JPH0136065B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention provides human chorionic gonadotropin (hCG)
It is sometimes abbreviated as. ) concerning immunochemical assay methods. hCG is a type of protein hormone produced by trophoblastic cells that are formed at the same time as pregnancy, and promotes the secretion of progesterone. Detection of hCG is commonly used for early diagnosis of pregnancy. In addition, hydatidiform mole,
In chorionic diseases such as destructive hydatidiform mole and choriocarcinoma,
Measurement of hCG in body fluids such as urine and blood has been found to be extremely important in terms of early detection of these diseases, determination of therapeutic effects, and prognosis management. However, for these diagnoses, a trace amount of hCG of about 100 IU or less is required.
It is necessary to measure hCG, and the problem here is hCG.
Protein hormones with a similar structure, namely luteinizing hormone (hereinafter sometimes abbreviated as hLH), follicle-stimulating hormone (hereinafter sometimes abbreviated as hFSH), and thyroid-stimulating hormone (hereinafter sometimes abbreviated as hFSH).
It is sometimes abbreviated as hTSH. ) is an immunological cross-reactivity with In particular, hLH is highly similar to hCG, and the physiological amount of hLH in urine can reach 150 IU/h, so in order to measure hCG in trace amounts of body fluids, it is necessary to differentiate hCG and hLH immunologically. It is necessary to. On the other hand, chemical analysis of these protein hormones revealed that their cross-reactivity is due to their α-subunit moieties, which have many common structures.
Therefore, attempts have been made to separate and purify the β-subunit of hCG (hereinafter sometimes abbreviated as hCG-β), which has relatively little similarity, and to produce anti-hCG-β antibodies to specifically detect hCG. There is. But hCG
The separation and purification of -β is complicated, and it is extremely difficult to avoid the mixture of hCG and the α-subunit of hCG (hereinafter sometimes abbreviated as hCG-α).
Due to the mixture of these and the common amino acid sequence that still remains between the β-subunits of hCG and hLH, cross-reactivity with hLH cannot be completely eliminated by using anti-hCG-β antibodies. however,
A peptide consisting of about 30 amino acids at the C-terminus of hCG-β shows an amino acid sequence that does not exist in hLH, and in this part, hCG and hLH
It was found that it is possible to completely distinguish between Based on the results of such structural analysis, Matsuura et al.
After synthesizing the C-terminal peptide of hCG-β and immunizing rabbits to obtain hCG-specific antiserum, we performed radioimaging assay (hereinafter abbreviated as RIA) using the competitive method [“Endocrinology”] , Vol. 104 (1979), p. 396], satisfactory results were obtained regarding specificity, but sensitivity was not sufficient. Furthermore, as described in Japanese Patent Applications No. 55-42484 and No. 55-80467, antibodies obtained by immunizing animals with hCG
The hCG antibody was purified using a solid phase obtained by insolubilizing the synthetic C-terminal peptide of hCG-β, and competitive enzyme immunoassay (hereinafter sometimes abbreviated as EIA) was performed to determine the specificity. Satisfactory results have been obtained regarding both sensitivity and sensitivity. However, RIA and EIA using competitive methods have the disadvantage that they are easily influenced by other components in the test solution and require a long time for measurement. Therefore, after repeated research in search of an even simpler method, it was discovered that hCG can be measured specifically and with high sensitivity by measuring it using a non-competitive method (hereinafter referred to as the Sanderutsch method).Based on this, As a result of further research, the present invention was completed. The present invention provides an immunochemical measurement method using an antibody held on a carrier, an antibody bound to an antigen, and a labeling agent, in which the antibody held on the carrier and the antibody bound to the labeling agent mutually have antigen-determining sites. Two types of antibodies that do not overlap, one of which has the general formula H-R-Pro-Ser-Asp-Thr-Pro-Ile-Leu-P
ro-Gln-OH [wherein R is 1 to 1 containing Gly at position 14 of the peptide shown in
Represents 14 partial peptide chains. Human chorionic gonadotropin obtained by inoculating a warm-blooded animal other than humans with a condensation product obtained by condensing a peptide represented by the following formula with a carrier protein in the presence of glutaraldehyde, and collecting the condensation product. An immunochemical measurement method for human chorionic gonadotropin, which is an antibody that specifically reacts with. In this specification, the measurement principles of the competitive method and the Sand-Deutsch method, which are often expressed, will be explained. (1) Competitive method: A test solution containing an unknown amount of antigen and a certain amount of antigen labeled with a labeling agent are subjected to a competitive reaction with a certain amount of the corresponding antibody, and the antibody binds to the labeling agent or the antibody. Measure the activity of the labeling agent. (2) Sand-Germany 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), then the excess amount of antibody labeled with a labeling agent is fixed. amount and react (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 measurement method using the Sanderutsch 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 Sanderuch method to specifically measure hCG, in order to eliminate measurement errors due to cross-reaction with protein hormones with similar chemical structures such as hLH, it is necessary to The antibody used in this reaction and the antibody used in the second reaction are two types of antibodies that do not have overlapping antigen-determining sites, and one of these antibodies is an antibody that specifically reacts with hCG. Examples of antibodies that specifically react with hCG include “Endocrinology”, Vol. 104 (1979),
