JPS6051662B2 - Method for producing antiserum for measuring 16α-hydroxy-5-ene steroids - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention is based on the following formula. 01[i]--■■, 16α-hydroxy represented by (in the formula, R'' represents an acetyl group, R'' represents a hydrogen atom, or R゛ and R゜ together represent an oxo group) The present invention relates to a novel reagent for immunological measurement of 5-ene steroids.

Conventionally, cholesterol is converted to dehydroepiandrosuarone in the body via pregnenolone, then undergoes hydroxylation at the 16α position in the adrenal glands of the fetus, undergoes aromatization in the placenta of pregnant women, and is excreted in the urine as estriol. It is known that
It is also known that the aforementioned 16α-hydroxylated dehydroepiandrosuarone is present in trace amounts in the urine or serum of pregnant women. Therefore, by quantifying both estriol and 16α-hydroxydehydroepiandokasten (3β, 16α-dihydroxy-5-androsten-17-one), we can confirm that the placenta of pregnant women or the adrenal glands of the fetus are functioning normally. You can know whether it is done or not. For example, if the amount of 16α-hydroxydehydroepiandrosterone is normal and the amount of estriol is low, there is an abnormality in the placenta.
If both hydroxydehydroepiandrosterone and estriol are lower than normal values, this indicates that the fetus has an abnormality in its adrenal glands. Thus, 16α-hydroxydehydroepiandrosterone is an important compound in the diagnosis of pregnant women and fetuses. In addition, 16α-hydroxypregnenolone is also present in the serum or urine of pregnant women, and quantifying this compound also provides important information for determining whether a pregnant woman is normal or not.

Therefore, there is a need for an antiserum for accurately and selectively quantifying 16α-hydroxy-5-ene steroids. There is a method in which 16-dihemisuccinate is used as a hapten (a type of incomplete antigen and also called an adherent).

However, since this compound also has a hemisuccinate hydroxyl group at the 16α position, the antiserum obtained when this compound is used as a hapten has extremely poor specificity for steroids having a hydroxyl group at the 16α position. In fact, for 16α-hydroxydehydroepiandrosterone and de5hydroepiandrosterone, this 3β●
When using an antiserum prepared with 16α-dihemisuccinate as a hapten, when the reaction rate against 16α-hydroxydehydroepiandrosterone is set as 100,
Against dehydroepiandrosterone! Then 166,
It has been found that the values are completely non-specific. However, since dehydroepiandrosterone is also present in large amounts in the serum or urine of pregnant women, when quantifying 16α-hydroxydehydroepiandrosterone, dehydroepiandrosterone coexisting in the serum or urine must be removed. However, the procedure is extremely difficult and involves various operations, making it almost impossible to selectively and accurately measure 16α-hydroxy-5-ene steroids. Therefore, as a result of research on this point, the present inventors found that only the hydroxyl group at the 3-position is 16 in hemiacylate.
A novel 5-ene steroid with a free hydroxyl group at the α position (
When we synthesized 1) and produced an antiserum using this steroid derivative as a hapten, we surprisingly obtained a new antiserum with unprecedented and excellent specificity for 16α-hydroxy-5-ene steroids. I found out.

The present invention provides 3.1 represented by the general formula (wherein R is an alkylene group or an alkenylene group, R1 is an acetyl group, R2 is a hydrogen atom, or R1 and R2 together represent an oxo group).
16α-hydroxy-5-ene steroid, which is characterized in that a warm-blooded animal is immunized using a combination product of 6α-dihydroxy-5-ene steroid-3-hemiaacylate and an antigen protein, and antiserum is collected from the blood. This is a method for producing antiserum used for quantitative determination.

