JPH0323151B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a clone capable of producing antibodies with high specificity and low cross-reactivity for use in immunoassays, etc., and a method for producing the same. Immunoassay is known as an analytical method for measuring in vivo active substances such as hormones with high sensitivity and specificity. This method utilizes a competitive antigen/antibody reaction and is an analytical method that uses a fixed amount of antibody and variable amount of antigen. For example, a radioimmunoassay method generally consists of the following four steps. (1) Creation of labeled antigen and specific antibody. (2) Reaction between labeled antigen and specific antibody in the presence of unlabeled antigen. (3) Separation of labeled antigen from the reaction product of labeled antigen and antibody. (4) Measurement of radioactivity followed by quantification using a standard curve. Labeled antibodies and unlabeled antigens can also be used. Step 1 is the most important, and the antibody used as a reagent for measurement is required to have strict specificity for the test substance (antigen). However, this requirement may not be met. In other words, even in immunoassays that are thought to have extremely high specificity, the antibodies used may cross-react with other substances that have similar structures to the test substance, and this can be one of the difficulties of immunoassays. be. Known means for overcoming these difficulties can be roughly divided into the following two methods. That is, (1) Measurement is performed after removing various cross-reactive substances from a sample of the test substance using a physicochemical method. (2) Cross-reactive antibodies in the antiserum are removed by pre-absorption using an immunoadsorbent. Alternatively, a highly purified antibody having as high a specificity as possible for the detection substance is used for the measurement. In order to obtain antibodies with the highest specificity for the target substance, the antigen can be conjugated to a carrier protein at a chemically appropriate site prior to administration to an animal host, so that the antigen can be recognized by the antibody. It has been proposed to expose the specific antigenic determinant site of the target antigen on the surface of a simple protein. Exposure of specific antigenic determinant sites induces the production of low cross-reactive antibodies. However, approaches of this type that have been used to date generally have the disadvantage of requiring complex techniques and synthetic processes. Moreover, the production of cross-reactive antibodies may not be avoided in some cases. Difficulties of this type define the limitations of immunoassay methods. The present invention provides, by using a copolymer of D-glutamic acid and D-lysine (hereinafter referred to as D-GL),
It is based on the knowledge that it is possible to reduce the amount of cross-reactive antibodies and to obtain antibodies that have excellent specificity for certain substances (for example, test substances). This copolymer (D-GL)
acts on B cells (antibody-producing precursor cells) to induce effective immune unresponsiveness (tolerance). That is, when an antigen bound to D-GL is administered to an animal, D-amino acids are generally not easily metabolized in vivo, and D-GL does not substantially induce immune responsiveness of T cells in vivo. . Therefore, D-GL
When an antigen bound to D-GL is administered to an animal, the antigen-D-GL conjugate binds to immunoglobulin receptors on the surface of B lymphocytes, thereby causing irreversible unresponsiveness within these cells. (tolerance). Therefore, the present invention provides a first method having a desired antigenic determinant.
Administering to a mammal a polymer of D-glutamic acid and D-lysine that is covalently bonded to a second antigen that has one or more antigenic determinants that are structurally similar to the antigen at a portion other than the antigenic determinant. And by this,
After inducing effective immune unresponsiveness in the mammal to the second antigen, the mammal is
High specificity for the first antigen obtained by a method of immunizing with an antigen to produce cells having the desired antibody-producing ability, isolating the cells from the mammal, and culturing the cells. Clones capable of producing antibodies with low cross-reactivity against two antigens are provided. According to the present invention, cells having a certain genetic structure (hereinafter referred to as clones) capable of producing the above-mentioned antibodies can be produced in a mammalian body. Therefore, by administering to a mammal a conjugate of D-GL and a cross-reactive antigen according to the present invention, substantially effective immune unresponsiveness to the cross-reactive antigen is induced, and then this By immunizing a mammal with a desired antigen, ie, an antigen containing a specific immunodeterminant, antibodies with low cross-reactivity and high specificity for the specific determinant can be produced. Antibodies with high specificity as described above can be produced in the form of purified antibodies or in the form of antisera containing said antibodies. Furthermore, clones extracted from living mammals can also be used for antibody production. That is, if desired, the clone can be kept alive forever by fusion (hybridization) with appropriate tumor cells, and antibodies can be produced from this clone. Thus, once a clone is obtained, the desired antibody can be produced in the absence of the original living mammal. Therefore, the present invention provides a method for producing the above antibody using the clone obtained by the above method. As is well known, if a suitable clone is obtained, a fused cell (hybridoma) can be easily obtained by combining it with a suitable tumor cell. For example, fusion cells (hybridomas) of certain clones and myeloma cells have been described in the literature, such as by Kohler et al. [Nature, vol. 256;
495−497 (1975), and European Journal of Immunology (Eur.J.Immunol.),
6, 511-519 (1976)] by Milstein et al. [Nature.266, 550-552 (1977)]
and Walsh [Nature,
266, 495 (1977)]. When the hybrid cells are then transplanted into another animal, they continue to proliferate in the animal's body (for example, in the ascites of mice) and produce large amounts of specific antibodies.
This hybrid cell can therefore serve as a new source of desired antibodies. Next, the present invention provides an immunoassay method that utilizes antibodies obtained by the above-described method or antisera containing antibodies of this type. Since the antibody has specificity for the substance, the assay is therefore carried out by reaction between the antibody and the substance. In this case, the substance to be assayed acts as an antigen. The immunoassay method according to the present invention is performed by a radioimmunoassay method or a non-isotopic immunoassay method (for example, a method using a label with an enzyme, a free radical, a cell, a virus, a metal ion, a fluorophore, a chemiluminescent material, etc.). be able to. The assay method according to the invention can also be used to detect the presence of cross-reactive antigens. The D-DL used for the purpose of the present invention preferably has a molecular weight of about 27,000 to about 120,000. A molar ratio of D-glutamic acid and D-lysine of 70:30 to 30:70 is used, for example, D-glutamic acid:D
- Lysine = 60:40. This type of copolymer is commercially available, for example from Miles-Yeda (USA), but on the other hand, a copolymer of D-glutamic acid-γ-methyl ester with N-carboxylic acid anhydride and ε-N-carboxylic acid is prepared by a conventional method. It can also be prepared by copolymerizing benzyloxy-L-lysine with N-carboxylic acid anhydride in the presence of a suitable amine and then removing the protective group. For example, a number of natural products can be used to produce antibodies for immunoassays. Examples of preferred materials include steroids and their glucuronide and sulfate conjugates, catecholamines, peptides and their subunits, related compounds (fragments), and other various drugs. Specific examples of steroids include testosterone (hereinafter abbreviated as T), 5α-dihydrotestosterone (hereinafter abbreviated as DHT), androsterone, etiocholanolone, progesterone, and 17α-hydroxyprogesterone (17α- hydroxyprogesterone), Pregnenolone, Dehydroepiandrosterone, Oestradiol, Oestrone,
Oestriol, Aldosterone, Deoxycorticosterone, Cortisol, Cortisone, Corticosterone, 11-deoxycortisol, Bile acids { glycocholic acid, cholic acid, deoxycholic acid, lithocholic acid, etc.} and conjugates thereof. Examples of catecholamines are Dopamine, Norepinephrine, Epinephrine. Examples of peptide hormones are gastrin, cholecystokinin-pancreozymin, insulin, proinsulin, C-peptide,
Glucagon, Follicle Stimulating Hormone (FSH), Luteinizing Hormone (LH), Human Chorionic Gonadotropin (HCG), Somatostatin, Thyroid Stimulating Hormone (TSH)
and their subunits and related compounds. An example of a drug is the beta-blocker propranolol (l
-Propranolol) and the analgesic L-type or D-type Cyclazocine. For example, when anti-T antibodies or antisera are produced by conventional immunization methods, DHT acts as a cross-reactive antigen because its structure closely resembles that of T. Similarly, when anti-DHT antibodies or antisera are generated, T acts as a cross-reactive antigen. In this way, various steroids act as cross-reactive antigens for other steroids. The above substances are generally of the hapten class. That is, although these substances can bind to antibodies, they do not have the ability to induce an immune response or are unable to induce a weak immune response unless they are bound to a carrier before being administered to a living body. Therefore, in order to induce antibody production, the above-mentioned substance must be bound to a suitable carrier. for example,
In order to induce antibodies specific to a certain antigen (for example, anti-testosterone antibody, hereinafter referred to as anti-T antibody), this antigen must be combined with a suitable carrier and used for immunization. However, the T clones and antibodies thus obtained usually have strong cross-reactivity with substances having similar structures (eg, DHT). According to the invention, D
- GL copolymer and cross-reactive antigen (in this case
The production of anti-DHT clones and antibodies with the above-mentioned cross-reactivity can be specifically suppressed by treating animals with a conjugation product with DHT (DHT). In addition, peptides similar to the desired antigen and D-
It has also been found that by treating animals with covalent conjugates of GL, antibodies capable of reacting with specific determinants of the desired antigen can be obtained (in this case, part of the structure of the peptide is (shares part of the structure of the desired antigen). An example of this is an octapeptide antibody consisting of the C-terminal eight amino acids of cholecystokinin-pancreothymine (hereinafter abbreviated as CCK-PZ), which is a type of gastrointestinal hormone. For reference, the structural formulas (amino acid sequences) of gastrin (human), CCK-PZ and related compounds are shown.

