 780 μg of m-MBHS.
was added and reacted at 30°C for 30 minutes. reaction mixture
Pass through a Sephadex G-15 column (0.9 x 55 cm) equilibrated with 0.02M phosphate buffer (PH6.3),
Excess reagent and maleimidized peptide were separated. Gradually add 0.5 ml of the obtained maleimide solution to 0.5 ml of β-Gal solution (1 mg/ml) diluted in 0.02 M phosphate saline buffer (PH7.5), and leave at 5°C overnight with occasional shaking. Made it react. After the reaction is complete, add 0.02M phosphate saline buffer (PH7.0)
Purify the enzyme using Sepharose 6B column chromatography and separate the enzyme-containing fraction.
hCG-βC-terminal fragment peptide (130-145)
A conjugate of β-Gal. and β-Gal. was obtained. The physical properties of this conjugate will be described below. This conjugate is a synthetic substrate used in EIA such as O-nitrophenyl-β-D-galactopyranoside and 4-
Methylumbelliferyl-β-D-galactopyranoside is decomposed to liberate O-nitrophenol and 4-methylumbelliferone, respectively. Synthetic substrate 4-methylumbelliferyl-β-
When D-galactopyranoside is used, the Michaelis constant of this conjugate shows the same value as the original β-D-galactosidase. The optimum pH for enzyme activity is 6.5 to 7.3. Approximately 90% or more of this conjugate with enzyme activity is anti-inflammatory.
It has reactivity with hCG antibodies, and its immunoactivity and enzyme activity are stable for more than 4 months when stored under refrigeration. The molecular weight is approximately 550,000, and the molar ratio of hCG-βC-terminal fragment peptide (130-145)/enzyme is approximately 9.
It is. It is easily soluble in aqueous solvents with a pH of 5 to 9. See Figure 1 for the ultraviolet absorption spectrum. See Table 1 for amino acid analysis values. Other physical properties are the physical properties of β-Gal.
Enzymes”, Volume 7 (1972), Page 617,
Written by Boyer, Academic Press, New York.
London).

[Table] Reference Example 4 Production of specific anti-hCG antibody: (1) Production of anti-hCG serum: Approximately 10,000 U/ml purified from human urine by a known method.
Dissolve 1 mg of hCG in 1 ml of physiological saline and add Freund's
Complete adijuvant, “Biochemistry of Immunity”, Tachibana et al., Kyoritsu Shuppan Co., Ltd. (1967)] Add 1 ml and mix well to make an emulsion, and inject this into the muscles of both thighs and several subcutaneous places on the back of rabbits. Inject. The above procedure is performed 5 times every 3 weeks, and one week after the final immunization, blood is collected and a pilot assay is performed. As a result, an anti-hCG serum with strong affinity for the C-terminal fragment peptide (130-145)-enzyme conjugate of hCG-β was obtained. (2) Production of specific anti-hCG antibody: 5 mg of hCG-β C-terminal fragment peptide (123-145) obtained by the method of Reference Example 1.
Dissolved in 8 _ml of 0.1M NaHCO containing 0.5M NaCl,
Bromcyan-activated Cepharose 4B (Pharmacia Huain, pre-washed with N/1000HCl)
Chemicals Co., Ltd.) and stirred overnight at 5°C. After the reaction, 0.1M containing the same 0.5M NaCl,
Wash thoroughly with NaHCO ₃ and then with HCl to pH 8.