The novel steroid compound of formula 1, which is the starting material for producing the antiserum used in the present invention, has the general formula (where R.R.
It is also possible to synthesize it purely chemically from the 5-ene steroid-3-hemiaacylate compound represented by (1 and R2 have the above meanings); By introducing a hydroxyl group at the 16α-position by allowing the bacterial cells of Streptomyces roseochromogenus, Streptomyces halstetdii or Streptomyces medeiosideicus or the enzyme produced by them to act in a conventional manner, It can be produced particularly advantageously (see Japanese Patent Application No. 50-37411).

These bacteria are common bacteria that are easily available in Japan and can be cultured by various methods, but aerobic methods such as shaking culture method or aerated agitation culture method are particularly preferred. Glucose,
Nitrogen sources such as saccharose, dextrin, and starch are used as nitrogen sources, such as peptone, chilitin, yeast scratches, and meat scratches, and other inorganic salts, such as sodium chloride, magnesium sulfate, ammonium phosphate, and ammonium nitrate, are used for the growth of bacteria. Cultivation is carried out by adding the necessary nutrients appropriately. The steroid compound of formula (■) may be dissolved or suspended in water or an organic solvent such as ethanol, acetone, dimethylformamide, dimethylacetamide, tetrahydrofuran, dioxane, ethylene glycol dimethyl ether, or diethylene glycol dimethyl ether, and in some cases, may be dissolved or suspended at an interface. It can be added to a bacterial culture or an enzyme system all at once or continuously or intermittently over a certain period of time as a solution or suspension containing an active agent, a dispersant, etc., and reacted.

The time of addition may be selected from the start of culture of the bacteria, during culture, or at any appropriate time after culture, but midway through culture is particularly preferred. The pH of the medium is usually 6.5 to 9.0, preferably 7.
．． 0 to 8.0, and the temperature is preferably 25 to 30°C.
A reaction period of 1 to 5 days, preferably 2 to 3 days is sufficient.
The compound of formula (1) can be collected separately by various methods, such as adsorbing it on an adsorbent and eluting it with an appropriate solvent, directly extracting it with an organic solvent that is immiscible with water, or Various separation means can be selected and used, such as a method of separation by flow distribution.

It is particularly preferred to use chromatography for purification. The lower alkylene group represented by R in formula (1) and formula (■) has 1 to 4 carbon atoms, such as methylene group, ethylene group, propylene group, butylene group, etc.
An alkylene group having 2 to 4 carbon atoms is preferable, and the alkenylene group is preferably an alkenylene group having 2 to 4 carbon atoms such as a vinylene group, a propenylene group, a butenylene group, and among them, an ethylene group is particularly preferable.

Representative examples of the novel hemiester represented by formula (1) are shown below.

3β・16α-dihydroxy-5-androstene-1
7-one-3-hemisuccinate, 3β●16α-dihydroxy-5-pregnene-20-one-3-hemisuccinate, 3β●16α-dihydroxy-5-androstene-17-one-3-hemiglutarate, 3β●1
6α-dihydroxy-5-pregnene-20-one-3
-Hemiglutarate, 3β・16α-dihydroxy-5
-androstene-17-one-3-hemimaleate,
3β●16α-dihydroxy-5-pregnene-20-
On-3-hemimaleate.

The hemiester of formula (1) is administered to warm-blooded animals in the form of a conjugation product with an antigenic protein.

Examples of antigen proteins used include bovine serum albumin, human serum albumin, rabbit serum albumin,
Examples include albumin such as horse serum albumin, bovine serum globulin, and globulin such as horse serum guprine. The method of bonding the carboxyl group of the hemiester of formula (1) with the amino group of these antigenic proteins is known per se, and a wide range of polypeptide synthesis methods can be used.For example, it can be carried out by the following method. be able to.
(1) Carbodiimide method: Bonding is performed using a condensing agent such as dicyclohexylcarbodiimide or N-(3-dimethylaminopropyl)-N''-monoethylcarbodiide hydrochloride.

(2) Alkyl carbonic anhydride method: A hemiester is reacted with an alkyl chloroformate to form an alkyl carbonic anhydride, which is bound to an antigen protein.