[Table] Cholecystokinin-pancreozymin (CCK-PZ)
is a gastrointestinal hormone secreted primarily from the small intestine that stimulates pancreatic enzyme secretion and causes gallbladder contraction, and is a polypeptide consisting of 33 amino acids. Its biological activity is due to its C-terminal eight amino acid moiety (CCK
octapeptide (hereinafter abbreviated as CCK-8-P). Also, CCK-8-P
The C-terminal 5 amino acid sequence of gastrin is a hormone that stimulates acid secretion in the stomach.
The amino acid sequence of Therefore, it is known that conventional anti-CCK-8-P antibodies exhibit high cross-reactivity with gastrin. On the other hand, it has recently been discovered that CCK-8-P and its reactive receptors also exist in the brain. This hormone's neurotransmitter
It is desired to investigate the function of this hormone and to learn about the secretion and actions of this hormone, which is related to diseases of the gastrointestinal tract. Therefore, it is important to provide antibodies that specifically react with CCK-8-P. According to the present invention, pentagastrin and or gastrin-like compounds can be produced by administering to an animal a covalent conjugate of D-GL and pentagastrin (i.e., gastrin and the C-terminal five amino acids of CCK-8-P). After inactivating clones producing antibodies that cross-react with CCK-
By immunizing with 8-P-KLH, CCK-
Clones capable of producing antibodies capable of specifically reacting with 8-P and antibodies of this type can be obtained. Therefore, according to the present invention, pentagastrin and D-GL as the C-terminal fragment of the desired antigen
If the production of clones (clones that cross-react with CCK-PZ) is suppressed by treating an animal with a covalently bound compound of CCK-PZ and then immunizing the animal with gastrin, it is possible to suppress the production of clones (clones that cross-react with CCK-PZ). Antibodies can be obtained. As can be seen from the examples below, the sharing of D-GL with cross-reactive peptides even when specific antigenic determinants cannot be easily removed from the whole peptide by fragmentation of the peptide. A desired specific antibody can be obtained by administering the conjugate to an immunized animal. For example, Atassi et al.
The composition of the antigenic determinants of lysozyme
[Atassi, MZ
Immunochemistry, Vol. 15, pp. 909-936 (1978)]. When the antigenic determinant is formed by amino acids that are present at irregular intervals, for example, if the antigenic determinant is formed from the amino acids ABCD, it has a completely different amino acid sequence. If a peptide happens to have a part that forms a sterically common antigenic determinant B-C, the antigenic determinant containing the amino acids B-C is
The substance obtained by binding to GL is administered to animals, thereby eliminating clones that have cross-reactivity with antigenic determinants containing amino acids B-C. As a result, antigenic determinants A-D It is possible to selectively increase the number of specific clones directed against specific sites and increase the production of only antibodies directed against specific sites. The conjugate of the above antigen and D-GL is prepared by (1) a method of directly binding the above antigen and D-GL, or (2) a method of indirectly binding the antigen and D-GL by crosslinking. be done. As crosslinked products, oxime compounds, succinyl compounds, and chlorocarboxylic acid compounds according to Erlanger et al. [e.g., BF
Erlanger et al. J. Biol. Chem. 228 . 713 (1957)],
Carboxymethyl thioether derivatives [Weinstein A et al. Steroids 20 , 789 (1972)],
Carboxymethyl ether derivatives [PNRao et
al, J. Steroid Biochem. 9 , 539 (1978)]. The binding methods include the carbodiimide method and the anhydride method (Mixed method).
Examples include the Anhydride Method, the Schotten-Baumann Method, and the Isoxazolium Method. In addition, if -NH ₂ group is introduced into the above compound,
It can be bonded using the glutaraldehyde method, and if _-NH2 group or -SH group is introduced, m
-maleimidobenzoyl-N-hydroxy succinimide-ester, succinimidyl-4-(N
-maleimidomethyl-cyclohexane)-1-carboxylate, succinimidyl-4-(P-malemidophenyl)butyrate, etc. or N-succinimidyl-3-(2-pyridyldithio)propionate, N-succinimidyl-(4-azidophenyl), etc. enyldithio)propionate, etc. are used. Other methods of bonding peptides with other substances commonly used in peptide chemistry include D-GL,
Can be used to bind to antigens. Furthermore, since the immunizing antigen used in the present invention is usually a hapten, it is used after binding the antigen to an appropriate carrier commonly used in a conventional immunization method. Suitable carriers include, for example, keyhole limpet haemocyanin.
(hereinafter abbreviated as KLH), serum γ-globulin and albumin of various foreign animals, such as humans and goats. The method of binding these carriers to the antigen, its subunit or related fragment is similar to the method of binding the cross-reactive antigen, its subunit or related fragment, to D-GL, such as direct binding,
A method of crosslinking and bonding using a suitable intermediate is used. In the latter case, the same intermediates and methods used for conjugation of D-GL and cross-reactive antigens are used. The immunization method is a conventional method. Depending on the animal, a physiological saline solution of the antigen (conjugate of hapten and carrier) is administered intraperitoneally or into the footpads and back, the first time with a complete Freund's adjuvant and the second time with an incomplete Freund's adjuvant. Thereafter, mice only need saline. In large animals such as rabbits, complete and incomplete Freund's adjuvant can be administered alternately. The dosage depends on the binding ratio of the antigen and carrier, the molecular weight of the carrier, etc., but it is usually 1/μg/~100μg/animal for small animals such as mice, and 0.1mg~1mg/dose/animal for large animals such as rabbits. 2 to 5 times
Repeat administration every 2-4 weeks. A conjugate of cross-reactive antigen and D-GL is usually administered 2-3 days before the first immunization. Of course, depending on the type of antigen, a conjugate of antigen and D-GL may be administered even after the first or second immunization. The dosage of the conjugate of crossantigen and D-GL is preferably 100 μg to 500 μg for small animals such as mice, and 2 to 10 mg for large animals such as rabbits. This dose is for D-
It varies somewhat depending on the amount of binding to GL, the binding position, the type of intermediate compound, etc. Practically, the conjugate of antigen and D-GL is administered intraperitoneally as a saline solution. It is preferable that the binding mode of the conjugate between the cross-reactive antigen and D-GL is the same as that between the immunizing antigen and the carrier. It is also preferable to use it for binding an antigen and a carrier. As described above, by administering a conjugate of D-GL and a cross-reactive antigen to an animal, it is possible to induce strong immunological unresponsiveness specifically to a particular cross-reactive antigen. Thereafter, immunization is performed with the desired antigen containing the specific desired antigenic determinant. Thereby, it is possible to obtain a clone, an antibody, or an antiserum containing the antibody that can produce an antibody with low cross-reactivity and extremely high specificity for a specific antigenic determinant of interest. The above findings were confirmed not only in small animals such as mice but also in large animals such as rabbits.
Although it is expected that the results would be different for different animal species, it is of great practical importance that the same results were obtained for both small and large animals. This is because large animals such as rabbits can be used to obtain larger amounts of antibodies than small animals such as mice. Conventionally, it has been difficult to create antibodies that can distinguish structurally similar compounds such as T and DHT, but the present invention has made it possible to produce antibodies that can distinguish one antigen from other cross-reactive antigens. A large number of clones can be selectively obtained. As a result of the increased frequency of specific antibody-producing clones in vivo, it becomes actually possible to pick out and isolate specific clones. According to known literature, the probability of extracting a specific clone capable of differentiating antigens from an animal is about 1 in 1 million to 1 in 10 million, and therefore this type of extraction and isolation is virtually impossible. It was considered impossible. However, even when a clone having the ability to produce a specific antibody obtained by the present invention is immunized with an antigen containing a cross-reactive determinant, the clone has a cross-antigenic determinant that is structurally similar to the desired antigenic determinant. It is possible to specifically identify the difference between The method of the present invention makes it possible to produce this kind of specific antibody with a high probability, and therefore also increases the probability of producing a clone capable of producing this kind of specific antibody. The method of the present invention can be applied to all antibody production methods by appropriately modifying the method of binding D-GL and a low cross-reactive antigen, the type of antigen to be bound, etc. Next, the present invention provides a method for easily quantifying a specific substance contained in a sample when the amount of this substance is unknown. This method is characterized by using antibodies or antiserum obtained by the method described below. The reagents and analytical methods used in the examples below are as follows. (1) Synthesis method of DHT-3-(o-carboxymethyl)oxime (hereinafter abbreviated as DHT-3-CMO). (a) Reaction formula: (b) Raw materials used ³ H-DHT (Radiochemical Center,
Amersham) 6μci (0.015μg as DHT) DHT (Sigma Chemical Co., USA)
0.3g (1mmole) (o-carboxymethyl)hydroxylamine hemihydrochloride (Tokyo Kasei Kogyo Co., Ltd.) 0.3g (2.7mmole) 2N-NaOH 1.25ml Ethanol 15ml (c) Procedure Transfer the above raw materials to a round bottom container with a reflux device. After heating under reflux for 3 hours, 45 ml of purified water was added to the reaction solution, and the pH was adjusted to 8 with diluted NaOH solution. This liquid was transferred to a separatory funnel, about 30 ml of ethyl acetate was added and shaken, and then the ethyl acetate layer was separated and removed to remove unreacted DHT. Next, add ice to the aqueous layer to cool it, and acidify it (about PH4) with dilute hydrochloric acid.