After adding 10 ml of 0.5M ethanolamine prepared in
0.1M acetate buffer (PH4.0) containing (2) 1M NaCl
0.1M borate buffer (PH8.0) containing (3)
0.02M borate buffer (PH
8.0) and packed into a column. 8 ml of the anti-hCG serum described in (1) above was subjected to salting-out precipitation using 1.5 g of anhydrous sodium sulfate, and the resulting γ-globulin fraction was applied to a C-terminal fragment peptide (123-145)-conjugated Sepharose 4B column (0.9 x 4 cm). 0.02M borate buffer (PH
8.0) to remove anti-hCG antibodies that cross-react with hLH, thyroid-stimulating hormone, and follicle-stimulating hormone. Then, by elution with 0.17M glycine-hydrochloric acid buffer (PH2.3), a specific anti-hCG antibody having strong affinity for the C-terminal peptide of hCG-β was obtained (protein amount: 1.8 mg). The physical properties of this antibody will be described below. This antibody is a hCG-β-Gal. conjugate (see Reference Example 5) and an hCG-BC end fragment peptide (130-145)-β-Gal. conjugate with an enzymatic activity of approximately 2 μU at a final dilution concentration of 80 ng/ml. (see Reference Example 3), respectively. The optimal pH for antigen binding activity of this antibody is 6-9. This antibody is stable for over 1 year when stored refrigerated. It has a molecular weight of approximately 150,000 and contains approximately 3% sugar. Easily soluble in aqueous solvents with a pH of 2-12. In electrophoresis, γ- moves toward the cathode.
Belongs to the globulin fraction. For ultraviolet absorption spectra, see Figure 2. For amino acid analysis values, see Table 2. For other physical properties, see Physical Properties of Immunoglobulin G ("Medical Immunology", p. 61, by Kikuchi et al., Nankodo (1976))
is the same as

[Table] Reference Example 5 Production of a conjugate of hCG and β-Gal. Dissolve 1 mg of hCG (approximately 10,000 IU/mg) in 1 ml of 0.05 M phosphate buffer (PH7.0), and add 78 μg of m-Gal.
Add 50 μl of THF containing MBHS while stirring.30
The reaction was carried out at ℃ for 30 minutes. The reaction mixture was immediately passed through a Sephadex G-25 column (0.9 x 55 cm) to remove excess reagent. Elution is performed with 0.05M citrate buffer (PH5.5), and maleimidated hCG is fractionated. β-Gal. solution (1 mL
mg/ml) was added dropwise to 0.5 ml and reacted at room temperature for 2 hours. After the reaction, the hCG-β-Gal
The conjugate was adsorbed and then eluted with 0.05M phosphate saline buffer (PH7.5) containing 0.2M α-methylmannoside, and fractions showing hCG immunoreactivity as well as enzymatic activity were pooled. This active fraction was further purified by Sepharose 6B column chromatography using 0.02M phosphate saline buffer (PH7.0), and the enzyme-containing fraction was separated to obtain a hCG-β-Gal conjugate. Reference example 6 (1) Anti-hCG-βC-terminal fragment peptide (123
-145) Production of antibody: Fragment peptide prepared in Reference Example 1 [hCG-βC-terminal fragment peptide (123-
145)] 25 mg and 50 mg of bovine thyroglobulin (abbreviated as BTG) in 0.2 M phosphate buffer (PH7.3)
Add 4 ml of 5% GLA aqueous solution, stir at room temperature for 3 hours, and then dialyze at 4°C (2 x 4 water).
The immunogen was obtained by freeze-drying. This hCG-βC
Terminal fragment peptide (123-145) -
Dissolve 1.5 mg of BTG condensate in 0.75 ml of physiological saline,
0.75 ml of Freund's complete adjuvant was added and mixed well to form an emulsion, which was injected intramuscularly into both thighs and subcutaneously at several points on the back of the rabbit. The above procedure was performed four times at four-week intervals, and one week after the final immunization, blood was collected, centrifuged, and antiserum was collected to obtain anti-hCG-βC-terminal fragment peptide (123-145) serum F5C. Next, the γ-globulin fraction obtained by salting out the antiserum F5C with ammonium sulfate in a conventional manner was
It was applied to a Sepharose 4B column (diameter 0.9 cm, length 4 cm) to which mg of hCG was bound. 0.02M borate buffer (PH
Anti-hCG-β C-terminal fragment peptide (123-145) F5CS with high affinity for hCG was prepared by washing the column with 8.0) and then eluting with 0.17M glycine-HCl buffer (PH2.3). did. (2) Preparation of antibody-bound solid phase Polystyrene balls (diameter 6.4 mm, Precision
Plastics Ball Co.Chicago, USA) 300 pieces
Add 50ml of 0.01M phosphate buffer (PH7.7) and heat at 56°C.