Antibodies such as those described on page 396 may be mentioned. That is, the hCG-specific peptide on the C-terminal side of the hCG-β chain and a carrier protein such as bovine albumin or bovine thyroglobulin are combined with 1-ethyl-
A condensate obtained using a water-soluble carbodiimide such as 3-(3-dimethylaminopropyl)carbodiimide is inoculated frequently with Freund's complete or incomplete adjuvant to a warm-blooded animal other than humans, such as a rabbit, to form antibodies, By collecting this, an antiserum that specifically reacts with hCG can be obtained. The anti-hCG antibody specifically reacting with hCG described in Japanese Patent Application No. 55-42484 and Japanese Patent Application No. 55-80467, that is, the general formula [] H-R-Pro-Ser-Asp-Thr- insolubilized on a carrier Pro−Ile−Leu−P
ro-Gln-OH [] [In the formula, R is represents 1 to 14 partial peptide chains containing Gly at position 14 of the peptide shown by Examples include anti-hCG antibodies obtained by contacting a peptide represented by the following with a body fluid containing anti-hCG antibodies, and then eluating the specifically absorbed anti-hCG antibodies. Indicated by R in the peptide used to produce the antibody that specifically reacts with hCG mentioned above. 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. In this specification, when amino acids, peptides, protective groups, active groups, etc. are indicated by abbreviations,
They are IUPAC-IUB Commission on
The abbreviations are based on Biological Nomenclature 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 Tyr: Tyrosine 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 metho-para-
Toluenesulfonate GLA: Glutaraldehyde m-MBHS: Meta-maleimidobenzoyl-N
-Hydroxysuccinimide ester p-MCHS: para-maleimidomethylcyclohexane-1-carboxyl-N-hydroxysuccinimide ester The various peptides used in the present invention 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. As a means of such peptide synthesis, for example, “The
Peptides”, Volume 1 (1966), Schro¨der and
by Lubke, Academic Press, New York, US
A. Or "peptide synthesis", methods described in Izumiya et al., Maruzen Co., Ltd. (1975), such as azide method, chloride method, acid anhydride method, mixed anhydride method, DCC method, active ester method, Woodward method. Method using reagent K, carbodiimidazole method, redox method, DCC/additive (e.g., HONB,
Examples include the HOBt, HOSu) method. Examples of carriers used in the method for producing antibodies described above include gel particles (e.g., agarose gel [e.g., Sepharose 4B, Sepharose 6B (manufactured by Pharmacia Fine Chemicals, Sweden)], dextran gel [e.g., Sepharose G75, Cephadex G100, Cephadex G200 (manufactured by Pharmacia Fine Chemicals)], polyacrylamide gel [e.g., biogel
P30, Biogel P60, Biogel P100 (Biorad Laboratories, Inc. (USA))], cellulose particles [e.g., Avicel (manufactured by Asahi Kasei), ion exchange cellulose (e.g., diethylaminoethylcellulose, carboxymethylcellulose)], physical adsorbents [e.g., Glass (e.g. glass bulb, glass rod,
aminoalkyl glass spheres, aminoalkyl glass rods), silicone pieces, styrene resins (e.g., polystyrene spheres, polystyrene particles)], ion exchange resins (e.g., weakly acidic cation exchange resins (e.g., Amberlite IRC-50) Rohm-Haas (USA)
), Zeocurve 226 (Palmchitz (West Germany)
(manufactured by Dow Chemical Company, USA)], weakly basic anion exchange resins (eg, Amberlite IR-4B, Dowex 3 (manufactured by Dow Chemical Company, USA)). Known conventional methods can be applied to insolubilize peptides on carriers, and such means for synthesizing insolubilized peptides are described, for example, in "Metabolism", Vol. 8 (1971).
(2010), p. 696. Examples include the bromcyan method, the GLA method, the DCC method, etc., and preferably a method in which a peptide is bonded to a carrier activated with bromcyanide is used. Next, we immunized the animal with hCG and obtained the anti-inflammatory agent.
A body fluid, particularly serum, containing an hCG antibody is brought into contact with a peptide represented by the general formula [ ] insolubilized on a carrier. In this method, the IgG fraction is precipitated by salting out with sodium sulfate or ammonium sulfate, etc., and the IgG fraction is fractionated directly or further by column chromatography such as DEAE-cellulose, and then brought into contact with the above-mentioned insolubilized peptide to immobilize only the specific anti-hCG antibody. absorb into the phase. This results in hLH,
Other anti-hCG antibodies that cross-react with hFSH and hTSH can be removed. Then, the specific anti-hCG antibody absorbed on the solid phase is eluted. To perform this elution, use a low pH or high pH buffer (e.g. pH 2.3).
0.17M glycine-hydrochloric acid buffer, aqueous ammonia at pH 11) or buffers containing high salt concentrations (e.g., 6M
guanidine hydrochloride solution, 7M urea solution) were used,
Elute and separate the specific antibody fraction. The above operation may be carried out in a batch manner, but is preferably carried out in a column. The physical properties of the antibody obtained in this way were determined by (1) precipitation between hCG and hCG-β using the Ouchterlony method [“Medical Chemistry Experimental Method Course” Vol. 4, p. 159 (1972) (Nakayama Shoten)]; (2) electrophoretic mobility belongs to the γ-globulin fraction, (3) hCG is mixed with sensitized red blood cells to agglutinate red blood cells, (4) molecular weight is about 14 to
170,000, contains approximately 2 to 7% sugar, (5) is easily soluble in aqueous solvents with a pH of 2 to 12, (6) is stable for more than a year when stored in a refrigerator, and (7) has the properties shown in Figure 1. It has an ultraviolet absorption spectrum, and (8) the amino acid composition is lysine 85-97, expressed as the number of moles of each amino acid per 100 moles of glycine.
Histidine 35-43, Arginine 38-45, Aspartic acid 110-132, Threonine 98-107, Serine 118
~135, glutamic acid 138~145, proline 92~
134, glycine 100, alanine 73-79, valine 129
~138, methionine 2-10, isoleucine 28-37,
Leucine 100-112, tyrosine 38-48, and phenylalanine 55-68, (9) each with two H chains and two L chains.