DHT-3 produced by adding cold ethyl acetate
-(o-carboxymethyl)oxime is extracted, and the ethyl acetate layer is dehydrated with anhydrous sodium sulfate. Ethyl acetate is removed by evaporation, and the residue is recrystallized from hot methanol to obtain the desired product. (2) A method for synthesizing testosterone-3-(o-carboxymethyl)oxime (hereinafter abbreviated as T-3-CMO). The desired product was obtained by carrying out the same operation as in (1) except that ³ H-T was used in place of ³ H-DHT, and T was used in place of DHT in the same molar amounts. (3) Synthesis method of hapten-D-GL (a) Synthesis method of DHT-3-(o-carboxymethyl)oxime-D-GL (hereinafter abbreviated as DHT-3-D-GL) (mixed acid method by Erlanger et al. (Based on anhydride method) Reaction formula: (a) Raw material DHT-3-CMO. (as a tracer)
^3H -labeled DHT-3-CMO)
20mg dry dioxane (1ml + 5ml) tri-n-butylamine 20μ Chlorformic acid isobutyl ester 10μ Purified water 6ml D-GL (Mw49000) 30mg 1N-NaOH 150μ (b) Procedure Add 20mg of DHT-3-CMO to anhydrous dioxane (dioxane mixed with molecular sieves) Add 5A and dehydrate) Dissolve in 1 ml and stir.
20μ of tri-n-butylamine was added and stirred for 5 minutes while maintaining the temperature at 11°C. Next, 10μ of isobutyl chloroformate was added, and the mixture was reacted for 1 hour with stirring while maintaining the temperature at 11°C. Separately D
-Dissolve 30mg of GL in 6ml of purified water and
150μ of NaOH was added and mixed, and 5ml of dioxane was gradually added to this solution. This D-GL solution was added all at once to the above reaction solution while stirring. After 10 minutes, check the pH and find 1N−
Add NaOH little by little to bring the pH to 6.5-7.0
Return to temperature and continue stirring at around 10°C for about 2 hours. Next, this reaction solution was dialyzed against water at 4° C. overnight, and the next morning the pH was adjusted to about 4.5 with diluted hydrochloric acid, and a precipitate was deposited. This solution was left at 4° C. for 7 hours to complete precipitation. Next, this solution was centrifuged (4°C, 3000 rpm, 20 minutes), the supernatant was discarded, and 10 c.c. of purified water was added to the precipitate, and 1N
-Adjust the pH to about 7.0 with NaOH to dissolve the precipitate. This solution was dialyzed against purified water at 4°C and then freeze-dried. ^3H -DHT-3 added as a tracer
-Radioactivity and reaction product 1 per mg of CMO
The number of DHT bound per mole of D-GL was calculated from mg of radioactivity. The molar ratio of DHT to D-GL in the resulting reaction product was 30:1.
It was hot. The amounts of water and dioxane relative to BSA calculated from the original report of the mixed acid anhydride method by Erlanger et al. [J.Biol.Chem. 228 , 713 (1957)] are such that when D-GL is added, it becomes gel-like. The amounts of dioxane and water were determined so that large amounts of water and dioxane were added to prevent D-GL from becoming gel-like in the reaction solution and to allow the reaction to proceed appropriately. This idea made it possible to bond D-GL and oxime using the mixed acid anhydride method, and also made it possible to bond a sufficient amount of oxime to D-GL. (b) Testosterone-3-(o-carboxymethyl)oxime-D-GL (hereinafter T-3-D-
Preparation of T-3-oxime (abbreviated as GL)
³ H-labeled T-3-oxime) was used to prepare T-3-D.
-GL37mg (T:D-GL molar ratio is 30:1)
I got it. (4) Synthesis method of hapten-KLH (a) DHT-3-(o-carboxymethyl)oxime-KLH (hereinafter abbreviated as DHT-3-KLH)
(a) Raw material used: DHT-3-CMO (as a tracer)
^3H -labeled oxime) 12mg Anhydrous dioxane 0.5ml + 2.5ml tri-n-butylamine 10μ Chlorformic acid isobutyl ester 5μ Purified water 4ml KLH (Mw100000) 150mg 1N NaOH 75μ (b) Procedure Same procedure as above using the above raw materials DHT-3-KLH54.7mg (DHT:
The molar ratio of KLH was 10:1). (b) Testosterone-3-(o-
carboxymethyl)oxime-KLH (hereinafter T-3
-KLH) preparation method T-3-CMO (contains ^3H -labeled oxime as tracer) 40mg anhydrous dioxane 2ml + 5ml tri-n-butylamine 40μ iso-butylchloroformate 20μ purified water 10ml KLH 300mg 1N NaOH 300μ Perform the same operation as above to obtain T-3-
KLH197mg (T:KLH molar ratio is 8:1)
I got it. (5) Radioimmunoassey operation method Reagents (1) ³ H-T standard solution Dilute 1, 2, ³ H-T (58 Ci/mmol) with ethanol to create an ethanol solution containing 20000 dpm/10 μ (45 Pg as T). . (2) ³ H-DHT standard solution 5α-dihydro-1,2,4,5,6,7
- ³ H-T (114Ci/mmol) was diluted with ethanol and 40000dpm/10μ (as DHT)
An ethanol solution containing 45Pg) was prepared. (3) 0.05M Tris buffer PH8.0 (0.05% BSA, 0.1
% BGG) (4) Saturated ammonium sulfate solution (5) Non-radiolabeled T standard solution (6) Non-radiolabeled DHT standard solution procedure (1) Measurement of the titer of anti-testosterone antiserum ³ H-T 10μ of the standard solution was placed in a glass tube and evaporated to dryness. Add 0.2 ml of antiserum serially diluted with Tris buffer to this and mix. 2
After standing at room temperature for an hour, 0.2 ml of saturated ammonium sulfate solution was added and mixed. Then, it was centrifuged (3000 rpm, 10 minutes). 0.2 ml of the supernatant was transferred to a vial, 2 ml of scintillator (2.0 g of PPO dissolved in 1 part of toluene) was added, and radioactivity was measured. Procedure (2) Measurement of cross-reactivity of anti-testosterone antiserum with DHT Place 10μ of each of the above ³ H-T standard solutions into two series of glass tubes, and one system contains a non-radiolabeled T standard solution and the other. One system has a non-radiative label
Each DHT standard solution was added, each stepwise increasing amount of unlabeled steroid was added, and each was then evaporated to dryness. Add 0.2 ml of antiserum (diluted with Tris buffer) to the glass tube at a concentration capable of binding to 60% of ³ H-T (45 pg) in the glass tube and mix. After leaving it in the greenhouse for 2 hours, perform the same operation as the above procedure (1). Cross-reactivity is Abraham's method.
GE: J. Clin. Endocr. 29 (1969) 866-870]. Procedure (3) Measurement of titer of anti-DHT antiserum Measurement was carried out in the same manner as in procedure (1) above, except that ³ H-DHT standard solution was used instead of ³ H-T standard solution. Procedure (4) Measurement of cross-reactivity of anti-DHT antiserum with T. ^3H -DHT was used instead of ^3H -T.
The measurement was carried out in the same manner as in the procedure (2) above. Example 1 Preparation of low cross-reactive anti-testosterone specific antibody in mice Three groups of 4-5 C57BL/6 8-10 week old female mice were used per group. The first group is the control group and DHT-3-D
- Physiological saline was administered without GL. The second group received 500μg of DHT-3-D-GL and T-3-D-GL.