(3) Active ester method: A hemiester is converted into an active ester derivative by reacting with p-nitrophenol, N-hydroxysuccinimide, etc., and then combined with an antigen protein.

(4) Imidazolid method: A hemiester is reacted with carbonyldiimidazole, thionyldiimidazole, etc. to convert it into an imidazolide derivative, which is bound to an antigen protein.

The thus obtained conjugation product of hemiester and antigenic protein is administered parenterally, especially by injection, to warm-blooded animals, especially mammals.

An injection solution of the conjugated product can be prepared by a conventional method, and, for example, a solution having the following composition is used. combined product
1-1 saliva volume adjuvant
0.5-2 volumes of saline
As the 0.1 to 2 parts by volume adjuvant, for example, Freund's adjuvant, complete Freund's adjuvant, etc. are used.

This injection solution is administered to mammals such as rabbits, sheep, goats, rats, monkeys, etc. by subcutaneous injection, intramuscular injection, intravenous injection, intraperitoneal injection, etc.

The dosage will vary depending on the type of animal to which it is administered, but is generally between 0.1 and 10 m91k9 of the conjugated product per dose.
The dosing interval is 5 days to 1 month, and usually at least 1 month to about 1 month until a sufficient rise in serum antibody titer is observed.
It is administered yearly, but in the case of rabbits, for example, it is sufficient to administer it once every 3 to 6 months. Blood is collected from the mammal that has been sufficiently immunized in this way, and serum is separated from it by a conventional method, for example, by centrifugation. The antiserum thus produced can be directly subjected to immunoassay, and can be stored for a long period of time by freezing or freeze-drying. This antiserum has the formula (4) corresponding to formula (1).
), a reagent containing this antiserum as an active ingredient can immunologically and sensitively quantify 16α-hydroxy-5-ene steroids, and is used in medicine. and veterinary endocrinological diagnosis and analysis.

Production example 1 3β◆16α-dihydroxy-5-androstene-1
Production of 7-one-3-hemisuccinate Glucose 1%,
Peptone 0.5%, meat scratches 0.25%, yeast scratches 0
．． 1%, NaClO. l% and MgSO4・71(20
An aqueous solution containing 0.05% of
100 ml of the mixture was dispensed into a 0 ml shaking container, and after heat sterilization, it was inoculated with Streptomyces roseochromogenus and cultured with shaking at 28° C. for 24 to 3 hours.

Then 600 m9 of dehydroepiandrosterone-3
- 15 mg of hemisuccinate in each corben (a)
．． 5 cc (7)N-N''-dissolved in dimethylformamide) was added, and further shaking was performed at the same temperature for a long time.After the cultivation was completed, the bacterial cells were removed by centrifugation, and the pH
After extracting twice with an equal amount of ethyl acetate, dehydrating with anhydrous sodium sulfate, and distilling off the solvent, approximately 1V
of oil was obtained. The reaction products were separated by adsorption chromatography using silica gel. As the eluent, a mixed solvent of benzene and ethyl acetate was used, and 15
A total of approximately 100 m9 of 3β●16 was obtained from each elution with % ethyl benzene and then 20% ethyl benzene acetate.
Crude crystals of α-,dihydroxy-5-androstene-17-one-3-hemisuccinate were obtained. This was suspended in a small amount of 8% aqueous sodium bicarbonate (PH 9.0), and impurities were extracted and removed with an equal amount of ethyl acetate (this operation was repeated several times), and the pH was adjusted to 3.0. Purified by extraction with an equal volume of ethyl acetate. Yield approximately 60m9. Melting point 211-2
18℃. This substance is IR. ．． The 3β
・16α-dihydroxy-5-androstene-17-
It was confirmed to be on-3-hemisuccinate.