The chains form a molecule through S-S bonds. Further, as an antibody that specifically reacts with hCG, (3) general formula [] H-R-Pro-Ser-Asp-Thr-Pro-Ile-Leu-P
ro-Gln-OH [] [In the formula, R is 1 to 1 containing Gly at position 14 of the peptide shown in
Represents 14 partial peptide chains. ] A condensation product obtained by condensing the peptide represented by [ ] and a carrier protein in the presence of glutaraldehyde (sometimes abbreviated as "GLA") is inoculated into warm-blooded animals other than humans to form antibodies. , and antibodies obtained by collecting this. Here, the carrier protein refers to a protein that is used in combination with a hapten to produce antibodies against haptens (low molecular weight substances) such as peptides that cannot induce antibody production alone. Examples include serum albumin, bovine gamma globulin, bovine thyroglobulin, tetanus toxoid, hemocyanin, and polyamino acids. In order to bind the peptide represented by the general formula [] and the carrier protein in the presence of GLA,
Known methods [e.g. “Hormone and Metabolic
Research”, Vol. 8 (1976), p. 241].The ratio of the peptide represented by the general formula [ ] to the carrier protein is suitably 1:1 to 2:1. , a reaction pH of around 7.3 often gives good results.Also, the time required for the reaction is often 2 to 6 hours, but 3 hours is particularly appropriate.The condensation product prepared in this way can be stored by dialysis against water at around 4°C in a conventional manner and lyophilized. The condensation product produced in the above manner is inoculated into warm-blooded animals other than humans. Examples of warm-blooded animals other than humans used for producing specifically reactive antibodies include warm-blooded mammals (e.g., rabbits, sheep, rats, mice, guinea pigs, cows, horses, pigs), birds (e.g.,
Examples include chickens, pigeons, ducks, quail, etc. As for the method of inoculating the condensation product into warm-blooded animals other than humans, the condensation product to be inoculated to the animal may be in an amount effective for producing antibodies, for example, 2 mg of the condensation product is administered to rabbits at a time in an equal volume (1 ml). Antibodies can often be produced when emulsified with saline and Freund's complete adjuvant and inoculated subcutaneously on the back and palms of the hind limbs 5 times at 4-week intervals. As a method for collecting antibodies formed in warm-blooded animals in this way, for example, in rabbits,
Blood is usually collected from the ear vein 7 to 12 days after the final inoculation and centrifuged to obtain serum. Examples of the carrier for the antibody supported on a carrier used in the measurement of hCG include carriers similar to those used in the production of the antibody described above. In order to bind the antibody to the carrier, known conventional methods can be applied, such as the Bromsian method and the GLA method described in "Metabolism", Vol. 8 (1971), p. 696. Alternatively, as a simpler method, the antibody may be physically adsorbed onto the surface of the carrier. Examples of test samples containing antigens to be measured in the present Sand Deutsch method include body fluids such as urine, serum, plasma, and cerebrospinal fluid, with urine and serum being particularly frequently used. Examples of the labeling agent in the labeling agent-conjugated antibody used in the measurement of hCG include radioactive isotopes, enzymes, fluorescent substances, luminescent substances, and the like. Examples of radioactive isotopes include ¹²⁵ I,
¹³¹ I, ³ H, ¹⁴ C, etc. The enzymes mentioned above are preferably those that are stable and have a large specific activity, such as (1) carbohydrases [e.g., glycosidases (e.g., β-galactosidase, β-glucosidase, β-glucuronidase, β-fructosidase, α-galactosidase, α-glucosidase, α-mannosidase), amylase (e.g.
α-amylase, β-amylase, isoamylase, glucoamylase, Takaamylase A), cellulase, lysozyme], (2) amidase (e.g., urease, asparaginase), (3) esterase [e.g., cholinesterase (e.g., acetylcholinesterase)] , phosphatase (e.g. alkaline phosphatase), sulfatase, lipase],
(4) Nucleases (e.g., deoxyribonuclease, ribonuclease), (5) Iron/porphyrin enzymes (e.g., catalase, peroxidase, cytochrome oxidase), (6) Copper enzymes (e.g., tyrosinase, ascorbate oxidase), (7) Dehydration enzymes (e.g., alcohol dehydrogenase, malate dehydrogenase, lactate dehydrogenase, isocitrate dehydrogenase), etc., and fluorescent substances such as fluorescamine, fluorescens isothiocyanate, etc. Examples include luminol, luminol derivatives, luciferin, and lucigenin. In order to bind the antibody that specifically reacts with hCG and the labeling agent, the chloramine T method ["Nature, Vol. 194 (1962), No. 495]
page], periodic acid method [“Journal of
"Histochemistry and Cytochemistry", Vol. 22 (1974), p. 1084], maleimide method ["Journal
of Biochemistry”, Volume 79 (1976), Page 233]
etc. are used. In order to carry out the specific immunochemical measurement method of hCG and hCG-β by the Sand-Deutsch method, (1) the test sample containing unknown amounts of hCG and hCG-β is physically or chemically A solid phase to which an antibody is bound is added and reacted (first reaction).
After washing the solid phase, a certain amount of antibody labeled with a labeling agent is added and reacted (second reaction). Then usually
The solid phase is thoroughly washed and the activity of the labeling agent bound on the solid phase is measured. If the labeling agent is a radioactive isotope, measure using a well counter or liquid scintillation counter. When the labeling agent is an enzyme, a substrate is added and left to stand, and the enzyme activity is measured by colorimetry or fluorescence. Even if the labeling agent is a fluorescent substance or a luminescent substance, the measurement is performed according to a known method. In the above assay method,
Washing between the first reaction and the second reaction may be omitted, or to further simplify the reaction, the test liquid, the antibody-bound solid phase, and the antibody labeled with a labeling agent may be added and reacted at the same time. That is, since the antibodies used in the present invention have different antigen-determining sites,
It has an extremely excellent feature that it is not affected by the order of addition of reagents, the timing of addition, the presence or absence of washing operations, etc. Thus, according to the present invention, (1) trace amounts of hCG and hCG-β can be measured without being completely influenced by peptide hormones similar to hCG, such as hLH; (2) Since it uses the Sand-Deutsch method, the measurement concentration range is wider than that of competitive methods. (3) Since the two types of antibodies used and the antigen can be reacted simultaneously, the operating method is simple and the time required for measurement is short. (4) Therefore, it is extremely applicable to early diagnosis and prognosis management of chorionic diseases such as choriocarcinoma. The quantitative kit used in the immunochemical measurement of hCG using the Sand-Deutsch method of the present invention mainly contains (1) an antibody retained on a carrier, (2) a labeled antibody, and (3) 0 to 100 IU of standard hCG. (4) Buffer solution used for diluting the reagents and test samples in (1) to (3) above (any buffer solution that can be used for diluting the reagents and test sample may be used, but one example is Examples include phosphate buffer or glycine buffer with pH 6 to 9.) (5) Buffer used for washing the carrier after incubation (any buffer that can be used for washing the carrier) (Any of these may be used, but examples include phosphate buffer or glycine buffer.) (6) When using an enzyme as a labeling agent, reagents necessary for measuring the enzyme. As an example, in the case of β-D-galactosidase, the 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 a glycine buffer). When a luminescent substance is used as a labeling agent, a material for measuring the luminescent substance. For example, Luminol uses an oxidizing agent (preferably hydrogen peroxide), a catalyst (preferably microperoxidase or hypochlorite), and a buffer used to dissolve the oxidizing agent and catalyst (preferably a sodium hydroxide solution or a carbonate buffer). liquid). Moreover, the above (1) and (2) may be mixed in advance. The kit described above is preferably used, for example, in the following manner. Standard hCG or test solution approximately 10-200μ reagent
Add and dilute (4), add a certain amount of reagent (1), then reagent
After adding about 10 to 300μ of (2), 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. If the labeling agent is a radioactive isotope, measure using a well counter or liquid scintillation counter. If the labeling agent is an enzyme, add about 10 to 1000μ of substrate solution and incubate at about 20 to 40℃ for about 0.5 to 24 hours.