It was administered intraperitoneally to mice 3 days before the initial immunization with KLH. T-3-KLH (100μg/mouse) for each group
A physiological saline solution was administered intraperitoneally with Freund's complete adjuvant. After 3 weeks, the same amount of T-3
- A saline solution of KLH was administered to each mouse with incomplete Freund's adjuvant. 5, 7,
After 9 and 17 weeks, the same amount of T-3-KLH in physiological saline was administered to each mouse. In the third group, DHT-3-D-GL (500 μg) was added to T-3-D-GL (500 μg).
They were administered 3 days before the second (3 weeks after the first time) and third (5 weeks after the first time) immunization with KLH, respectively. Blood was collected every two weeks to measure antibody titer and cross-reactivity. Some of the results are shown in Table 1. Cross-reactivity was determined by the Abraham method.

[Table] As is clear from Table 1, although the antibody titer of the second group was slightly lower than that of the first control group, it was found that the second group exhibited antibody productivity similar to that of the control group. On the other hand, in the third group, no significant antibody production was observed over the entire course. Furthermore, it can be seen that in the second group pretreated with DHT-3-D-GL, the cross-reactivity with DHT was significantly reduced compared to the control group. Furthermore, when the results in Table 1 were examined by plotting the relationship between the antibody titer and the cross-reactivity of the antibody for each mouse, no correlation was observed between the antibody titer and the cross-reactivity. Generally speaking
When pretreated with DHT-3-D-GL, DHT
The cross-reactivity with the antibody decreased, and no correlation was observed between it and the decrease in antibody titer. In other words, there is no correlation between low cross-reactivity and low antibody titer. As is clear from these facts, when pre-administration of DHT-3-D-GL eliminates clones that exhibit cross-reactivity with DHT, subsequent immunization with T-3-KLH results in higher levels of testosterone. Generation of clones capable of producing specific antibodies is selectively enhanced. The selective production of antibodies with low cross-reactivity as described above represents a practical advantage of the present invention. Example 2 Preparation of anti-dihydrotestosterone specific antibody with low cross-reactivity in mice In this Example 2, 5 groups of C57BL/6 female mice (5 to 7 mice, 8 weeks old) were used. The first group is a control group, without pretreatment with T-3-D-GL, and with DHT.
-3- Perform the first immunization with KLH, the second immunization 3 weeks after the first, and the third immunization 5 weeks after the first,
Booster immunizations are then given every two weeks. The timing and method of immunization of mice in other groups were the same as in the control group. Group 2 is 3 days before the first immunization with DHT-3-KLH.
The day before, 500 μg of T-3-D-GL was intraperitoneally administered to each mouse. The third group is another control group, each
500μg of DHT-3-D-GL500μg of DHT-3
- Administered intraperitoneally to each mouse 3 days before the first immunization with KLH. Each mouse in groups 4 and 5 was treated with DHT-3-KLH.
The first immunization was carried out by immunization, followed by the second immunization three weeks later, and three days before the second immunization, 500 μg of T-3 was administered each.
-D-GL and DHT-3-D-GL were each administered intraperitoneally. Blood was collected every 2 weeks from 7 weeks after the first immunization for the above 5 groups, and the antibody titer and cross-reactivity in the serum were examined. Table 2 shows the results 13 weeks after the first immunization.

[Table] Note 1) Both *1 and *2 are the same as the footnotes in Table 1.
The results in Table 2 are similar to those shown in Example 1.
Pre-administration of -3-D-GL resulted in the production of low cross-reactive anti-DHT antibodies, as shown in the second group. T-3-D-GL and DHT-3 after initial immunization
- In groups 4 and 5 treated with D-GL, anti-
No significant production of DHT antibodies was observed. After pretreatment with DHT-3-D-GL, DHT-3-
The cross-reactivity of anti-DHT antibodies produced in the third group immunized with D-KLH with T was as high as in the control group. Furthermore, the antibody titer was similar to that in the second and third groups. This shows that when treated with T-3-D-GL, an anti-DHT antibody with low cross-reactivity with T can be obtained. Example 3 Preparation of anti-T or anti-DHT antibodies in rabbits (domestic rabbits) In this example, 1 group of female rabbits weighing approximately 3 kg were used.
Six groups of animals were used. For the first immunization of rabbits in the first group, DHT-3-KLH (each
100μg), and 3 days before that, T-3-D-
GL (10 mg each) was administered intraluminally. Three weeks after the first immunization, a second immunization was performed by subcutaneously injecting DHT-3-D-KLH (200 μg each) together with incomplete Freund's adjuvant. Thereafter, every month, complete Freund's adjuvant and incomplete Freund's adjuvant were used alternately, and DHT
The third and fourth immunizations with -3-D-KLH (200 μg each) were performed. The second group is the control group, T-3-D-
The mice were immunized with DHT-3-KLH in the same manner as in the first group without administering GL. The third group received 10 mg of T-3-D-3 days before the second (3 weeks later) and third (7 weeks later) administration of DHT-3-KLH.
GL was administered intraperitoneally to rabbits. The fourth group received 10 mg each of DHT-3-D-GL in T-3.
- Administered intraperitoneally to rabbits 3 days before the first immunization with KLH. The fifth group was a control group, in which DHT-3-D-GL was not administered, and immunization was performed with T-3-KLH in the same manner as in the fourth group. Group 6 is the second (3 weeks after the first immunization) and third (7 weeks after the first immunization) with T-3-KLH.
10 mg each of DHT-3-D-GL 3 days before immunization
was administered intraperitoneally to each rabbit. Blood collection after each immunization
I did it on the 10th day. As a result, the third and sixth groups
In the group, no significant antibody production was observed across all routes. The anti-DHT antibody from the second control group and the anti-testosterone antiserum obtained from the fifth group (control group) did not react well with the hapten used for immunization or with the cross-reactive hapten. They showed almost the same level of reactivity. That is, the second group of anti-
The cross-reactivity of DHT antibodies to testosterone was 100%, and the cross-reactivity of the fifth group of anti-testosterone antibodies was 100%.
Cross-reactivity to DHT was 95.2%. However, Groups 1 and 4 showed significantly lower reactivity to cross-reacting substances. In other words, the cross-over of the first group of anti-DHT antibodies to testosterone is
11.9%, and of group 4 anti-testosterone antibodies.
The crossover to DHT was 22.0%. Example 4 Measurement of human blood T and DHT using rabbit anti-T-specific antibodies and anti-DHT-specific antibodies (1) Anti-T-specific antibodies with low cross-reactivity in the rabbits of groups 1 and 4 of the above example, respectively and anti
Now that DHT-specific antibodies have been obtained, in order to examine the practicality of these antibodies in detecting T and DHT in human serum, known amounts of testosterone and DHT were added to human serum.
We investigated the relationship between the amounts of T and DHT measured by radioimmunoassay using the above antiserum and the amounts actually added. When measuring blood testosterone levels, it is more appropriate to
1 ml of pooled serum (serum from a woman with a low serum T level was used) contains 1, 2, 4 ng of T1, 2, 4 ng, or
Add 0.1, 0.2, and 0.3 ng of DHT, respectively, and further add to measure the recovery rate during the extraction process.
³ H−T2000dpm or ³ H−DHT2000dpm
was added and mixed. 3 ml of hexane:ether (3:2) was added to each sample and shaken for 1 minute using a Vortex mixer. After shaking, the test tube was placed in a dry ice-acetone bath for 10 seconds, and the organic solvent layer was transferred to another test tube. The organic solvent was evaporated off using an evaporator, 2 ml of ethanol was added to the residue, mixed, and the ethanol solution was transferred in an amount corresponding to the measurable range of the inhibition curve using each antiserum to a small test tube, and evaporated to dryness. , DHT−
It was measured by radioimmunoassay using a low cross-reactive anti-T antibody obtained by D-GL pretreatment. On the other hand, transfer 0.5 ml of the above ethanol solution to a vial, evaporate the ethanol, add a scintillator to the residue, measure the cant, use it to calculate the recovery rate in the extraction process, and use the value obtained by radioimmunoassay. Corrected. In addition, when measuring blood DHT levels, 0.2 and 0.5 ng of DHT or 0.2 and 0.5 ng of T were added to 1 ml of human female pool serum, and ³ H-
2000 d.pm of DHT or 2000 d.pm of ³ H-T was added and mixed well. Hexane: ether (3:
2) Add 3 ml to the mixture, perform extraction in the same manner as above, add 2 ml of ethanol to the extraction residue, transfer a portion of this ethanol solution to a small test tube, and calculate the amount of DHT inhibition curve by each antiserum. It was adjusted to fall within the measurable range of , evaporated to dryness, and measured by radioimmunoassay using a low cross-reactive antiserum obtained by treatment with testosterone-D-GL. On the other hand, the ethanol solution was transferred to a 0.5 ml vial, the ethanol was evaporated, a scintillator was added to the residue, the count was measured, and the value obtained by radioimmunoassay, which was used to calculate the recovery rate in the extraction process, was corrected. The results showed that when 1, 2, and 4 ng of T was added to normal female serum using a low cross-reactive anti-T antibody, the T titer of normal serum before the addition was 0.30 ng, but after each addition. The corresponding T number is
They were 1.15ng, 2.30ng, and 4.60ng. In addition, DHT was used instead of T in a similar experimental system.