It was heated to Next, 2 mg of F5CS prepared in (1) above was added and incubated at 56°C for 2 hours. 0.1
After washing with 0.05M phosphate buffer (PH 7.0) containing % BSA, it was stored in a cool place until use. (3) Production method of conjugate of anti-hCG antibody and β-Gal. Approximately 10,000 IU/
The anti-hCG serum obtained by immunizing a rabbit with 1 mg of hCG using the method (1) above was salted out with ammonium sulfate, and 5 mg of hCG-βC-terminal fragment peptide (123-145) was bound to a Sepharose 4B column ( A fractionated resistor T7CS was prepared that can pass through affinity chromatography with a diameter of 0.9 cm and a length of 4 cm. T7CS4mg 0.05M
Dissolved in 2 ml of phosphate buffer (PH7.0), add 200 μl of THF containing 400 μg of m-MBHS, and incubate at 30°C.
was allowed to react for 30 minutes. Sephadex G-25 in which the reaction mixture was equilibrated with 0.02M phosphate buffer.
(diameter 0.9 cm, length 55 cm) to separate excess reagent and maleimidized antibody. Gradually add 0.5 ml of the obtained maleimidized antibody solution to 0.3 ml of β-D-galactosidase solution (1 mg/ml) diluted in 0.02 M phosphate saline buffer (PH 7.5).
The mixture was allowed to react overnight at 5°C with occasional shaking. After the reaction is complete, add 0.02M phosphate saline buffer (PH7.0)
The fractions containing enzyme activity and antibody activity were purified by column chromatography using Sepharose 6B, and the fractions containing anti-hCG antibody and β
- Obtained a combination with Gal. Reference Example 7 Using the hCG enzyme immunoassay kit described below, the hCG concentration in the urine or serum of normal subjects and pregnant women was measured by EIA according to the procedure described below. The results are shown in Table 3. Enzyme immunoassay kit for hCG (1) A portion that binds about 40% of the enzymatic activity of the peptide-β-D-galactosidase conjugate added to the reaction system of the specific anti-hCG antibody obtained in Reference Example 4. (2) A portion of the freeze-dried product of the hCG-β chain C-terminal fragment peptide (130-145)-β-D-galactosidase conjugate obtained in Example 3, which has an enzymatic activity of about 0.4 μU. (3) O-100 IU standard human chorionic gonadotropin. (4) 0.15M NaCl, 0.5% human serum albumin, used for diluting the reagents and test samples in (1) to (3) above;
0.02M of PH7.3 containing 0.5% EDTA, 0.1% _NaN3
Phosphate buffer. (5) 2.5 (W/V)% suspension anti-rabbit IgG-conjugated cellulose. (6) 10μg4-methylumbelliferyl-β-D-
Galactoside (7) 0.1M NaCl, 1mM MgCl ₂ used for washing the cellulose in (5) and dissolving the enzyme substrate in (6),
PH with 0.1% bovine serum albumin, 0.1% _NaN3
7.0 of 0.02M phosphate buffer. (8) 0.1M carbonate buffer with pH 10.5. Procedure: Add 250 μl of reagent (4) and 50 μl of standard hCG or test sample to 100 μl of reagent (1), then add 100 μl of reagent (2), and react at 4°C. After 40 hours of reaction,
Add 100 μl suspension of reagent (5), stir thoroughly, and then heat to 20°C.
Let it react for 4 hours. After the reaction is completed, the cellulose is washed with reagent (7), and then 500 μl of reagent (6) is added to start the enzyme reaction. After reacting at 20°C for 16 hours, 3 ml of reagent (8) was added to stop the reaction. Measure the fluorescence intensity in the reaction solution and determine the hCG concentration in the test solution.

