JPS6073358A - Antigen and anti-serum for measuring 17alpha- hydroxyprogesterone - Google Patents

Abstract

PURPOSE:To enable the simple high sensitivity measurement of 17alpha-hydroxyprogesterone, by preparing antigenic protein of 17alpha-hydroxyprogesterone-11- hemixasuccinate, and immunizing an animal by using this protein as an antigen to obtain anti-serum. CONSTITUTION:By using anti-serum specifically reacted with 17alpha-hydroxyprogesterone obtained by immunizing a mammal with a steriod compound represented by formula (wherein Y is antigenic protein), 17alpha-hydroxyprogesterone in blood can be measured specifically with high sensitivity. As the antigenic protein Y to be coupled, bovine serum albumin, rabbit serum albumin or egg albumin are used. In coupling, a known acid anhydride mixture method or a carbodiimide method may be used. By using this anti-serum, only 17alpha-hydroxyprogesterone can be measured with high sensitivity. By this method, the diagnosis of congenital adrenal hyperplasia or adrenal androsterone excessive formation is certainly conducted.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel steroid compound, more particularly having a hemisuccinate-antigenic protein at position 77.
The present invention relates to an antigen for measuring 7α-hydroxyprogesterone and an antiserum derived from the steroid compound.

Cortisol, an adrenal cortical hormone, is produced in the body from glogesterone/7α-hydroxyglogesterone, //-
It is known to be biosynthesized via desoxycortisol. That is, the biosynthesis of cortisol from /7α-hydroxyprogesterone requires the presence of the 2/-sugar fermentation cord.

Congenital adrenal hypermorphism (CΔH) is the most common adrenogenital syndrome observed in children, and is a disease caused by a congenital defect in the cortinol-producing enzyme in the adrenal cortex. Generation is the cause of this. Most of these CAH are known to have 2/-hydrogenase deficiency, and as a result, the biosynthesis of /2α-hydroxyprogesterone cara//-desoxycortisol is inhibited. /2α-hydroxyprogesterone in rIL increases.

Measuring /7α-hydroxyprogesterone is extremely important in diagnosing CAH.

Conventionally, /7α-hydroxyprogesterone-3-carboxymethyloxime-BSA or /7α-hydrogineprogesterone-oα-saxonate-BSA has been used as an antigen for producing an antiserum that specifically reacts with /7α-hydroxyprogesterone. The method of using it was known [see Hormones and Clinical Practice 2zago 773 (/9za0)].

However, in the above method, antibodies are produced using a compound in which the 3rd position of /7α-hydroxyprogesterone is oximeated and bound to BSA (bovine serum albumin), or a compound in which hemisaxine)=BSA is bound to the 6th position as an antigen. However, these antibodies react with glogesterone, /7α-hydroxypregnenolone, and 2α-hydroxypregnenolone contained in the test solution.
It has the drawback of reacting with 0α-hydroxyprogesterone and the like, resulting in low specificity.

The present inventors have conducted intensive research on a method for detecting /7α-hydroxyprogesterone that does not have the above-mentioned drawbacks, and have found a new /7. If α-hydroxyprogesterone-//-hemisaxuccinate-antigen protein conjugate is used as an antigen and /7α-hydroxyprogesterone is measured immunologically, it can be detected and quantified with extremely high sensitivity and specificity. I found out.

/2α-Hydroxyprogesterone-//-hemisuccinate used in the present invention can be produced by a known method (for example, see US Patent No. θ/3 Otsuzaza). In this way, /7α-hydroxyprogesterone-//-hemisuccinate represented by the following formula (1-a) is obtained.

OOH Next, to obtain the /7α-hydroxyprogesterone-//-hemisuccinate antigenic protein conjugate of the above formula (1), /7α-hydroxyprogesterone-//-hemisuccinate of the above formula (1-a) is obtained. Binds antigenic proteins.

In this specification 71), "antigenic protein" refers to 1Qf
i refers to a protein that has the ability to form antibodies against it when immunized in mammals; for example, bovine serum albumin (BOA), rabbit serum albumin (BSA),
), human serum albumin (BSA), egg albumin (E
A), among others, BSA and BSA are preferably used in the present invention.

Methods of binding the steroid compound of formula (1-a) to the antigenic protein are known per se, for example E,
F, Erlanger et al, J, Biol
It can be carried out according to the method described in known documents such as Chem-W"l 1090 (/9 Pa). For example,
(1) Mixed acid anhydride method The steroid compound of formula (1-a) is first treated with a haloalkyl carbonate ester (e.g. isobutyl chloroformate).
Activate the carboxyl group of the side chain at the /- position, and then
This can be carried out by dropping an antigenic protein such as sA or RsA into a water-organic solvent solution.

(,2) Carbodiimide method After dissolving the steroid compound of formula (1-a) in a solvent such as dimethylformamide or dioxane, a carbodiimide compound (e.g. dicyclohexylcarbodiimide), BSA, and 4% R8A antigen protein are added and dehydrated. An amide bond is formed by condensation.

(3) Active ester method After dissolving the steroid compound of formula (1-a) in a solvent such as dimethylformamide or dioxane, an active ester (e.g. p-nitrophenol) is added to the carboxyl of the chain at the //- position. After changing the group to an active ester derivative,
This can be carried out by adding an antigenic protein such as BSA or R8A and condensing it.

The /7α-hydroxyprogesterone-//-hemisuccinate-antigenic protein conjugate thus formed can then be used to immunize a mammal and prepare an antiserum according to methods known per se. , Examples of mammals for human use include domestic rabbits, goats, sheep, guinea pigs, etc.
Immunization with the antigen described above is carried out according to conventional methods. that time,
For example, complete Freund Aji band, etc.
Antiserum can be effectively produced by using Panto in combination.