When 0.1, 0.2 and 0.3 ng were added and the effect of DHT on the measured testosterone level was examined, it was found that the level was almost the same as that of serum without additives, and there was no significant effect. On the other hand, normal female serum contains DHT 0.2 and 0.5n
g to reduce the amount of DHT in the serum with a low cross-reactive anti-inflammatory agent.
The results of measurements using DHT antibodies showed that 0.21 ng of DHT was measured in normal serum before addition, but 0.41 ng and
0.72 ng of DHT was measured, and each value was in good agreement with the added DHT. In addition, in the same experimental system, 0.2 and 0.5 mg of T were added instead of DHT.
When looking at the effect of T on the amount of DHT measured, it was found that the level was almost the same as that of serum without additives, and there was no significant effect. This result shows that using the low cross-reactive antiserum of Example 3, serum T or DHT levels can be accurately measured. (2) Measurement in a mixed system of cross-reactive antigen and specific antigen DHT in human blood is measured using a rabbit anti-DHT antibody with low cross-reactivity. DHT and T in 0.1ml of female pool serum as shown in Table 3.
(5ng, 10ng) and (10ng, 5n), respectively.
g) or (10 ng, 10 ng) were added and the measurement was carried out according to the example of (1) above. As a result, even if the sample contains various amounts of the cross-reactive antigen T, the amount of DHT measured in each case is
They were 5.67ng, 10.17ng and 9.20ng. This revealed that the low cross-reactive anti-DHT antibody obtained in Example 3 can accurately detect DHT even if it contains a large amount of T, a cross-reactive antigen.

[Table] Similarly, 0.1ml of serum contains testosterone.
DHT as shown in Table 4 (5ng, 10ng),
(10ng, 5ng) and (10ng, 10ng) were added, and the amount of stetosterone was measured using a low cross-reactive rabbit anti-stetosterone antibody.
6.80ng, 10.3ng and
The value of stetosterone was 10.59 ng. In this way, even in the case of a low cross-reactive anti-testosterone antibody, testosterone can be accurately measured in the presence of cross-reactive DHT.

[Table] (3) Measurement of actual human specimens (direct method) The direct method refers to a method of analysis without performing any operation to separate T and DHT in advance. On the other hand, for comparison, values measured by a conventional method, that is, a method in which T and DHT are separated and then measured using paper chromatography, are shown. (3.1) Measurement of T in serum (a) Common processing Take 0.1 ml of male serum sample or 0.5 ml of female serum sample into test tubes, and extract 3000 d.pm of ³ H-T during the extraction process or by extraction and chromatography. It was added to measure the recovery rate during the fractionation process and mixed well. Additionally, 0.5 ml of pure water was added to the male serum sample.
Add. Hexane:ether (3:2)
3 ml of was added and shaken for 1 minute using a Vortex mixer. Immerse the test tube in a dry ice-acetone bath for 10 seconds to freeze the serum portion.
The organic solvent layer was transferred to another test tube, and the organic solvent was evaporated off using an evaporator. (b) Direct method Add ethanol to each residue of male serum sample.
0.4ml was added and mixed. In order to put this ethanol solution in the range of 50 to 400 pg,
100μ in the normal range, and 1 in Table 5.
In the case of high testosterone serum such as Nos. 2 and 3, 50μ was transferred to each small test tube. For measuring the recovery rate during the extraction process
100μ was transferred to a vial, and a scintillator was added to the evaporated dry residue for counting. For female serum samples, 0.7 ml of ethanol was added to the hexane-ether extraction residue.
Transfer 0.5 ml of the solution to a small test tube. To measure the recovery rate in the extraction process, 100μ was transferred to a vial, and after evaporation to dryness, a scintillator was added to the residue and counted. The ethanol in the ethanol solution, both of which was transferred to a small test tube, was removed by evaporation under a stream of nitrogen gas, and the residue was used for radioimmunoassay. (c) Paper chromatography method 50μ of chloroform was added to the hexane-ether extraction residue obtained in the same manner as the above-mentioned direct method, and the mixture was coated on paper.
This operation was performed a total of three times. Separately, 10 μg of testosterone was coated on the same paper as a standard marker. PushA paper
(Cyclohexane: methanol: water = 10:
The sample was placed in a chromatographic tank containing a 8:2) solvent. After 2 hours, a top layer of BushA was added and developed for 16 hours. After development, spots of testosterone as a standard marker were detected by UV absorption at 254 nm. A 3 cm length of the sample paper corresponding to the spot of this standard marker was cut and extracted with 3 ml of ethanol. Ethanol was removed by evaporation under a nitrogen stream. The procedure was then carried out in the same manner as the direct method and used for radioimmunoassay. (d) Radioimmunoassay To create a T standard curve, two T/ethanol solutions (containing 0, 20, 50, 100, 200, or 400 pg of T) were placed in test tubes. To each of these small test tubes and the small test tube containing the sample, 10 µ of ³ H-T ethanol solution (20,000 dpm/10 µ) was added, and the ethanol was removed by evaporation. Rabbit anti-T antiserum ^3H -T20000dpm (45pg as T)
Concentration that can bind 60% of (Bo = 60%)
0.05M Tris buffer [PH
8.0, 0.05% BSA (bovine serum albumin),
Contains 0.1% BGG (bovine serum γ-globulin)], added 0.2 ml to each small test tube, and mixed with a vortex mixer. Separately,
³ Add 0.2ml each of Tris buffer to two small test tubes containing only H-T20000dpm, mix,
Bo = 100%. Each solution was left at room temperature for 2 hours, and 0.2 ml of a saturated aqueous ammonium sulfate solution was added and mixed. Centrifugation was performed at 3000 rpm for 10 minutes, 0.2 ml of the supernatant was transferred to a vial, 2 ml of a scintillator was added, and the mixture was counted. The measured counts were then converted into B/Bo (%). (3.2) Measurement of DHT in female serum The amount of serum sample, extraction, and separation of DHT and T by paper chromatography were performed in the same manner as those used for measuring T in female serum samples. DHT was detected by spraying a mixed solution of equal amounts of 1% m-dinitrobenzene in ethanol and 2.5N sodium hydroxide in ethanol. DHT radioimmunoassay was performed in the same manner as T's radioimmunoassay method using a DHT/ethanol standard solution and a ³ H-DHT/ethanol solution (40,000 dpm/10μ). The values obtained by radioimmunoassay were corrected by the recovery rate for both the direct method and the conventional paper chromatography method.
In the case of the direct method, the recovery rate of T or DHT is 95
Since it was within the range of ~100% and there was no variation in the measured values, recovery correction by addition of ³ H-T or ³ H-DHT is considered unnecessary in normal cases. The results are shown in Tables 5 and 6. Results and analysis

【table】

[Table] As is clear from the above table, the measured values of both measurement methods clearly agree with each other.

[Table] As is clear from the above table, the measured values of both measurement methods show good agreement. As is clear from the above experimental facts, the target substance can be easily and accurately measured even in the presence of cross-reactive antigens by the direct method using the low cross-reactive antibodies provided by the present invention. Unlike the conventional method of paper chromatography, it is not necessary to pre-separate cross-reactive antigens, and the target substance can be easily measured. According to the invention, pretreatment of animals with a conjugate of a cross-reactive antigen and D-GL thereby induces immune unresponsiveness to the cross-reactive antigen, resulting in specific antibodies and said antibodies. Clones with production ability can be obtained. This method uses T-3-D-GL and DHT-
The present invention is not limited to the above-mentioned example using 3-D-GL, but can be applied to other examples as shown in the following Example 5. That is, in Example 5, although the binding sites of the carrier with T and DHT are changed, substantially the same results are obtained in this case as well. In this example, T and DHT are again used as model antigens, and their binding site with the carrier is changed from position 3 to position 15, thereby
The structure of the hapten exposed on the surface of the carrier molecule is changed. Example 5 15β-carboxyethylmercaptotestosterone (hereinafter abbreviated as 15β-CEM-T) and 15β-carboxyethylmercapto-5α-dihydrotestosterone (hereinafter abbreviated as 15β-CEM-5α-DHT)
Regarding the properties of antiserum obtained using the conjugate of 15β-CEM-T and KLH or D-GL, Rao, PN and PHMoore
Jr: Steroids 28 (1976) P101, 15β−CEN−
5α−DHT is Rao.PN, AHKhan and PH
Moore. Jr.: Steroids 29 (1977) P. 17, synthesized by the methods described in each case were used. 15β
- Method of binding CEM-T and D-GL or KLH and 15β-CEM-5α-DHT and D-GL or KLH
The bond with DHT-3-CMO or T
-3-COM and D-GL or KLH were combined in the same manner. The respective conjugates obtained are T-15-
D-GL, T-15-KLH, DHT-15-D-GL,
It is abbreviated as DHT-15-KLH. In this example, 6 groups of 7 female C57BL/6 mice (8-10 weeks old) were used. In the second group, 500 μg/mouse of DHT-15-D-GL was administered intraperitoneally to the mice 3 days before the first immunization. The first group is a control group and is similarly administered physiological saline not containing DHT-15-D-GL. The third group is another control group with 500μ of T-15-D-GL.
g/1 animal for 3 days after the initial immunization with T-15-KLH.
