JPS6316710B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel enzyme immunoassay. In more detail, the general formula [S is 6β-hydroxycortisol or an OH-containing steroid derivative of testosterone S-OH
Shows the parent compound of persimmon. n is an integer of 1 to 6] A novel active ester represented by the following is reacted with an enzyme to form the general formula [S and n are the same as in the above general formula (), E: enzyme]
This invention relates to a method for producing a novel enzyme-labeled antigen represented by: Next, the above S will be explained in detail. For example, in the case of 6β-hydroxycortisol, S-OH
What is

[Formula] and its What is the parent compound of

[Formula]. On the other hand, in the case of testosterone, what is S-OH?

[Formula] and its mother What is a body compound?

[Formula]. Measuring steroid compounds in body fluids is useful for various diagnoses. For example, the amount of urinary 6β-hydroxycortisol is of great significance in measuring cancer, glucose-6-phosphate dehydrogenase abnormality, or as an indicator of the drug metabolic function of the liver. However, no literature regarding measurement using the enzyme immunoassay method (hereinafter abbreviated as EIA method) has yet been found. Traditionally, steroid derivatives have been measured using gas chromatography (hereinafter referred to as GLC) or radionomy assay (hereinafter referred to as RIA), but the GLC method is difficult to operate in order to measure a large number of samples. The RIA method is complicated and has problems in handling radioactive materials, so it is difficult to use as a general measurement method. On the other hand, the EIA method is used to measure the concentration of various hormones or administered drugs, and is known to be a useful method in clinical tests. In other words, the EIA method performs enzyme labeling of the substance to be measured using the mixed acid anhydride method, carbodiimide method, or glutaraldehyde method.
This is a method of measuring in vivo drug concentration using this as a labeled antigen and utilizing an antigen-antibody reaction. Therefore, the preparation of enzyme-labeled antigens is very important. The present inventors have also attempted various enzyme labeling methods for steroid derivatives using known methods, but these methods do not allow for self-bonding (self-bonding) between enzymes or antigens.
There have been problems such as difficulty in obtaining only the enzyme-labeled product of the antigen of interest, and fluctuations in the amount of enzyme that binds to the antigen. As a result of intensive research into developing an EIA reagent that does not have these drawbacks, we synthesized an enzyme-labeled antigen via a new active ester compound represented by the general formula (). The present invention was completed by discovering that the enzyme-labeled antigen represented by the general formula (') obtained in this manner is an EIA reagent that has been improved from the above-mentioned drawbacks. In the present invention, a steroid compound serving as an antigen is dissolved in a solvent such as pyridine, and succinic anhydride is added and reacted to form a hexuccinate compound. and then a dehydration condensation agent, e.g. 1-ethyl-3
-(3-dimethylaminopropyl)carbodiimide hydrochloride is added and reacted with N-hydroxysuccinimide, and then extracted with a solvent to obtain hemisuccinate N-hydroxysuccinimide ester (). This active ester is stable for a long period of time, and the active ester for enzyme labeling thus obtained has (a) excellent reactivity with enzymes. (b) The amount bound to the enzyme can be regulated. That is, it has excellent reproducibility. (c) It has advantages such as not causing self-bonding between enzymes. In order to enzymatically label this active ester, it is dissolved in a commonly used solvent such as tetrahydrofuran, dioxane, acetone or dichloromethane to form an ester solution. The enzyme to be bound is dissolved in a buffer such as a phosphate buffer, added to the above ester solution, and reacted at low temperature for about 12 hours. If necessary, the enzyme-labeled antigen (' ) solution is obtained. When the product thus obtained is freeze-dried, dichloromethane is most preferably used as the solvent for dissolving the active ester. Furthermore, if necessary, a preservative such as sodium titanate can be added. The reaction formula of the present invention is as follows. [S, E, and n are the same as in the general formula (') above] Any enzyme can be used as the labeling enzyme, but β-D-galactosidase can be measured with high sensitivity, is stable, and has high substrate specificity. or peroxidase is particularly preferred. Furthermore, the antibodies required for the EIA method can be obtained by conventional methods. For example, 6β-hydroxycortisol 21-hexuccinate is bound to bovine serum albumin and injected together with adjuvant into an animal such as a rabbit to produce antibodies in the serum. The measurement method of the present invention involves performing an antigen-antibody reaction between an enzyme-labeled antigen, a specimen (unlabeled antigen), and an antibody against these in a buffer solution, adding a second antibody against the resulting antigen-antibody complex, and centrifuging. After dissolving the precipitate in a buffer solution, enzyme activity is measured by a known method. The concentration of the analyte is determined by performing the same procedure using a sample with a known concentration to determine the enzyme activity, and then quantifying the enzyme activity using a calibration curve prepared therefrom. Next, the present invention will be explained with examples. Example 1 Process for producing testosterone 17-hemisuccinate N-hydroxysuccinimide ester 25 mg of N-hydroxysuccinimide, 280 mg of 1-ethyl-3-(3-diethylaminopropyl)carbodiimide, and 1.0 ml of dioxane solution containing 200 mg of testosterone 17-hemisuccinate. water