After reacting for a period of time, the enzyme reaction is stopped and the absorbance or fluorescence intensity in the reaction solution is measured. Even if the labeling agent is a fluorescent substance or a luminescent substance, the measurement can be performed by a known conventional method. 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 uses a silica gel plate 60F ₂₅₄ manufactured by Merck Co., Ltd. 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 Reference example 1 H-Ala-Pro-Pro-Pro-Ser-Leu-Pro-
Ser−Pro−Ser−Arg−Leu−Pro−Gly−Pro
−Ser−Asp−Thr−Pro−Ile−Leu−Pro−
Gln-OH [hereinafter referred to as peptide (). ]
Production of [hCG-β(123-145) C-terminal peptide moiety] (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 filtering off the catalyst 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. The reaction solution was mixed with 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 C60.95;
H7.21; N9.69. Analysis value C61.04; H7.20; N9.49. (b) Production of Z−Leu−Pro−Gln−OBu ^t : Z−
13 g of Pro-Gln-OBu ^t is dissolved in 500 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, 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. The reaction solution was diluted with 0.2N hydrochloric acid, 4
%-sodium bicarbonate solution, washed with water, dried over anhydrous sodium sulfate, and the solvent was distilled off. 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: C61.52;
H7.74; N10.25. Analysis value: C61.61; H7.94;
N9.92. (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°C (decomposed), [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: C61.89;
H8.10; N10.62. Analysis value: C62.35; H8.31;
N10.16. (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 100 ml of DMF, and add 4.7 g of Z-Pro-OH and 3.0 g 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, heated, and 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: C61.88;
H7.99; N11.10. Analysis value: C62.04; H8.21;
N10.70. (e) Z−Thr−Pro−Ile−Leu−Pro−Gln−
Manufacture of ^OBut : Z-Pro-Ile-Leu-Pro-Gln
- 12.5 g of OBu ^t 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 is dissolved in 100 ml of DMF. In this solution, Z-
Add and dissolve 4.2 g of Thr-OH and 2.7 g of HOBt,
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% aqueous sodium bicarbonate, 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: C60.19;
H7.87; N11.43. Analysis value: C59.54; H7.91;
N11.19. (f) Z−Asp(OBu ^t )−Thr−Pro−Ile−Leu−
Production 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. 4.5 g of Z-Asp(OBu ^t )-OH and 2.3 g of HOBt were added and dissolved therein, and the mixture was cooled 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 and filter the precipitated gel.
Crystallize from ethyl acetate and ether and filter with ether. Yield 12.15g (85.9%), melting point 94-96°C (decomposed), [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:
C58.49; H7.89; N10.70. Analysis value: C58.60;
H8.07; N10.71. (g) Z−Ser−Asp(OBu ^t )−Thr−Pro−Ile−
Production 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 combined 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.
The residue was dissolved in 500 ml of ethyl acetate, washed with 0.2N hydrochloric acid, 4% aqueous 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℃,
[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: C57.17;
H7.73; N11.11. Analysis value: C57.34; H7.77;
N11.14. (h) Z−Pro−Ser−Asp(OBu ^t )−Thr−Pro−
Production 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,
Reduction is carried out in a hydrogen stream 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:
C57.54; H7.69; N11.38. Analysis value: C57.44;
H7.69; N11.38. (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 4g of Z-Gly-ONB
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°C (decomposed), [α] ²² _D -119.4° (c = 0.35, methanol),
^Rf2 0.27. Elemental analysis C ₆₁ H ₉₅ O ₁₈ N ₁₁ H ₂ O Calculated value:
C56.86; H7.59; N11.96. Analysis value: C56.65;
H7.68; N12.02. (j) Production 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 0.2N hydrochloric acid to the reaction solution,
Wash with 4% aqueous sodium bicarbonate, water, dry over anhydrous sodium sulfate and evaporate 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 : 13.8 g of Z-Leu-Pro-OBu ^t was added 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 )-OH11.7
Dissolve 6.7 g of HOBt in 200 ml of DMF and cool to 0°C. Add 7.5 g of DCC to this, stir at 0°C for 4 hours and at room temperature for 12 hours, filter off the precipitate, and distill off the solvent. The residue was extracted into 600 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. The residue is left to stand, and the precipitated crystals 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: C56.20;
H7.32; N15.82, analysis value: C55.79; H7.16;
N15.85. (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 the mixture was 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. 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 the precipitate was filtered off and the solvent was distilled off, the residue was extracted with 500 ml of ethyl acetate, washed thoroughly with water saturated with sodium chloride, dried over anhydrous sodium sulfate, and the solvent was distilled off to leave a candy-like residue. get. ^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),
^Rf3 0.41. Elemental analysis C ₃₇ H ₅₈ O ₉ N ₈・HCl・H ₂ O Calculated value:
C54.63; H7.56; N13.78; Cl4.36. Analysis value:
C54.50; H7.70; N14.11; Cl4.21. (n) Z－Ser－Pro－Ser－Arg－Leu－Pro－
Manufacture of ^OBut : Z-Pro-Ser-Arg-Leu-Pro
-Dissolve 3.8 g of OBu ^t in 200 ml of methanol, 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) 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°C (decomposition), [α] ²² _D -83.5° (c = 0.38, methanol),
^Rf3 0.34. Elemental analysis C ₄₀ H ₆₃ O ₁₁ N ₉・HCl・H ₂ O Calculated value:
C53.35; H7.39; N14.00; Cl3.94. Analysis value:
C53.67; H7.45; N13.72; Cl3.52. (o) Production of Z−Ser−Leu−Pro−OBu ^t : Z−
13.8 g of Leu-Pro-OBu ^t is dissolved in 700 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, 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.
0.2N-hydrochloric acid, 4%-sodium bicarbonate water,
Wash with water, dry over anhydrous sodium sulfate, and evaporate the solvent to obtain 17 g of an oil. Add this to 15ml of a mixed solvent of chloroform and methanol (200:3).