It was administered intraperitoneally one day before. The 1st, 2nd, and 3rd groups are T-15-KLH (100μ
The first immunization was carried out at a dose of 1.5 g/mouse), the second immunization was carried out 3 weeks later, and the booster immunization was carried out every 2 weeks after the second immunization. Groups 4, 5 and 6 were immunized with DHT-15-KLH. On the other hand, in the fifth group, T-15-D-GL500μg/1
Animals were administered intraperitoneally 3 days before the first immunization with DHT-15-KLH. Similarly to the fifth group, physiological saline was administered to the mice of the fourth group (control group) instead of T-15-D-GL. Group 6 is another control group that receives DHT-15-D-GL.
500 μg/mouse was administered intraperitoneally 3 days before the first immunization with DHT-15-KLH. Blood was collected every two weeks from 7 weeks after the first immunization for the six groups as described above, and the antibody titer and cross-reactivity in the serum were examined. The results of the antiserum obtained 13 weeks after the first immunization with the highest antibody titer are as follows. Properties of anti-T antisera obtained from groups 1, 2, and 3. ³ The antibody titer of the control group (group 1) is expressed as the reciprocal of the dilution factor of the antiserum that can bind 50% of H-T45pg.
It is between 1600 and 9000, and the antibody titer of the second group is
It is between 1000 and 3500. No antibody production was observed in the third group administered with T-15-D-GL. On the other hand, when determined by Abraham's method (described above), the cross-reactivity of the antisera from mice in the first group to DHT was in the range of 4.1 to 8.2%, indicating that DHT-
The cross-reactivity of Group 2 mouse antisera treated with 15-GL ranged from 0.27 to 0.94%. This number is higher than that of the antitestosterone antisera reported in the literature to date.
The cross-reactivity with DHT is better than the value. Therefore, the cross-reactivity of the antiserum according to the invention with DHT could be practically ignored. The lowest cross-reactivity of anti-T antiserum with DHT reported so far is 1.81%. To obtain this value, Rao et al. used 15β-CEM-T-BSA [P.
N.Rao and PH Moore Jr. Steroids 28
(1976)]. This example also uses a 15β-CEM-T derivative. The cross-reactivity obtained from the first group of this example is approximately equal to the minimum value mentioned above. The results of anti-DHT antisera obtained from groups 4, 5, and 6 were as follows. ³ Expressed as the reciprocal of the dilution factor of the antiserum that can bind 50% of H-DHT45pg, the antiserum of the fourth group (control group)
DHT antibody titer ranges from 1500 to 5600. The corresponding antibody titer of the fifth group administered with T-15-D-GL falls within the range of 1000-7600. No antibody production was observed in the 6th group administered with DHT-15-D-GL. According to the Abraham method, the cross-reactivity with testosterone in group 5 treated with T-15-D-GL was as follows:
The control group (group 4)
The cross-reactivity of was in the range of 48.3-68.5%. This result shows that cross-reactivity with testosterone is significantly suppressed by T-15-D-GL treatment (administration). Furthermore, the reason why no antibody production was observed in the 6th group administered with DHT-D-GL is that the clones capable of producing DHT antibodies were specifically inactivated by the administration of DHT-D-GL. Conceivable. The reagents and analytical methods used in Examples 6-9 (peptide examples) below are as follows. (1) Synthesis of hapten-D-GL Creation of pentagastrin-D-GL conjugate Liu
[Biochemistry, Vol. 18, p. 690 (1979)]. (a) Synthesis of S-acetyl mercapto-succinyl-D-GL (hereinafter abbreviated as Ac-SD-GL) D-GL (molecular weight 49,000) 40 mg (1.166 μmol)
Dissolve in 900μ of 0.125M phosphate buffer (PH7.2) and adjust the pH to 7.2 with 1N NaOH.
Add dimethylformamide (hereinafter referred to as DMF) of S-acetylmercaptosuccinic anhydride to this solution.
) solution (200 mg of acid anhydride in 1 ml of DMF)
Add 50 μH (57.4 μmol) (dissolved in
Stir at room temperature for 30 minutes. The pH was maintained at 7.2 during the reaction. After the reaction, the reaction solution was transferred to Sephadex G-
25 column and eluted with phosphate buffered saline (PH7.2) containing 0.01M Na ₂ -EDTA to separate the reaction product and unreacted reagent. 0.5M in 100μ of a solution containing this reaction product
N-Hydroxyamine (PH7.3) aqueous solution 100μ
(50 μmol) was added and incubated at 37°C for 20 minutes. Ellman et al.'s method [Arch.Biochem.Biophys.No. 82]
Volume P70 (1959)], the number of S-acetylmercapto groups introduced into D-GL was calculated.
That is, 50μ of this reaction solution was deoxygenated.
Add 0.1 ml of 0.01M [5,5′-dithiobis(2-nitrobenzoic acid) methanol solution],
React for 20 minutes in 1 ml of Tris buffer at pH 8.0, and then measure the absorbance at 412 nm. The number of S-acetylmercapto groups introduced per molecule of D-GL was about 15. (below,
The resulting acetylmercapto-succinyl-D-GL is designated as Ac-S ₁₅ -D-GL) and the recovery rate is approximately 77%. D-GL is
Since it has no absorption at 280 nm, the recovery rate of the fraction containing the D-GL derivative cannot be determined by absorbance measurement. Therefore, N-succiimidyl-3-(4-hydroxyphenyl)propionate and D-
GL was reacted, and the reaction product was passed through a Sephadex G-25 column as a monitor.
The recovery rate was determined by measuring absorbance (280 nm). (b) Preparation of m-maleimidobenzoyl-pentagastrin (hereinafter abbreviated as MB-pentagastrin) 11 mg (16.1 μmol) of pentagastrin was added.
Dissolve in 9.5ml of 0.1M phosphate buffer (PH8.0),
This was added to m-maleimidobenzoyl-N-hydroxysuccinimide ester (hereinafter referred to as
(abbreviated as MBS) 25.3 mg (80.5 μmol, 1 ml)
(dissolved in DMF) at once and stir. Thin layer chromatography [Developing solvent cyclohexane: ethyl acetate (1:1), volume ratio]
After confirming that the reaction has progressed sufficiently, 25 minutes after the start of the reaction, add 3 ml of dichloromethane to the reaction solution, shake thoroughly, and centrifuge. Transfer the upper buffer solution to another test tube. Add a small amount of phosphate buffer to the lower dichloromethane layer, shake it, centrifuge, and then add the upper layer to the previous solution. It was confirmed by thin layer chromatography that unreacted MBS was almost completely removed from the buffer solution containing MBS-pentagastrin. On the other hand, 20 μ (70 n mol) of 2-mercaptoethanol aqueous solution deoxygenated with a nitrogen stream
20 µ of the above-mentioned buffer containing MBS-pentagastrin was added to the mixture, and the mixture was allowed to react at room temperature for 20 minutes, and then the number of moles of 2-mercaptoethanol consumed was determined by Eman's method. This number of moles corresponds to the maleimidobenzoyl group introduced into pentagastrin. The maleimidobenzoyl group (MB) introduced per molecule of pentagastrin was 0.9. (Hereinafter, this compound will be referred to as MB _0.9 -pentagastrin.) (c) Creation of pentagastrin-D-GL conjugate The Ac-S ₁₅ -D-GL solution prepared in section (a) above and section (b) The MB _0.9 -pentagastrin solution prepared above was mixed and reacted as follows. After sufficient deoxidation under a nitrogen stream, 500μ of 5M hydroxylamine aqueous solution (PH7.3) was added to the mixture.