【table】

[Table] Reference Example 8 hCG immunochemical measurement kit and hCG measurement Using the hCG immunochemical measurement kit below, measure the hCG concentration in urine or serum of normal people and pregnant women according to the following procedure. It was measured. The results are shown in Table 2. Immunochemical measurement kit for hCG (1) 6.7 μg of anti-hCG per kit obtained in Reference Example 6
hCG-βC-terminal fragment peptide (123-
145) Polystyrene balls 6.4 mm in diameter sensitized with antibodies. (2) About lyophilized product of β-D-galactosidase standard anti-hCG antibody conjugate obtained in Example 6
Part with 400 μU of enzymatic activity. (3) 0 to 100 IU of standard hCG (4) Used for diluting the reagents and test samples in (2) to (3) above. 5% normal rabbit serum, 0.1% _NaN3 ,
0.1M phosphate buffer at PH7.4 containing 1mM _MgCl2 . (5) 20μg4-methylumbelliferyl-β-D-
Galactoside. (6) Contains 0.1% bovine serum albumin, 0.1% NaN ₃ and 1mM MgCl ₂ used for dissolving the enzyme substrate in (5).
0.02M phosphate buffer with PH7.0. (7) Used for cleaning the polystyrene balls in (1)
PH7.0 containing 0.1M NaCl, 1mM _MgCl2
0.05M phosphate buffer. (8) 0.1M carbonate buffer with pH 10.5. Procedure: Add reagent (4) to 100 μl of standard hCG or test sample
Add 400 μl, 1 reagent (1), and 100 μl of reagent (2), and react at room temperature. After 2 hours of reaction, wash the polystyrene ball with reagent (7), then add 400 μl of reagent (5) to start the enzyme reaction. After reacting at 37°C for 2 hours, 2.5 ml of reagent (8) was added to stop the reaction. Measure the fluorescence intensity in the reaction solution and check the hCG in the test solution.
Determine the concentration. The hCG concentration in the test solution was measured by the above method. The results are shown in Table 4.

[Table] Example 1 β-D-galactosidase (manufactured by Boehringer Mannheim, West Germany) was mixed with the following buffer (aqueous solution).
Dissolved in (a) and (b). The β-Gal concentration in each solution was 500 μg/ml. (a) 5% (W/V) sugar or sugar alcohol (types of sugar or sugar alcohol are shown in Table 5), 0.2
% bovine serum albumin, containing 0.001M _MgCl2
0.05M phosphate buffer (PH7.0). (b) Same as (a) but without sugars. The β-Gal.-containing aqueous composition was frozen at -40°C and then freeze-dried at 10°C and below 10 mmHg to obtain a lyophilized product. Thereafter, it was left at room temperature for two weeks. Thereafter, the lyophilized product was opened, and the β-Gal enzyme activity of the resulting composition was measured (4
-Methylumbelliferyl-β-D-galactopyranoside was reacted at 25°C for 10 minutes), and the results shown in Table 5 were obtained.

[Table] Values.
As is clear from the above results, all compositions obtained by opening the freeze-dried product of β-Gal.-containing aqueous composition (a) retained 76% or more of the initial enzyme activity. . On the other hand, β-Gal.-containing aqueous composition (b)
In the composition obtained by opening the freeze-dried product, the enzyme activity was reduced to 8% of the initial level. Example 2 Pancreatic glucagon C-terminal fragment peptide (21-
29) and β-Gal (see JP-A-55-39702) was dissolved in the following buffers (aqueous solutions) (a) and (b). The β-Gal concentration in each solution was 100 ng/ml. (a) 0.05M phosphate buffer containing 5% (W/V) sucrose, 0.2% bovine serum albumin, 0.001M _MgCl2 (PH7.0). (b) Same as (a) but without sucrose. After freezing the above β-Gal.-containing aqueous composition at -40°C and freeze-drying at 10°C and 10 mmHg or less, the lyophilized product was sufficiently replaced with nitrogen gas, and sealed tightly under a nitrogen gas flow. It was sealed and a lyophilized product was obtained. This was then stored at 10°C. After 20 weeks, each freeze-dried product was opened. There was almost no change in the enzyme activity of the composition obtained by opening the freeze-dried product (a). ] 100 μl of antiserum at various dilutions (N6E:
100 μl of JP-A No. 55-39702), 50 μl of Antagosan (manufactured by Hoechst, 10,000 units/ml), assay buffer (0.5% human serum albumin), 0.5
Mix with 250 μl of 0.02 M phosphate buffer (PH7.3) containing % ethylenediamine, disodium tetraacetate, _2H2O , 0.1% _NaN3 and 0.1M NaCl,
By keeping the lyophilized product for 4 days and nights at ℃, the enzymatically active part of the β-D-galactosidase-containing aqueous composition after opening the freeze-dried product is bound to the antiserum. Next 5
% anti-rabbit IgG antibody conjugated cellulose suspension
Add 100 μl and further react at 30°C for 4 hours. This mixture was centrifuged, the precipitate was washed, and the enzyme substrate solution [0.1% bovine serum albumin, 0.1% NaN ₃ ,
Suspend in 500 μl of 20 μg/ml 4-methylumbelliferyl-β-D-galactopyranoside dissolved in 0.01 M phosphate buffer (PH 7.0) containing 0.1 M NaCl and 1 mM MgCl ₂ and stir at room temperature. Let it react overnight.