Its physical constants are shown below. Nuclear magnetic resonance spectrum (C
5D5N) δ: 0.89 (aiming for 18th place, single line), 0.9
7 (Aiming at the 19th position, singlet), 4.65 (H1 multiplet of 1 only 0H), 2.90 (4H1 singlet of succinate) Infrared absorption spectrum (KBr) ν: 3440CTfi-1
(Hydroxyl group), 1738 cm-1 (CO absorption) Mass Svectani (M+-COCH2CH2COO) 304 Production example 23β・16α-dihydroxy-5-pregnene-2
Production of 0-on-3-hemisuccinate 100 ml of medium 4e having the same composition as in Production Example 1 was placed in a 500 ml shaking kolben.
After dispensing and sterilizing, Streptomyces roseomogenus was inoculated, and the temperature was 24-3ea at 28°C. Interculture was performed.

At this point, 600 ml of pregnenolone-3-hemisuccinate was added to each colben at 15 mg (dissolved in 1 ml of ethanol) and incubated with shaking for an additional period of time to allow substrate conversion. The culture solution was then extracted with ethyl acetate, and the extract was concentrated to obtain approximately 1V of oil. This was subjected to column chromatography using silica gel,
From the 20% ethyl acetate eluate, 3β・16α-dihydroxy-5-pregnen-20-one and 3β●
Approximately 200 m9 of mixed crude crystals of 16α-dihydroxy-5-pregnene-20-one-3-hemisuccinate were obtained. By suspending this crude crystal in 8% sodium bicarbonate water (PH9.O) and extracting with ethyl acetate, 85 m9 of 3β
-16α-dihydroxy-5-pregnene-20-one (melting point 240-247C) was obtained.

On the other hand, by adjusting the pH of the aqueous layer to 3.0 and extracting and concentrating the precipitated crystals with ethyl acetate, 52m9 of crystals of 3β·16α-dihydroxy-5-pregnene-20-one-3-hemisuccinate were obtained (melting point: 151~ 15
3℃). This product was prepared using commercially available pharmacopeia diastase.
Since it was hydrolyzed to β◆16α-dihydroxy-5-pregnen-20-one, it was confirmed that it was 3β·16α-dihydroxy-5-pregnen-20-one-3-hemisuccinate. Its physical constants are shown below. Nuclear magnetic resonance spectrum (CDCl3) δ: 0.65
(18th place 4, singlet), 1.05 (19th place aim, singlet), 2.18 (21st place aim, singlet), 2.57 (succinate 4H1 singlet), 4.70 (Tokui 0H (7
) H1 multiplet) Infrared absorption spectrum (KBr) ν:3
450cm-1 (hydroxyl group), 1740cr! l-1, 1
700C1ri-1 (acid and CO at position 20) The 16α monohydroxylation reaction of each 3-hemisuccinate of dehydroepiandrosterone and pregnenolone was carried out in place of Streptomyces roseochromogenus in Production Examples 1 and 2.
When the same procedure was carried out using Streptomyces halstetdii and Streptomyces medeiosidicus, almost the same results as in Production Examples 1 and 2 were obtained.

3β・16α-dihydroxy-5 obtained by Example Production Example
- To a solution of 65 m9 of androstene-17-one-3-hemisuccinate and 30 m9 of tri-n-butylamine in 1.9 mL of dioxane is added 23.0 m9 of isobutyl chlorocarbonate with stirring at 10°C.