It is 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
−Leu−Pro−OBu ^t production: Z−Ser−Pro−
Ser−Arg−Leu−Pro−OBu ^t 1.0g in 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, it was immediately added to the above DMF solution together with 407 mg of HONB to dissolve, and the mixture was cooled to 0°C. 466 mg of DCC 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 450 mg (33.8%), melting point 110-120°C (decomposed), [α] ²⁴ _D -106.6° (c = 0.31, methanol),
^Rf5 0.71. (q) Manufacturing of Z-Pro-Pro-OBu ^t : Z-Pro-
Dissolve 10.1 g of ^OBut in 500 ml of methanol, reduce using palladium black as a catalyst in a hydrogen stream, remove the catalyst by filtration, evaporate the solvent, and remove the residue from 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 0.2N 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,
^Rf2 0.72. Elemental analysis C ₂₂ H ₃₀ O ₅ N ₂ Calculated value: C65.65;
H7.51; N6.96. Analysis value: C65.42; H7.38;
N7.20 (r) Manufacturing of Z−Pro−Pro−Pro−OBu ^t : Z−
9 g of Pro-Pro-OBu ^t was dissolved in 300 ml of methanol, reduced in a hydrogen stream using palladium black as a catalyst, the catalyst was filtered off, the solvent was distilled off, and the residue was dissolved 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,
^Rf2 0.69. Elemental analysis C ₂₇ H ₃₇ O ₆ N ₃・1/2H ² O Calculated value:
C63.76; H7.53; N8.26. Analysis value: C63.77;
H7.53; N8.62. (s) Manufacture of Z−Ala−Pro−Pro−Pro−OBu ^t :
5g of Z-Pro-Pro-Pro-OBu ^t 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.
233 mg of Z-Ala-Pro-Pro-Pro-OBu ^t is dissolved 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 430 mg (82.1%), melting point 152-155°C (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: C55.42; _H7.43 ; _N14.37 ; _Cl2.27 . Analysis value: C56.21; H7.54; N14.04; Cl2.30. (u) Z−Ala−Pro−Pro−Pro−Ser−Leu−Pro
−Ser−Pro−Ser−Arg−Leu−Pro−Gly−
Pro−Ser−Asp(OBu ^t )−Thr−Pro−Ile−Leu
-Production of Pro-Gln-OBu ^t : Z-Gly-Pro-
Ser−Asp(OBu ^t )−Thr−Pro−Ile−Leu−Pro
-Gln-OBu ^t 363mg was dissolved in methanol 30ml,
Reduction is carried out in a hydrogen stream using palladium black as a catalyst. Filter off the catalyst, distill off the solvent, and remove the residue.
Dissolve in 10ml DMF. Z-Ala-Pro-Pro-
Pro−Ser−Leu−Pro−Ser−Pro−Ser−Arg
430 mg of -Leu-Pro-OBu ^t is dissolved in 3 ml of trifluoroacetic acid and left at room temperature for 45 minutes. The solvent was distilled off, ether was added to the residue, and the powder was collected by filtration.
After drying, add and dissolve in the above DMF solution together with 130 mg of HONB, and cool to 0°C. next
Add 118 mg of DCC and stir at 0°C for 6 hours and at room temperature 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 700 mg (95.5%), melting point 120-125°C (decomposed), [α] ²³ _D -127.7° (c = 0.12, methanol),
Rf ⁵ 0.62, Rf ⁶ 0.44. Elemental analysis _{C121H191O34N27}・HCl・_2H2O Calculated _value : C53.12; _H7.80 ; N14.94; _Cl1.40 . Analytical values: C53.39; H7.62; N15.10; Cl1.54. (v) H-Ala-Pro-Pro-Pro-Ser-Leu-Pro
−Ser−Pro−Ser−Arg−Leu−Pro−Gly−
Pro−Ser−Asp−Thr−Pro−Ile−Leu−Pro
-Gln-OH [peptide ()...hCG-β (123-
145) Production of C-terminal peptide moiety: 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 -OBu ^t 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 make Amberlite.
IRA-410 (acetic acid type) resin column (1 x 5 cm)
and collect the eluate. Wash the resin thoroughly with 50 ml of water, combine the washing solution with the previous eluate, and freeze-dry.
Obtain 390 mg of white powder. Next, this was added to a Sephadex LH-20 column (2
×83cm) and develop with the same solvent. 70~100
ml fractions are collected, freeze-dried, and rechromatographed on the same column, and fractions containing the desired product are collected and freeze-dried 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 H-Ser-Pro-Ser-Arg-Leu-Pro-Gly-
Pro−Ser−Asp−Thr−Pro−Ile−Leu−Pro
-Gln-OH [hereinafter referred to as peptide (). ]
Production of [hCG-β(130-145) C-terminal peptide moiety] Z-Gly-Pro-Ser-Asp(OBu ^t )-Thr-Pro
2.16 g of -Ile-Leu-Pro-Gln-OBu ^t is dissolved in 100 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 25 ml of DMF. In this solution,
Z−Ser−Pro−Ser−Arg−Leu−Pro−OBu ^t 1.5
Z-Ser- obtained by treating g with trifluoroacetic acid
Pro−Ser−Arg−Leu−Pro−OH and HONB1.22
g and cooled to 0°C. Next, 701 mg of DCC was added, and the mixture was stirred at 0°C for 6 hours and at room temperature for 40 hours, 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
Dissolve in 5 ml of Rf ³ solvent, apply to a silica gel column (5.7 x 9 cm) packed with the same solvent, and develop with the same solvent. A portion of 534-914 ml was collected, the solvent was distilled off, ether was added to the residue, and the mixture was filtered. Yield 1.1g (33.9%), Rf ³ 0.20. Next, 70 mg of this protected peptide was added to methanol.
Dissolve in 10 ml and reduce using palladium black as a catalyst in a hydrogen stream, remove the catalyst by filtration, and distill off the solvent. Dissolve the residue in 1 ml of 90% trifluoroacetic acid,
After a few minutes, the solvent was distilled off and the residue was dissolved in 3 ml of water. Add this aqueous solution to Amberlite IRA−410.
(acetic acid type) column (1 x 1 cm) and lyophilize. Next, dissolve this in 0.5 ml of N-acetic acid and
Apply to a Sephadex LH-20 column packed with acetic acid and develop with the same solvent. The sections containing the target product are 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) Arg−1.01(1),
Asp1.01(1), Thr0.93(1), Ser2.57(3), Glu0.96(1),
Pro4.85(5), Gly1.00(1), Ile0.96(1), Leu2.04(2),
Average recovery rate was 81.1%. Reference example 3 H-Gly-Pro-Ser-Asp-Thr-Pro-Ile-
Leu−Pro−Gln−OH [hereinafter referred to as peptide ()
It is called. ] Production of [hCG-β(136-145) C-terminal peptide moiety] Z-Gly-Pro-Ser-Asp(OBu ^t )-Thr-Pro
Dissolve 150 mg of -Ile-Leu-Pro-Gln-OBu ^t in 2.5 ml of 90% trifluoroacetic acid and leave at room temperature for 60 minutes.