After the reaction was completed, the fluorescence intensity of 4-methylumbelliferone liberated as a result of enzymatic decomposition was measured using an excitation wavelength of 365 nm and a fluorescence wavelength of 450 nm. Next, for the determination of pancreatic glucagon in the test solution, each of the antiserum and the β-D-galactosidase-containing aqueous composition at a certain dilution determined as above was used.
100 μl was added to a mixture of 50 μl of test solution, 50 μl of antagosane and 200 μl of assay buffer and then quantified as above. The measurement results of EIA using the above competitive binding method are shown in Figure 3 (in Figure 3, -Γ- is the lyophilized product.
(a) represents the results of the composition obtained after opening, and -Γ- represents the results of the β-Gal.-containing aqueous composition before lyophilization. ). As is clear from Figure 3, the composition obtained by opening the freeze-dried product (a) gave the same measurement values as the aqueous β-Gal.-containing composition before freeze-drying. Ta. The enzyme activity of the composition obtained by opening the freeze-dried product (b) was less than 10% of the initial activity, making it impossible to apply it to EIA. Example 3 The conjugate of hCG-β chain C-terminal fragment peptide (130-145) obtained in Reference Example 3 and β-Gal was treated in the same manner as in Example 2 to obtain freeze-dried products (a) and (b). ) was obtained. After 16 weeks, each freeze-dried product was opened. There was almost no change in the enzyme activity of the composition obtained by opening the freeze-dried product (a). ] 100 μl of various dilutions of antiserum (specific anti-hCG antibody obtained in Reference Example 4), assay buffer (0.5% human serum albumin, 0.5% ethylenediamine tetraacetate disodium 2H ₂ Contains O, 0.1% _NaN3 and 0.1M NaCl
The enzymatically active portion of the aqueous β-D-galactosidase-containing composition is bound to the antiserum by mixing with 300 μl of 0.02M phosphate buffer (pH 7.3) and keeping at 5°C for two days and nights. Next, 100 μl of a 2.5% anti-rabbit IgG antibody-conjugated cellulose suspension is added, and the mixture is further reacted at 20° C. for 4 hours. The mixture was centrifuged, the precipitate was washed, and the enzyme substrate solution (0.1% bovine serum albumin, 0.1% _NaN3 , 0.1M NaCl and 1mM
20 μg/ml 4-methylumbelliferyl-β dissolved in 0.02 M phosphate buffer at PH 7.0 containing MgCl ₂
-D-galactopyranoside solution) Suspend in 500 μl and react overnight at room temperature. After the reaction, the excitation wavelength
The fluorescence intensity of 4-methylumbelliferone liberated as a result of enzymatic decomposition was measured at 365 nm and a fluorescence wavelength of 450 nm. Next, to quantify hCG in the test solution, add 100 μl each of the antiserum and β-D-galactosidase-containing aqueous composition at a constant dilution determined above to 50 μl of the test solution and 250 μl of the assay buffer. and then quantified as above. The measurement results by EIA using the competitive binding method described above are shown in Figure 4 (in Figure 4, -Γ- indicates the result of the composition obtained by opening the freeze-dried product (a), -・--represents the results of the β-Gal.-containing aqueous composition before freeze-drying.) As is clear from Figure 4, the freeze-dried product
The composition obtained by opening (a) is β before lyophilization.