Add 0.2ml and stir at room temperature for 3 hours, then add 10ml of water.
was added to crystallize the product, which was dissolved in ethyl acetate, washed with water, and dehydrated with sodium sulfate. The solution was concentrated under reduced pressure, and the residue was dissolved in hexane-ethyl acetate and purified by silica gel column chromatography. After recrystallization with dichloromethane-methanol, testosterone 17-hemisuccinate N-
Obtain 170 mg of hydroxysuccinimide ester. Elemental analysis value Calculated value C66.78% H7.27% N2.88% Experimental value 66.67 7.32 2.88 Example 2 Process for producing 6β-hydroxycortisol 21-hemisuccinate N-hydroxysuccinimide ester 6β-hydroxycortisol 21-hexuccinate 100mg N- in 1.0 ml of dioxane solution containing
Hydroxysuccinimide 150mg and 1-ethyl-
150 mg of 3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.2 ml of water were added, stirred at room temperature for 3 hours, dissolved in ethyl acetate, washed with water, dried over sodium sulfate, concentrated under reduced pressure, and the residue was dissolved in chloroform. - After dissolving in methanol and purifying with silica gel column chromatography, recrystallizing with water-containing methanol, 6β-hydroxycortisol
70 mg of 21-hemisuccinate N-hydroxysuccinimide ester are obtained. Elemental analysis value Calculated value C60.51% H6.48% N2.43% Experimental value 60.48 6.52 2.38 Example 3 Testosterone enzyme-labeled antigen The active ester prepared in Example 1 was dissolved in dichloromethane (1.85mM), 1 of these
After putting 10μ into a test tube and distilling off the solvent,
-D-galactosidase (manufactured by Sigma, USA) 1
Add 0.2 ml of 0.05 M phosphate buffer (PH 7.3) containing 0.0 mg, stir immediately, and leave at 4°C overnight. After that, dialysis was performed for 2 days against the same buffer, and then 0.05M phosphate _buffer (PH
7.3) to obtain the testosterone-hemisuccinate-β-D-galactosidase enzyme-labeled antigen. (Stored at 4°C) Reference Example 1 EIA of Testosterone The enzyme-labeled antigen obtained in Example 3 was added to a phosphate buffer (0.05M, PH7.3) containing 0.5% normal rabbit serum.
Phosphate buffer (0.05M, PH7.3) containing 100μ diluted 50-100 times and 0-10ng of testosterone
100μ and 4000μ of rabbit anti-testosterone antiserum
Add 100μ diluted solution and leave at 4℃ for 4 hours, then goat anti-rabbit γ-globulin antiserum (secondary antibody)
Add 100μ and leave at 4°C for another 16 hours.
Next, add 1 ml of the same buffer containing 0.1% gelatin and 0.1% NaN ₃ and centrifuge (3000 rpm, 15 minutes).
After removing the supernatant, 1.0 ml of 2mMO-nitrophenyl-β-D-galactopyranoside and 0.1
% gelatin, 0.1% _NaN3 , 10mM _MgCl2 ,
0.05M phosphate buffer (PH7.3) containing 0.1M NaCl
Add 1.0 ml and incubate at 37°C for 60 minutes, stop the reaction by adding 2.0 ml of 1M sodium carbonate solution (PH 11.9), and measure the enzyme activity by measuring the O-nitrophenol produced at absorbance at 420 nm. Quantify. Example 4 6β-hydroxycortisol enzyme-labeled antigen The active ester produced in Example 2 was dissolved in dichloromethane (3.8mM), of which 4μ
was put into a test tube, the solvent was distilled off, and the E. coli β-D-
Add 0.5 ml of 0.05 M phosphate buffer (PH7.3) containing 2 mg of galactosidase (Sigma, USA), stir immediately, and then react with gentle stirring overnight at 4°C, then incubate with the same buffer for 2 days. After dialysis, NaN ₃ is added to obtain 6β-hydroxycortisol-hemisuccinate-β-D-galactosidase enzyme-labeled antigen. (Stored at 4°C) Reference Example 2 EIA of 6β-hydroxycortisol The enzyme-labeled antigen obtained in Example 4 was added to a phosphate buffer (0.05M, PH7.3) containing 0.5% normal rabbit serum.
Add 100μ of a 2000-fold dilution, 100μ of a phosphate buffer (0.05M, PH7.3) containing 0 to 1 ng of 6β-hydroxycortisol, and 100μ of a 2000-fold dilution of rabbit anti-6β-hydroxycortisol antiserum, and stir at 4°C. After standing for an hour, 100μ of goat anti-rabbit γ-globulin anti-blood serum (secondary antibody) was added, and the mixture was further left at 4°C for 16 hours. Then 0.1% gelatin,
After adding 1 ml of the same buffer containing 0.1% NaN ₃ and centrifuging, the supernatant was removed and the enzymatic activity was determined in the same manner as in Example 4.

[Brief explanation of the drawing]

Figure 1 is a calibration curve for testosterone, and Figure 2 is a calibration curve for 6β-hydroxycortisol. The vertical axis shows the ratio of the absorbance of each steroid-added sample to the steroid-free sample as a percentage (%), and the horizontal axis shows the amount of steroid (antigen) added. FIG. 3 is a correlation diagram between RIA measurement values of 6β-hydroxycortisol and the method of the present invention. N=43 and y=
It showed a correlation of 1.02x+4.7, γ=0.96.

Claims (1)

[Claims] 1. General formula [S is 6β-hydroxycortisol or an OH-containing steroid derivative of testosterone S-OH
and its parent compound is shown. n is an integer of 1 to 6] N-hydroxysuccinimide is reacted with hemisuccinate, a steroid derivative represented by the general formula [S, n are the same as the general formula ()] A method for producing an enzyme-labeled antigen [formula], which is characterized in that it is an active ester represented by the following formula, and then the ester is reacted with an enzyme. [S and n are the same as in the general formula (), and E represents an enzyme. ]