Next, the solvent is distilled off, and the residue is dissolved in 30 ml of 50% acetic acid and reduced in a hydrogen stream using palladium black as a catalyst. The catalyst was filtered off, the solvent was distilled off, and the residue was dissolved in water.
Dissolve in 10 ml and apply to a column (1 x 3 cm) of Amberlite IRA-410 (acetic acid form), then wash the resin thoroughly with water. Combine the eluate and wash solution and lyophilize. Cephadex filled with N-acetic acid
Apply to LH-20 column (2 x 83 cm) and elute with the same solvent. Aliquots of 90-105 ml are collected and lyophilized to obtain a white powder. Yield 70 mg (57.9%), [α] ¹⁹ _D −150.5° (c = 0.2
,
0.1N-acetic acid), Rf ⁵ 0.29, Rf ⁴ (cellulose) 0.55. Amino acid analysis values: Asp1.02(1), Thr0.98(1),
Ser0.92(1), Glu1.04(1), Pro3.17(3), Gly0.99(1),
Ile0.99(1), Leu1.00(1). Average recovery rate 82.6%. Reference example 4 Production of antibody Approximately 10,000 IU/mg purified from human urine by a known method
Dissolve 1 mg of hCG in 1 ml of physiological saline and add Freund's complete adjuvant (Freund's complete adjuvant) to this.
Complete adjuvant, “Biochemistry of Immunity”, Tachibana et al.
Kyoritsu Shuppan Co., Ltd. (1967)) and mix well to form an emulsion, which is injected intramuscularly into both thighs and subcutaneously at several points on the back of the rabbit. 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, hCG
- C-terminal peptide of β (), () and ()
Obtain an anti-hCG antibody that also has affinity for Reference Example 5 Production of specific anti-hCG antibody 5 mg of the peptide () obtained in Reference Example 1 was
Dissolved in _{8ml of} 0.1M NaHCO3 containing 0.5M NaCl,
The mixture was added to 1 g of bromcyan-activated Sepharose 4B (manufactured by Pharmacia Fine Chemicals) which had been washed with N/1000 HCl in advance, and stirred overnight at 5°C.
0.1M _NaHCO3 containing the same 0.5M NaCl after the reaction is completed.
and then adjusted the pH to 8 with HCl.
1 at room temperature by adding 10 ml of 0.5M ethanolamine.
After reacting for an hour, (1) 0.1M acetate buffer (PH4.0) containing 1M NaCl, (2) 0.1M borate buffer (PH8.0) containing 1M NaCl, and (3) 0.15M NaCl were added. include
It was washed sequentially with 0.02M borate buffer (PH8.0) and packed into a column. 8 ml of the anti-hCG serum obtained in Reference Example 4 was subjected to salting-out precipitation using 1.5 g of anhydrous sodium sulfate, and the resulting γ-globulin fraction was added to the above peptide ().
It was applied to a coupled Sepharose 4B column (0.9 x 4 cm). 0.02M borate buffer containing 0.15M NaCl (PH
Wash the column with hLH, hFSH and
Anti-hCG antibodies that cross-react with hTSH were removed. Next, by elution with 0.17M glycine-hydrochloric acid buffer (PH2.3), a specific anti-hCG antibody with strong affinity for hCG-βC-terminal peptide was obtained (protein amount
1.8mg). The physical properties of this antibody will be described below. This antibody has a final dilution concentration of 80ng/ml, approximately 2μU
Approximately 95% of hCG-enzyme labeled products have enzymatic activity of
It has the ability to combine. 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. See Figure 1 for the ultraviolet absorption spectrum. See Table 1 for amino acid analysis values. Other physical properties are those of immunoglobulin G (“Medical Immunology”, p. 61, Kikuchi et al., Nankodo (1976).
year)).

【table】

[Table] Number of moles of amino acid Example 1 (1) Production of anti-hCG-βC-terminal fragment peptide antibody Peptide prepared in Reference Example 1 () [hCG
-β(123-145) C-terminal peptide moiety] 25 mg and bovine thyroglobulin (abbreviated as BTG) 50
mg in 4 ml of 0.2 M phosphate buffer (PH7.3), added 4 ml of 5% GLA aqueous solution, stirred at room temperature for 3 hours, dialyzed at 4°C (2 x 4 water) and lyophilized to produce the immunogen. I got it. 1.5 mg of this hCG-βC-terminal peptide (123-145)-BTG condensate was dissolved in 0.75 ml of physiological saline, and Freund's complete adjuvant was added to this solution.
0.75 ml 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 4 times at 4-week intervals, blood was collected 1 week after the final immunization, centrifuged to collect antiserum, and anti-hCG-βC-terminal peptide (123
−145) Serum F5C was obtained. Next, 2 mg of the γ-globulin fraction obtained by salting out the antiserum F5C with ammonium sulfate using a conventional method.
of hCG was attached to a Sepharose 4B column (0.9 cm in diameter, 4 cm in length). 0.02M borate buffer (PH
A specific antibody F5CS with high affinity for hCG was prepared by washing the column with 8.0) and then eluting with 0.17M glycine-hydrochloric acid buffer (PH2.3). (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) Preparation method of β-D-galactosidase-labeled anti-hCG antibody complex Approximately 10,000 IU/
The anti-hCG serum obtained by immunizing rabbits with 1 mg of hCG using the method described in (1) above was salted out with ammonium sulfate, and 5 mg of peptide () was bound to a Sepharose 4B column (0.9 cm in diameter, 4 cm in length).