This was stirred at 4°C for 20 minutes, and the resulting cold reaction solution was mixed with 7ml of water containing 0.250f of calf serum albumin (BSA).
It is added dropwise to a solution in 7 ml of dioxane and 0.25 ml of 1N caustic soda (pH 9.5) at 8° C. with stirring. After stirring for 1 hour while cooling, 5 drops of 1N caustic soda was added to adjust the pH to 8.5, and the mixture was further stirred for 2 hours. This reaction solution was dialyzed overnight, then placed in a cooling centrifuge tube, and 1N monohydrochloric acid was added dropwise to adjust the pH to 4.5, producing a precipitate. The precipitate was aged overnight at 4°C and centrifuged (1000
0 rpm x 1 powder at 4°C), remove the supernatant liquid, add a small amount of water twice, add 10 ml of water, cool and stir while adding 5% aqueous sodium hydrogen carbonate solution dropwise to dissolve the precipitate. Perform dialysis overnight. The resulting solution is freeze-dried to obtain 306 mg of 3β·16α-dihydroxy-5-androstene-17-one-3-hemisuccinate-3-albumin conjugate as a colorless feather-like powder. Add 1 mg of this albumin conjugate to 0.5 ml of physiological saline for injection.
0.5 mt of Complete Freund's Adjuvant was added to form an emulsion, which was then injected subcutaneously in four or more points on the back of a male rabbit (body weight approximately 2.0 kg) in divided doses.

Injections are given twice a month for the first month, then once a month. From 10 days after the injection, 10 m of blood was collected from the ear vein over time.
1 is collected, the serum is separated by centrifugation, and the antibody titer is measured using the method described below. As a result, the antibody titer can be diluted 100 times after 2 months of administration, and diluted 10,000 times after 5 months. Five months later, whole blood was collected and serum was separated.
Store at -80°C and use for immunoassay.

Method for measuring antibody titer: Antibody titer is determined by preparing a 3β·16α-dihydroxy-5-androsten-17-one standard curve and expressing the dilution factor of the antiserum that can be prepared.

That is, the collected antiserum was adjusted to pH8. 0.05r of O!
4 borate buffer (0.06% bovine serum albumin, bovine γ
- containing globulin at a rate of 0.05%). Separately, each of 3β●16α-dihydroxy-5-androsten-17-one 0, 20, 5011001200
, 500 pg and 3β・1α-dihydroxy-5-androstene-17-one-7α monotritium (20.0
Ci17T1. M) Take 10,000 dpm into a small centrifuge tube, dry it, and then add 0.25 ml of the above diluted antiserum. After shaking the mixture to prevent foaming, the mixture was left at room temperature for 30 minutes, and then 0.25 ml of a saturated aqueous ammonium sulfate solution was added and mixed.

After leaving it for another 10 minutes, 1 at 3000r'Pm.
Centrifuge for 0 minutes to precipitate the protein portion, place 0.2 ml of supernatant in a measuring bias of a liquid scintillation counter, add 12 ml of dioxane scintillator, and measure radioactivity using a liquid scintillation counter. The binding rate of 3β·16α-dihydroxy-5-androstene-17-one-7α-monotritium to the protein moiety was calculated from the measured radioactivity value, and the amount of 3β·16α-dihydroxy-5-androstene-17-one added and the corresponding amount were calculated. The optimal standard curve prepared based on the binding rate is determined, and the dilution factor of the antiserum at this time is used to indicate the antibody titer. To test the cross-reactivity of this antiserum to various steroids, a standard curve similar to that for 3β·16α-dihydroxy-5-androsten-17-one was created for each steroid, and the amount of steroid that inhibited the binding rate by 50% was determined. The cross-reactivity rate is determined as a percentage of the amount of β·16α-dihydroxy-5-androsten-17-one that inhibits the binding rate by 50%.

Claims (1)

[Claims] 1. General formula ▲ Numerical formula, chemical formula, table, etc. ▼ (I) (In the formula, R represents an alkylene group or an alkenylene group, R^1 represents an acetyl group, R^2 represents a hydrogen atom, or R^1 and R^2 together represent an oxo group) 3.16
16α-hydroxy-5-, which is characterized in that a warm-blooded animal is immunized using a conjugation product of α-dihydroxy-5-ene steroid-3-hemiaacylate and an antigen protein, and an antiserum is collected from the blood thereof. A method for producing antiserum used for quantifying ensosteroids.