The obtained antiserum is absorbed with the same antigenic protein used in producing the antigen and fractionated by alcohol precipitation or 1-separation, according to a method known per se.
Can be purified.

In particular, in the present invention, the antibody titer obtained by monthly boosting of rabbits or goats with the antigen of the formula (1) is /12θθθ to J゛4θθθ times, preferably 2 subjects θ00 to Preferably, the antibody has a binding affinity constant of /θ7 to 10 l/mol, preferably 10″ to 70″l/mo.

Here, "1-antibody titer" refers to the R
In the IA method, it represents the optimal multiple that yields the optimal seed curve, and the [antibody binding affinity constant] is the affinity when an antigen and an antibody bind together, which is generally called binding affinity, and is experimentally determined. is the concentration of antigen expressed as a value when the ratio of bound antigen to unbound antigen is /.

As shown in the Examples below, the above antibodies provided by the present invention have a cross-reactivity of approximately 10θ with each acute compound.
The cross-reaction rate with other metabolites or structural analogues is approximately 3 or less, and it has extremely specific anti-blood properties. It can be very advantageously used as a reagent for the detection and quantification of /7α-hydroxyprogesterone by radioimmunoassay, immunological agglutination inhibition reaction, and photoimmunoassay.

These radioimmunoassays, immunological agglutination inhibition reactions,
The operation of photoimmunoassay itself is well known; for example, Tochigi Shiki et al., Hormone and Clinical Practice,
Name 1.9 (797 Otsu); Hanada Yoshibe et al., Hormone and Clinical Ze-, f9 (/97 Otsu); Tokko Shota t-//, 3j,
These methods are described in documents such as Publication No. 3', and these methods can be directly applied to the antibody of the present invention.

Next, the present invention will be further explained with reference to Examples.

Leprosy case//Production of 7α-hydroxyprogesterone-//-hemisuccinate-BSA/7α-hydroxyprogesterone-//-hemisuccinate 30r1 with N, N-dimegul dilumamide 0
After adding 1.1 n-butylamine/μl to this solution at 73°C, isophyl chlorocarbonate and μl of glyph were added and stirring was continued for 30 minutes. 79. Add BSA (bovine serum aluminum) to the solution in advance. μl>1q 2. The mixture was dissolved in 1 lnl of purified water, and 23 µl of sodium chloride solution and dimethylformamide were sequentially added, and the solution was heated to a temperature of 50°C and mixed. This was then stirred at 3°C and after 7 hours was added with /N sodium hydroxide ((y 7.J-p
(1) was added, and the mixture was further stirred for 33 hours. Then, gel iM filtration was performed using Sephadex G-, 2J' to remove unreacted /7α-hydroxyprogesterone-//- hemisuccinate and tri-n-butylamine. Mr. Molecular reagent was isolated. The solution was then dialyzed against purified water for 17 minutes and then lyophilized to obtain /7α-hydroxyprogesterone.
//-Hemisuccinate-BSA was obtained as a powder.

The number of moles of hemisuccinate bound per 1 mole of BEIA is /7α-hydroxyprogesterone-//-
Hydroxyprogesterone-//-hemisuccinate-
The difference was determined from the ultraviolet absorption spectra of BSA, /7α-hydroxyprogesterone-//-hemisuccinate, and BSA solutions at a constant concentration of 2 moles.

Example - Preparation of anti/7α-vitroxyprogesterone-//-hemisuccinate antibody Example/Prepared with/7α-hydroxyprogesterone-//-hemisuccinate)-BSA, 3*y in physiological & salt water of /go The mixture was dissolved in bath, emulsified with the same amount of Complete Freund's Adjuvant, and injected subcutaneously and into the heel of an adult rabbit. This injection was carried out at intervals of 1/2 month, and after confirming that the It titer had increased, blood was collected to obtain anti-blood fn1.This antiserum was inactivated at 56°C for 30 minutes, then absorbed with BSA, and the anti-/7α -Hydroxyprogestolone-//-hemisuccinate antibody was obtained.

Anti/7α-hydrochimiprogesterone obtained above
//-Hemisuccinate antibody has very high specificity, with almost no cross-reactivity with other steroid hormones, when the cross-reactivity with /7α-hydroxyprogesterone is /θ0(ly).

The cross-reactivity rate is shown in Table 1 below. However, the specificity of the above antibody was determined by Kobe et al.
/7α-Hydroxyprogesterone-3-carboxymethyloxime-BSA and /7 produced by /9 and θ)
α-Hydroxyprogesterone-O-α-hemisuccinate-BSA was used as an antigen and compared with the antiserum. The results are also shown in Table 1.

From Table 1 above, the antiserum prepared using a hemisuccinate-antigenic protein conjugate at position 77 has lower cross-reactivity than the antiserum prepared using an antigenic protein at position 3 or 6. In particular, the cross-reactivity with respect to 77α-hydroxypregnenolone and glogesterone is extremely low at about '/~'/ compared to the conventional method. Therefore, it is recognized that the antiserum according to the present invention has extremely high specificity.
/2α-hydroxyprogesterone in the test liquid can be accurately measured.

Applicant: Teikoku Zoki Pharmaceutical Co., Ltd.

Claims (1)

[Claims] An antigen for measuring /7α-hydroxyglogesterone consisting of a steroid compound represented by the formula 7, wherein Y represents an antigenic protein. An antiserum capable of specifically reacting with /7α-hydroxyprogesterone obtained by immunizing a mammal with a steroid compound represented by the following formula, wherein Y represents an antigenic protein.