A fractional antibody T7CS that passed through affinity chromatography (cm) was prepared. T7CS4mg
Dissolve in 2 ml of 0.05M phosphate buffer (PH7.0),
200μ of THF containing 400μg of m-MBHS was added and reacted at 30°C for 30 minutes. reaction mixture
The mixture was passed through Sephadex G-25 (diameter 0.9 cm, length 55 cm) equilibrated with 0.02 M phosphate buffer 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 (PH7.5), and heat at 5°C with occasional shaking. The mixture was allowed to react overnight. After completion of the reaction, purification was performed using Sepharose 6B column chromatography using 0.02M phosphate saline buffer (PH7.0), fractions containing enzyme activity and antibody activity were fractionated, and β-
A D-galactosidase labeled anti-hCG antibody complex was obtained. (4) Measurement 5% normal rabbit serum, 0.1% NaN ₃ , 1mM
hCG in 400μ of 0.1M phosphate buffer containing _MgCl2 ,
Add 100μ of standard solutions of hCG-α, hCG-β, hLH, hFSH, and hTSH, and
hCG-β C-terminal peptide (123-145) antibody-conjugated polystyrene ball, β produced in (3) above
-D-galactosidase labeled anti-hCG antibody solution
100μ was added and allowed to react at room temperature for 2 hours. After the reaction is complete, add 0.1M NaCl to the polystyrene ball.
3 in 0.05M phosphate buffer containing 1mM _MgCl2 .
After washing twice, enzyme substrate solution (0.1% bovine serum albumin, 0.1% NaN ₃ , 0.1M NaCl and 1%
20 μg/ml 4-methylumbelliferyl-β-D-galactopyranoside solution dissolved in 0.02 M phosphate buffer at pH _7.0 containing mM MgCl2)
400μ was added and reacted at 37°C for 2 hours. Fluorescence intensity was measured at 450 nm with an excitation wavelength of 365 nm. The respective standard curves are shown in FIG.
In Figure 2, ● indicates hCG, × indicates hCG-β, and □ indicates
hCG-α, 〇 is hLH, △ is hFSH, 〓 is
Standard curves of hTSH are shown, respectively. In this way, the measurement method of the present invention can be used to measure hCG and
hCG-α, which specifically reacts only with hCG-β,
There was no cross-reactivity with hLH, hFSH, and hTSH. Example 2 (1) Preparation method of alkaline phosphatase-labeled anti-hCG antibody complex Example 1 ( Anti-hCG antibody (T7CS) 2 obtained in 3)
Dissolve mg. Next, 0.1 ml of 2% GLA solution was added and the mixture was reacted at room temperature for 60 minutes, followed by dialysis overnight against 0.02M phosphate saline buffer (PH6.8).
Furthermore, it was fractionated using a Sephadex G-200 column,
An alkaline phosphatase-labeled anti-hCG antibody complex was obtained. (2) Measurement 5% normal rabbit serum, 0.1% NaN ₃ , 0.1M
hCG in 400μ of 0.1M phosphate buffer containing NaCl;
Add 100μ of standard solutions of hCG-α, hCG-β, hLH, hFSH, and hTSH, add one polystyrene ball conjugated with the anti-hCG-β C-terminal peptide (123-145) antibody prepared in Example 1 (2), and leave at room temperature. The reaction was allowed to proceed for 1 hour. Next, add 0.1M polystyrene balls.
After washing with phosphate buffer, alkaline phosphatase-labeled anti-hCG complex solution prepared in Example 2(1)
100μ was added and reacted at room temperature for 1 hour. After the reaction, the polystyrene ball was washed with a phosphate buffer solution and an enzyme substrate solution [containing 1mM MgCl2 _] was added.
2mg/dissolved in 0.05M carbonate buffer (PH9.8)
Add 500μ of para-nitrophenyl phosphoric acid (ml) and react at 37°C overnight. 405n after reaction completion
The absorption intensity of para-nitrophenol at m was measured, and a standard curve for each was obtained. A relationship similar to that shown in Figure 2 was observed. hCG and hCG−
It specifically reacts with β, hCG-α, hLH,
There was no cross-reactivity with hFSH and hTSH. Example 3 (1) Preparation method of ¹³¹ I-labeled anti-hCG antibody 1.5 mCi of Na ¹³¹ I was mixed with 0.25 M phosphate buffer (PH
7.5) Add 20μ to the antistress obtained in Example 1(3).
10 μg of hCG antibody (T7CS) in 20 μg of phosphate buffer
Add the dissolved ingredients. This includes chloramine
Add 20μg of T dissolved in 20μ of phosphate buffer, shake for 30 seconds at room temperature, immediately add 120μg of sodium metabisulfite dissolved in 50μ of phosphate buffer, and then add potassium iodide solution (10mg). /ml) and then purified the reaction mixture with Sephadex G-75.
¹³¹ I-labeled anti-hCG antibody was obtained. (2) Measurement 5% normal rabbit serum, 0.1% NaN ₃ , 0.1M
hCG in 400μ of 0.1M phosphate buffer containing NaCl;
Example 3 Add 100μ of standard solutions of hCG-α, hCG-β, hLH, hFSH, and hTSH to one anti-hCG-β C-terminal peptide (123-145) antibody-conjugated polystyrene ball prepared in Example 1 (2). 100μ of the iodine-labeled anti-hCG solution prepared in (1) was added and allowed to react at room temperature for 2 hours. After the reaction is completed, 0.1M
After washing with 0.05M phosphate buffer containing NaCl, the polystyrene balls were transferred to other test tubes and radioactivity was measured with a well counter to obtain a standard curve for each. A relationship similar to that shown in Figure 2 was observed. It reacted specifically only with hCG and hCG-β, and showed no cross-reactivity with hCG-α, hLH, hFSH, or hTSH. Example 4 (1) Preparation of anti-hCG-α antibody-bound solid phase hCG-α [Boehringa Mannheim (West Germany)
A rabbit was immunized with 100 μg of the anti-hCG-α serum produced by the method of Example 1 (1), and the anti-hCG-α serum produced was salted out with ammonium sulfate and dialyzed against distilled water.
hCG-β [Behringa Mannheim (West Germany)
[anti-hCG-α antibody (F9CS)] was obtained by affinity chromatography using a Sepharose 4B column bound with [Anti-hCG-α antibody (F9CS)]. 2 mg of anti-hCG-α (F9CS) was dissolved in 2 ml of 0.05M phosphate buffer (PH7.0), and a β-D-galactosidase-labeled anti-hCG-α antibody complex was obtained by the method of Example 1 (3). (2) Measurement 5% normal rabbit serum, 0.1% NaN ₃ , 1mM
0.1M phosphate buffer containing MgCl ₂ , 0.1M NaCl
400μ hCG, hCG-α, hCG-β, hLH,
Add 100μ of the standard solution of hFSH and hTSH, and add one polystyrene ball bound to the anti-hCG-β C-terminal peptide (123-145) antibody produced in Example 1 (2).