Stirring was continued at room temperature. After 1 hour, a portion of the reaction solution was taken and checked by Ellman's method, and no unreacted SH groups were found. This indicates that there are no SH groups that did not react with MB-pentagastrin;
That is, all SH groups on D-GL are MB
-Means that it has reacted with pentagastrin. After adding hydroxylamine aqueous solution 2
After an hour, 2-mercaptoethanol (final concentration 1mM) was added, and the mixture was stirred for an additional 20 minutes. This reaction solution was diluted with 0.01M phosphate buffer (PH7.2) using a dialysis membrane (cellophane membrane) for about 24 hours.
Dialyzed for hours. (The solution obtained after dialysis was used directly or diluted.) The pentagastrin-D-GL conjugate thus prepared had a molar ratio of pentagastrin to D-GL of 15:1. (Hereinafter referred to as pentagastrin ₁₅ -D-GL.) (2) Preparation of CCK-8-P-keyhole limpent hemocyanin (hereinafter abbreviated as CCK-8-P-KLH) (a) S-acetyl- Mercaptosuccinyl
KLH (hereinafter abbreviated as Ac-S-KLH) was prepared in the same manner as Ac-S-D-GL described in section (1) (a) above. Prior to synthesis, add 70 mg of KLH to 1.4 ml of 1%
It was dissolved _{in K2CO3} solution and dialyzed against 0.125M phosphate buffer (PH7.2). The amount of KLH was calculated by measuring the absorbance of this solution at 280 nm. 0.7ml of this KLH solution (26.0mg as KLH,
If the molecular weight is approximately 100,000, this corresponds to 260 n mol. The pH of the solution is 7.2. ), 6.5 μmol of S-acetylmercaptosuccinic anhydride was added, and synthesis was performed in the same manner as in section (1) (a). The number of S-acetylmercapto groups bonded to each KLH molecule was 6.8. (Hereinafter referred to as acetyl-S _6.8 -KLH.) (b) m-maleimidobenzoyl-CCK-8-
P (hereinafter abbreviated as MB-CCK-8-P) was produced in the same manner as in the production of MB-pentagastrin. 0.42mg synthesized by fragment condensation method
(385 n mol) of CCK-8-P was dissolved in 0.5 ml of 0.2 M phosphate buffer, and 600 μg (1.9 μmol) of CCK-8-P was dissolved in 0.5 ml of 0.2 M phosphate buffer.
Reacted with MBS. 1.2 mg MBS for reaction
50μ of MBS solution dissolved in 100μ of DMF
was used. The resulting product had a CCK-8-P:MB ratio of 1:1.0. (hereinafter MB _1.0 −CCK-8
-Display as P. ) (c) CCK-8-P-KLH Acetyl-S obtained in section (2) (a) above _6.8 −
Mix 2.3 ml (75.9 r mol) of the KLH solution and the MB _1.0 -CCK-8-P solution obtained in section (2) (b) above, and add 0.5 M NH ₂ OH0.3 c.c. (0.15 mmol). In addition, the same operations as in section (1) (c) were repeated. The ratio of CCK-8-P and KLH obtained was approximately 5:1. (3) CCK-8-P-Bovine serum albumin (Bovine
serum albumin) (hereinafter abbreviated as CCK-8-P-BSA) (a) S-acetylmercaptosuccinyl-
Preparation of BSA 25 mg (351 n mol) of BSA and 1.5 mg of S-acetylmercaptosuccinic anhydride
mg (8.8 μmol) and performed the same procedure as in section (1) (a) to synthesize. The ratio of BSA:S-acetylmercapto group is
The ratio was 7.6:1. (b) Preparation of MB-CCK-8-P 0.5 mg (457 n mol) of CCK-8-P was added to 0.5 ml of
Dissolve in 0.1M phosphate buffer PH8.0 and add 700μ
2.29 μmol of MBS. For the reaction
Solution of 1.4mg MBS dissolved in 100μ DMF
Using 50μ, synthesis was performed in the same manner as in section (1) (b). The ratio of MB:CCK-8-P was 1.0:1. (Hereinafter referred to as MB _1.0 -CCK-8-P.) (c) Preparation of CCK-8-P-BSA 1.9 ml (84 n mol) of the solution in (3) (a) above and (3) (b)
Mix the liquid obtained in Section 1 and make 0.5M
0.35 c.c. (175 μmol) of NH ₂ OH was added, and the same operation as in section (1) (c) was performed. The resulting CCK-8-P:BSA ratio was approximately 5:1. Below CCK-8-P ₅ -BSA
is displayed. (4) Preparation of pentagastrin-BSA (a) Preparation of MB-pentagastrin Add 0.5 mg (750 n mol) of pentagastrin to 0.5
Dissolved in ml 0.1M phosphate buffer (PH8.0),
This was reacted with 1.05 mg (3.3 μmol) of MBS. For the reaction, 50μ of a solution of 2.1mg of MBS dissolved in 100μ of DMF was used, and the same operation as in section (2) (b) above was performed. The MB:pentagastrin ratio was 1.0:1. Hereinafter, it will be indicated as MB _1.0 − Pentagastrin. (b) Preparation of pentagastrin-BSA 2.54 ml of solution obtained in the same manner as in (3) (a)
(112n mol) and (4)(a) were mixed, and 0.5M
0.4 cc (200 μmol) of NH ₂ OH was added, and the same operation as in section (1) (c) was performed. The resulting pentagastrin-BSA had a pentagastrin:BSA ratio of 5:1.
Hereinafter, it will be indicated as pentagastrin ₅ -BSA. (5) In the above item (1), using D-GL with a molecular weight of 115,000 instead of D-GL with a molecular weight of 34,300, and performing the operation with the same reaction amount as in item (1), pentagastrin ₃₃ -D-GL was obtained. The density of the antigenic determinant (pentagastrin) associated with D-GL on the molecular surface of these was similar. Example 6 (a) Immunization schedule and serum collection Six animals in each group (C57BL/6J×
DBA/2) F ₁ mice were immunized. Each mouse in all groups was treated with 10 μg of CCK-8-P-
Immunization with KLH (0.2 ml each of complete Freund's adjuvant emulsion) and 3 weeks later, 10 μg each
of CCK-8-P-KLH (0.2 ml each of incomplete adjuvant emulsion). Two weeks after the second immunization, 10 μg of each CCK-8
-P-KLH adsorbed on aluminum hydroxide gel (2 mg of aluminum) was used for boosting. 2 and 4 weeks after boosting, 10μ each
The cells were further immunized with 0.2 ml physiological saline containing 1 g of CCK-8-P-KLH. All immunizations were administered intraperitoneally. All mice in the second group were given 0.5 cc of saline containing 300 μg of pentagastrin ₁₅ -D-GL per mouse intraperitoneally 3 days before the first immunization. 1st
Physiological saline for each mouse in the group (control)
0.5ml (excluding Pentagastrin ₁₅ -D-GL)
was administered only. Group 3 mice received intraperitoneal administration of 0.5 cc of physiological saline solution containing 300 μg of pentagastrin ₁₅ -D-GL each 3 days before the second immunization and 3 days before the third immunization. Serum from each immunized individual was collected from the retroorbital venous plexus. (b) Measurement of antibody titer Antibody titer was measured by radioimmunoassay method. Each well of a polyvinyl round-bottom microplate for solid-phase radioimmunoassay was injected with 0.01 M phosphate buffer (0.15 at pH 7.2) containing 10 μg/ml of antigen (CCK-8-P-BSA or pentagastrin-BSA). (containing M NaCl),
The antigen is bound to the surface of the polyvinyl plate by adding 10μ aliquots and incubating at room temperature for 2 hours. After this, the solution in each well is discarded and the wells are rinsed thoroughly with tap water four times. After that, drain the water thoroughly and use 1%
The protein binding ability of polyvinyl was completely masked with BSA by adding 200 μl of physiological saline containing BSA to each well and leaving it at 4° C. overnight. Thereafter, the solution in each well was discarded, the wells were thoroughly rinsed four times with tap water, the water was thoroughly drained, and the wells were then used for immunoassay. Dilute each antiserum with 1% BSA-PBS,
Add 0.1 ml of it to the wells of the above plate. After standing overnight at 4°C, rinse thoroughly with tap water three times, wash six times with 2M NaCl-PBS, and finally rinse with tap water and drain thoroughly. A rabbit anti-mouse IgG antibody specifically purified using an immunoadsorbent labeled with ¹²⁵ I was diluted with 1% BSA-PBS, and 0.1 ml was added to each well and left overnight. By this operation, if there is an antibody that reacts with the antigen on the polyvinyl, it binds to it, and this antibody can be determined by the degree of binding of the ¹²⁵ I-rabbit anti-mouse IgG antibody. After removing the isotope solution from each well with a pipette and thoroughly washing each well with water, each well was sectioned with a hot nichrome wire, and the radioactivity of each well was measured. (c) Results The amount of antibodies in the pooled serum was measured for each group at the time of blood collection, and the amount of antibodies was further measured 11 weeks after the first immunization, when the amount of antibodies was the highest. The results obtained are shown in FIG. The standard serum used was pooled antiserum from Group 1 7 weeks after the first immunization. The amount of antibody in the sample obtained from each mouse after the 11th week was expressed as a ratio, with the amount of antibody contained in the standard antiserum as 1. The pooled antisera used as standards had the following titers: This pooled antiserum at a 600-fold dilution was used to detect cholecystokinin (CCK-8-P) in a liquid-phase radioimmunoassay system using CCK-8-P as described below.