-The same measured values as those obtained with the Gal.-containing aqueous composition were obtained. The enzyme activity of the composition obtained by opening the freeze-dried product (b) was less than 10% of the initial activity, and the EIA
It was impossible to apply it. Example 4 The conjugate of hCG and β-Gal obtained in Reference Example 5 was treated in the same manner as in Example 2 to obtain freeze-dried products (a) and (b).
I got it. After 20 weeks, each freeze-dried product was opened. There was almost no change in the enzyme activity of the composition obtained by opening the freeze-dried product (a), and hCG was then measured by EIA using the competitive binding method described in Example 3. (In Figure 5, -Γ- is the result of the composition obtained by opening the freeze-dried product (a). - - is the result of β before freeze-drying.
-represents the results for the aqueous compositions containing Gal. ). As is clear from FIG. 5, the composition obtained by opening the freeze-dried product (a) contains β-Gal before freeze-drying.
The same measurements were obtained as those obtained with the aqueous composition containing The enzymatic activity of the composition obtained by opening the freeze-dried product (b) was less than 10% of the initial activity, making it impossible to apply it to EIA. Example 5 Binding of β-adrenergic agonist (trans-5-hydroxymethyl-6-hydroxy-2-isopropylamino-1,2,3,4-tetrahydronaphthalen-1-ol) and β-Gal. The product obtained by Immunopharmacology
Vol. 1, page 3 (1979)] was treated in the same manner as in Example 2 to obtain freeze-dried products (a) and (b). After 12 weeks, each freeze-dried product was opened. There was almost no change in the enzyme activity of the composition obtained by opening the freeze-dried product (a), and the β-adrenergic agonist was measured by EIA using the competitive binding method described in Example 3. The results are shown in Figure 6 (in Figure 6, -
Γ- represents the result of the composition obtained by opening the freeze-dried product (a), and --.- represents the result of the β-Gal.-containing aqueous composition before freeze-drying, respectively. ). Freeze-dried product (a)
The composition obtained by opening the package contains β-
The same measurements were obtained as those obtained with the aqueous composition containing Gal. The enzymatic activity of the composition obtained by opening the freeze-dried product (b) was less than 10% of the initial activity, making it impossible to apply it to EIA. Example 6 The product obtained by binding the anti-hCG antibody obtained in Reference Example 6 with β-Gal. was treated in the same manner as in Example 2 to obtain lyophilized products (a) and (b). Obtained. After 23 weeks, each freeze-dried product was opened. There was almost no change in the enzyme activity of the composition obtained by opening the freeze-dried product (a). 100 μl of 5% normal rabbit serum, 0.1% _NaN3 ,
400 μl of 0.1M phosphate buffer containing 1mM _MgCl2 ,
100 μl of a test solution containing hCG was added to one polystyrene ball bound to an anti-hCG-βC-terminal fragment peptide (123-145) antibody (see Reference Example 6), and the mixture was allowed to react at room temperature for 2 hours. After the reaction, the polystyrene ball was washed three times with 0.05M phosphate buffer containing 0.1M NaCl and 1mM _MgCl2 , and then washed with enzyme substrate solution (0.1%
Bovine serum albumin, 0.1%, _NaN3 , 0.1M NaCl
and 20 μg/ml 4-methylumbelliferyl-β-D-galactopyranoside solution dissolved in 0.02 M phosphate buffer at pH _7.0 containing 1 mM MgCl2).
400 μl was added and reacted at 37°C for 2 hours. excitation wavelength
The fluorescence intensity was measured at 365 nm and the fluorescence wavelength was 450 nm. The measurement results of EIA using the above-mentioned Sanderch method are shown in Figure 7 (In Figure 7, -Γ- indicates the result of the composition obtained by opening the freeze-dried product (a).