100μ of the β-D-galactosidase-labeled anti-hCG-α antibody solution prepared in Example 4(1) was added and reacted at room temperature for 2 hours. After the reaction was completed, the polystyrene ball was washed with a 0.05M phosphate buffer containing 0.1M NaCl and 1mM MgCl ₂ , followed by enzymatic reaction and the fluorescence intensity was measured. The respective standard curves are shown in FIG. In Figure 3, ● indicates hCG, × indicates hCG-β, □
is hCG-α, 〇 is hLH, △ is hFSH, 〓
represent the standard curve of hTSH, respectively. In this way, the measurement method of the present invention specifically reacts only to hCG, and can be used to detect hCG-α, hCG-β, hLH,
There was no cross-reactivity with hFSH and hTSH. Example 5 (1) Preparation of antibody complex of horseradish peroxidase-labeled anti-hCG Method of Nakane et al. [“Journal of
“Histochemistry and Cytochemistry”, No. 22
(1974), p. 1084].
7 mg horseradish peroxidase in 1 ml
The mixture was dissolved in 0.3M sodium bicarbonate solution (PH8.1), 0.1ml of 1% 1-fluoro2,4-dinitrobenzene was added, and the mixture was reacted at room temperature for 1 hour.
Next, 1 ml of 0.06M NaIO ₄ was added and stirred at room temperature for 30 minutes, and then 1 ml of 0.16M ethylene glycol aqueous solution was added and left at room temperature for 1 hour. Specific hCG antibody obtained in Reference Example 5 after overnight dialysis against 0.01M sodium carbonate buffer (PH9.5)
Add 1 ml of 1.8 mg of 0.01M carbonate buffer (PH9.5) and react at room temperature for 3 hours, then add 5 mg of _NaHB4.
was added and reacted overnight at 4°C. 0.15M NaCl
After dialysis overnight at 4°C against 0.01M phosphate buffer (PH7.1) containing
The horseradish peroxidase-labeled anti-hCG complex was obtained by fractionation using 200 column chromatography. (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, prepared in Example 1 (3)
2.5 mg of T7CS was added and incubated at 56°C for 2 hours. 0.05M phosphate buffer (PH7.0) containing 0.1% BSA
After washing, it was stored in a cool place until use. (3) Measurement Add hCG, hCG-α, hCG-β, hLH,
100μ of the standard solutions of hFSH and hTSH were added, and one anti-hCG antibody-conjugated polystyrene ball prepared in Example 5(2) was added, followed by reaction at room temperature for 1 hour. Next, after washing the polystyrene ball with 0.1M phosphate buffer, 100μ of the horseradish peroxidase-labeled specific anti-hCG antibody solution prepared in Example 5(1) was prepared.
was added and reacted at room temperature for 2 hours. After the reaction, the polystyrene ball was washed with phosphate buffer and 244 mg/d of 4-aminoantipyrine was added to it.
After adding 2 ml of a substrate solution (dissolved in phosphate buffer of pH 7.0) containing 800 mg/d of phenol and 30 μ/d of 30% hydrogen peroxide solution and reacting at room temperature for 1.5 hours, 20% NaN ₃ solution was added. The reaction was stopped by adding 25μ, and the absorbance intensity at 507 nm was measured to obtain a standard curve for each. The same relationship as shown in Figure 2 was observed, and it specifically reacted only to hCG and hCG-β, while hCG-α, hLH,
There was no cross-reactivity with hFSH and hTSH. Reference Example 6 Kit for Immunochemical Measurement of hCG and Measurement of hCG Using the kit for immunochemical measurement of hCG shown below, the hCG concentration in urine or serum of normal subjects and pregnant women was measured according to the following procedure. The results are shown in Table 2. Immunochemical measurement kit for hCG (1) 6.7μg of anti-hCG per kit obtained in Example 1
Polystyrene balls with a diameter of 6.4 mm sensitized with hCG-βC-terminal fragment peptide antibody. (2) A portion of the β-D-galactosidase-labeled anti-hCG antibody complex having an enzymatic activity of approximately 400 μU. (3) 0 to 100 IU of standard hCG (4) 5% normal rabbit serum, 0.1% NaN ₃ , 1 m, used for diluting the reagents and test samples in (2) to (3) above.
0.1 M phosphate buffer at PH _7.4 containing MgCl2. (5) 20 μg 4-methylumbelliferyl-β-D
- Galactoside. (6) A 0.02M phosphate buffer solution with a pH of 7.0 containing 0.1% bovine serum albumin, 0.1% NaN ₃ and 1mM MgCl ₂ used for dissolving the enzyme substrate in (5). (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 100μ of standard hCG or test sample
Add 400 μ, 1 reagent (1), and 100 μ of reagent (2),
Allow to react at room temperature. After 2 hours of reaction, wash the polystyrene ball with reagent (7), then add 400μ of reagent (5).
to start the enzymatic 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
Determine hCG concentration. The hCG concentration in the urine or serum of normal people and pregnant women was measured using the above method. The results are shown in Table 2.

【table】 [Brief explanation of drawings]

Figure 1 shows the ultraviolet absorption spectrum of the antibody specifically reacting with hCG obtained in Reference Example 5, Figure 2 shows the standard curve obtained in Example 2, and Figure 3 shows Example 4.
The standard curves obtained are shown respectively.

Claims (1)

[Scope of Claims] 1. In an immunochemical measurement method using an antibody held on a carrier, an antibody bound to an antigen, and a labeling agent, the antibody held on the carrier and the antibody bound to the labeling agent are bonded to each other. These are two types of antibodies that do not have overlapping antigen-determining sites, and one of these antibodies has the general formula H-R-Pro-Ser-Asp-Thr-Pro-Ile-Leu-P.
ro-Gln-OH [wherein R is 1 to 1 containing Gly at position 14 of the peptide shown in
Represents 14 partial peptide chains. Human chorionic gonadotropin obtained by inoculating a warm-blooded animal other than humans with a condensation product obtained by condensing a peptide represented by ] with a carrier protein in the presence of glutaraldehyde, and collecting the condensation product. An immunochemical measurement method for human chorionic gonadotropin, which is an antibody that specifically reacts with. 2. The method for immunochemical measurement of human chorionic gonadotropin according to claim 1, wherein the antibody held on a carrier and the antibody bound to a labeling agent are reacted simultaneously with an antigen.