33) was able to bind 0.0015p mol. Antibodies obtained from mice that were not administered pentagastrin ₁₅ -D-GL (group 1, indicated by circles in Figure 1) cross-reacted with pentagastrin to almost the same extent as they reacted with CCK-8-P. showed his sexuality. pentagastrin
Antibodies obtained from mice administered with ₁₅ -D-GL (groups 2 and 3. In Figure 1, ▲
It was found that the titers of antibodies cross-reacting with pentagastrin (represented by marks and ●) were very low. In particular, the cross-reactivity with pentagastrin of antibodies obtained from group 3 mice administered with pentagastrin ₁₅ -D-GL after the first immunization (3 days before the second immunization and 3 days before the third immunization) was significantly low. It was. From the above results, according to the present invention, CCK-
For the amino acid sequence specific to 8-P, i.e.

It was found that an antibody specific for [Formula] was obtained. Example 7 Using a liquid phase immunoassay system in which CCK-8 and pentagastrin coexist, the cross-reactivity of the antibody obtained by the above method was measured as follows. CCK-8 with ^125I using Bolton-Hunter reagent
-P labeled [(H. Sankara et al., J. Biol.
Chem., Vol. 254, pp. 9349-9351, 1979); hereinafter, this labeled compound will be abbreviated as ¹²⁵ I-BH-CCK-8. ]. Add 0.02M phosphate buffer (PH) to each small test tube.
CCK by adding various amounts of unlabeled CCK-8-P dissolved in 50μ each (containing 8.0, 0.1% gelatin).
A series for constructing a standard curve for -8-P was created, and in another series, a series for constructing a pentagastrin standard curve was created by adding 50 .mu. of each buffer solution containing various amounts of pentagastrin to each test tube. In each case 50 mμ of antiserum diluted with normal mouse serum was added to the test tube and further ¹²⁵ I-BH-CCK-8 (approx.
50μ of the same phosphate buffer containing 5000 cpm) was added, the tube was mixed by shaking on a vortex mixer, then kept at 4°C for 24 hours, and then diluted (1:2) with the same phosphate buffer. rabbit anti mouse
Add 50μ of IgG Fab antibody to the test tube, mix, and
Keep at ℃ for 24 hours. The test solution was then centrifuged at 3000 rpm for 25 minutes. The supernatant was aspirated using an aspirator, and the radioactivity of the precipitate was measured using a counter. CCK-8-
All ¹²⁵ I−BH−CCK using the same phosphate buffer instead of P standard solution and pentagastrin standard solution.
-8 radioactivity (count, Bo as 100) was measured, and the antibody and ¹²⁵ I-BH-CCK-8 were tested in the presence of various concentrations of CCK-8-P or pentagastrin.
The binding rate (B/Bo%) was calculated. The molar amounts of various peptides required to inhibit the binding of ¹²⁵ I-BH-CCK-8 and antibody by 50% were as follows. In group 1, CCK-8 was 1.1 pmol and pentagastrin was 20.1 pmol. accordingly
Cross-reactivity calculated by Abraham's method is 5.5%
(1.1/20.1×100). In group 2, where pentagastrin ₁₅ -D-GL was administered before the first immunization, the cross-reactivity was 2.7% (4p
mol/150p mol×100). No cross-reactivity with pentagasupentagastrin was observed. That is, when using the above antibody, 1.7 pmol of CCK-8-P was required for 50% inhibition, but
No inhibition was observed even with 10000 pmol of pentagastrin. Pentagastrin was practically unable to react with the antibodies produced in group 3 mice. It was unable to actually react with antibodies produced in mice. From the above results, pentagastrin ₁₅ -D-
When using antibodies from group 3 mice obtained by administration of GL, CCK-8 in the sample was not significantly affected by coexisting pentagastrin.
It was found that -P can be specifically measured. Example 8 In place of the mouse used in Example 6, a domestic rabbit was used to carry out the same immunization procedure. However, 100μ
complete or incomplete Freund's adjuvant emulsion containing g of CCK-8-P-KLM and saline containing 10 mg of pentagastrin ₁₅ -D-GL.
10ml was used. The results obtained were approximately similar to those obtained using mice. Example 9 Pentagastrin ₁₅ − used in Example 6
Approximately the same results as in Example 6 were obtained by carrying out the same procedure except that pentagastrin ₃₃ -D-GL having a molecular weight of 115,000 was used in place of D-GL. Example 10 Production of clones capable of producing anti-CCK-8-P specific antibodies. (B) Production method The first group (control group) and the third group (pentagastrin D-GL) were immunized by the method described in Example 6(A).
2X10 ⁸ splenocytes were removed from each mouse in treatment group). Each was conventionally fused with 2×10 ⁷ myeloma cells P3-X63-Ag80-U1 (P3-U1) in polyethylene glycol 4000. Cell clusters were mixed with PEG4000 (9g) and HANKS (20g).
ml) to create a cell suspension. The cell suspension was allowed to stand at room temperature for 8 minutes, then MEM (15 ml) was added over 5 minutes, and then MEM was added to bring the total volume to 50 ml. Remove cells from suspension and add 10% to 50ml
The cells were treated in FCS-RPM1 and cultured overnight at 37°C using 1 ml/well culture plates. An incubator containing 7% _CO2 in air with 85-95% humidity was used. Each morning for the next two days, the HAT medium (1 ml) was aspirated and replaced with fresh HAT medium (1 ml).
Similar exchanges were made every 3 days for the next 2 weeks. Thereafter, the culture was removed and antibody production was examined using the solid-phase radioimmunoassay method described in Example 6B. The fused cells in which antibody production was observed were cloned by the limiting dilution method (using 10% FCS-RMPI). The specificity of the antibody produced by the clone thus obtained was examined by the phase radioimmunoassay method described in Example 6B. (b) Results By using splenocytes from mice of the first group (control group), 18 clones capable of producing anti-CCK-8-P specific antibodies were obtained. All of these obtained clones reacted with both CCK-8-P and pentagastrin. Therefore,
It was confirmed that a monoclonal antibody specific to the pentagastrin portion of CCK-8-P was obtained. On the other hand, by using the splenocytes of the third group, 15 clones capable of producing anti-CCK-8-P specific antibodies were obtained. Of these 15 clones, 12 produced anti-
CCK-8-P-specific antibodies do not react with pentagastrin and related peptides, and CCK-8-P does not react with pentagastrin and related peptides.
- It was specific for P. However, antibodies produced by the remaining three clones had cross-reactivity with pentagastrin. Therefore, by using mouse splenocytes treated with pentagastrin-D-GL according to the present invention, a greater number of desired anti-CCK-8 cells can be obtained than by using conventionally treated splenocytes. -Clones capable of producing P-specific antibodies are obtained. obtained
The CCK-8-P specific antibody showed no cross-reactivity with pentagastrin.

[Brief explanation of drawings]

The figure shows that the antibody according to the present invention has low cross-reactivity, and the horizontal axis represents the amount of antibody that reacts with pentagastrin, and the horizontal axis represents the amount of antibody that reacts with CCK-8-P. ○ indicates pentagastrin ₁₅
-D-GL represents antibodies obtained from mice that were not administered (group 1). ▲ indicates antibodies obtained from mice (group 2) administered with pentagastrin ₁₅ -D-GL before the first immunization. ● marks represent antibodies obtained from mice (group 3) administered with pentagastrin ₁₅ -D-GL after the first immunization. Each unit represents the ratio of the antibody content when the amount of each antibody contained in the pooled serum of Group 1 mice 7 weeks after the first immunization is conveniently assumed to be 1.0.

Claims (1)

[Claims] 1. D-glutamic acid and D-covalently bonded to a second antigen having one or more antigenic determinants that are structurally similar to the first antigen having the desired antigenic determinant in parts other than the antigenic determinant. immunizing the mammal with the first antigen after administering the polymer with lysine to the mammal, thereby inducing effective immune unresponsiveness in the mammal to the second antigen;
High specificity for the first antigen and low cross-reactivity for the second antigen, obtained by a method of generating cells with the desired antibody-producing ability, isolating the cells from the mammal, and culturing them. A clone that has the ability to produce antibodies with specific characteristics.