-- and -- represent the results of the β-Gal.-containing aqueous composition before freeze-drying, respectively. ). As is clear from Figure 7, the composition obtained by opening the freeze-dried product (a) gave the same measurement values as the β-Gal-containing aqueous composition before freeze-drying. . Freeze-dried product (b)
The enzymatic activity of the composition obtained after opening the package was less than 10% of the initial activity, making it impossible to apply it to EIA. Example 7 Product obtained by binding anti-human IgG antibody [manufactured by Miles Laboratory (USA)] with β-Gal. [Journal of Immunology Vol. 116]
1554 (1976)] was treated in the same manner as in Example 2 to obtain freeze-dried products (a) and (b). After 12 weeks, the freeze-dried product was opened. There was almost no change in the enzyme activity of the composition obtained by opening the freeze-dried product (a), and human IgG was then measured by EIA using the Sandersch method described in Example 6, but the results were as follows.
Shown in the figure. (In Figure 8, -Γ- is a freeze-dried product (a)
The results for the composition obtained by opening the package are shown, and the symbols ``-'' and ``-'' represent the results for the aqueous composition containing β-Gal. before lyophilization. ). As is clear from FIG. 8, the composition obtained by opening the freeze-dried product (a) gave the same measured values as the aqueous β-Gal.-containing composition before freeze-drying. The enzyme activity of the composition obtained by opening the freeze-dried product (b) is 10% or less of the initial activity;
Application in EIA was impossible. Example 8 The product obtained by binding the anti-hCG antibody obtained in Reference Example 6 with β-Gal. was added to 5% (W/V) in the buffer (a) shown in Example 2. Lyophilized products (a) and (b) were obtained by the same treatment using 3% (w/v) lactose or galactose instead of sucrose. After 23 weeks, the freeze-dried product was opened. There was almost no change in the enzyme activity of the composition obtained by opening the freeze-dried product (a) obtained using lactose or galactose.
hCG was measured. The results are shown in Figure 9 (in Figure 9, -△- indicates the freeze-dried product (a) obtained using lactose).
-□- is the result of the composition obtained by opening the freeze-dried product (a) obtained using galactose, -・- is the result before freeze-drying. -×- represents the results for the composition obtained by opening the freeze-dried product (b), respectively. ). As is clear from Figure 9, the composition obtained by opening the freeze-dried product (a) gave the same measurement values as the aqueous composition containing β-Gal. before freeze-drying. However, the enzyme activity of the composition obtained by opening the lyophilizate (b) obtained using a sugar-free buffer decreased to less than 10% of the initial activity;
It was not possible to create a calibration curve using EIA.

[Brief explanation of drawings]

Figure 1 shows the UV absorption spectrum of the conjugate of hCG-βC-terminal fragment peptide (130-145) and β-Gal. Figure 2 shows the UV absorption spectrum of the specific anti-hCG antibody, and Figure 3 shows the pancreatic glucagon Figures 4 and 5 show the standard curves of pancreatic glucagon (PG) obtained using the conjugate of C-terminal fragment peptide (21-29) and β-Gal. Figure 6 shows the standard curve of hCG obtained using the conjugate and the conjugate of hCG-βC-terminal fragment peptide (130-145) and β-Gal. Figures 7 and 9 show the standard curve of a β-adrenergic agonist obtained using a conjugate of anti-hCG antibody and β-Gal. FIG. 8 shows a standard curve of human IgG obtained using a conjugate of anti-human IgG antibody and β-Gal.

Claims (1)

[Scope of Claims] 1.1 ml of β-D-galactosidase or β-D-galactosidase in an aqueous composition containing β-D-galactosidase or a conjugate thereof with an immunoactive substance and sugar or sugar alcohol.
100pg to 10μg per sugar or sugar alcohol
A method for producing a lyophilizate for labeling, which comprises subjecting the aqueous composition containing the aqueous composition at a concentration of 0.2 to 5 w/v% to lyophilization. 2 Furthermore, add albumin to the aqueous composition from 0.01 to
The manufacturing method according to claim 1, wherein the content is 5 w/v%.