JPS589068A - Immunochemical determination of estriol-16alpha- glucuronide - Google Patents

Abstract

PURPOSE:To make it possible to measure a trace amt. of a titled material specifically, easily and quickly with high sensitivity by using an antibody obtd. by immunizing a complex of the titled material and protein and said complex which are respectively sensitized with latex particles. CONSTITUTION:An antibody obtd. by immunizing the E3-16alpha-G-protein complex wherein estriol-16-glucuronide (E3-16alpha-G) and protein are bound by Mannich reaction with mammals and said complex are sensitized with latex particles and these are used in determining the E3-16alpha-G in bodily fluid or eliminated fluid. For example, the E3-16alpha-G-RSA obtd. by reaction of E3-16alpha-G with rabbit serum alubmin (RSA) in a buffer soln. is injected to rabbits and after 10 months, an anti-E3-16alpha-G antibody is obtd. The E3-16alpha-G in sample is determined by the use of the reaction for inhibiting latex clotting using both of the respective sensitized latex suspensions obtd. by reaction of said antibody and E3-16alpha-G-RSA respectively with the latex suspensions.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an immunochemical assay method for striol-16α-glucuronide. More details: Nistriol-16α- in body fluids or excreted fluids
This invention relates to a method for specifically measuring glucuronide (hereinafter referred to as E, -16α-G) using a latex agglutination inhibition reaction.

Estrogen (yolk hormone), a type of steroid hormone, is mainly estrone and estradiol-17β in the gonads and adrenal cortex. : It is produced and secreted in the liver and converted to IJol), inactivated, and further conjugated with glucuronic acid, monosulfate, etc., and excreted from the body in the urine. Furthermore, in pregnant women, a large amount of 1-estriol is synthesized in the feto-placental system as the gestation period increases, undergoes conjugation in the maternal liver, and is then excreted from the body in the urine. Particularly during pregnancy, estriol is synthesized and secreted. Unlike non-pregnant states, the majority of urinary estriol is 9-16α-G, with glucuronic acid bonded to the 16α hydroxyl group. . Therefore the pregnancy process,
) Measurement of urinary E and -16α-G is important for diagnosing abnormalities in the fetus and placenta, especially in late pregnancy.

The purpose of the present invention is to immunochemically measure E, -16α-G, which exists in trace amounts in body fluids or excreta of humans and mammals in trace amounts, with high sensitivity, specificity, and simplicity. The present invention provides an immunochemical measurement method for

The analytical chemical measurement method for estriol (hereinafter referred to as 1E) that has been conventionally used in horses is 1E, in which avian-16α-G, which is generally present in body fluids, is hydrolyzed with an enzyme such as β-galactosidase.

It is indirectly quantified by subtracting the value isolated and fixed before hydrolysis treatment from the value isolated and quantified by various chromatography after liberating.1 These operations are complicated and Moreover, in addition to requiring a long time, a considerable portion of the chromatographic process is adsorbed to the adsorbent.
The collection warehouse is bad and 1 trace amount of Ebi-46a-G cannot be measured 1
There are problems such as: Furthermore, the method for measuring E, which utilizes an agglutination inhibition reaction, which has been conventionally used as an immunochemical measurement method, cannot specifically measure E, -16α-G. That is, the hapten antigen used in the conventional method is E8
+ 16α-G-protein complex 1 or E, -16,1
A 7-disuccinate protein complex synthesized by a mixed acid anhydride method was used.

The antisera obtained by immunizing mammals with these by the conventional method (these are bound to proteins via the D-ring of steroids) are specific for the D-ring of Aku or 4-16α-G. Only extremely low levels of antibodies were obtained. In the agglutination inhibition reaction system using these hapten antigens) and antibodies, 1 estrone (hereinafter referred to as ゛0.2), ゛A'''1''-X)59
*--17β (hereinafter referred to as S-bird) and other birds-
Since it is measured in the same way as 16α-G, 1 inevitably E
,, it was not possible to specifically measure only -16α-G.

One of the inventors, Toshio Nambara, first developed the radioimmunoassay (hereinafter referred to as
A patent application was filed for an antigen for producing antibodies for measurement using RIA (Japanese Patent Application No. 52-26509). Furthermore, we also reported a method for measuring E, -16α-G using RIA (T, -1E2M, -Sukeichi, T, Ronne, T.

ChJdoo: Prepazatianba-1fi
16
glu-crcnide by imounozati
an with 8-chest attaahai to pr
oteinthrot$ poeiticrm in
ring A, , r, b, D%,, 1
．． 55-61197g). However, since RIA uses radioactive isotopes, a high level of knowledge and skill is required to handle them, and special equipment and measuring instruments are required, resulting in a lack of simplicity. - In order to solve the various shortcomings of the conventional measurement methods as mentioned above,
The present inventors have conducted extensive research and found that E,
The present invention has been completed based on the discovery of a method for measuring 6α-G with high sensitivity, quickly and easily.

In order to obtain an antibody that specifically reacts only with -18-16'a-G, the present invention uses a complex in which an antigen protein is bound to the 2λ or 4 position of the A ring of F, -16α-G by Mannich reaction. 9F using a latex suspension obtained by sensitizing latex particles by a known method with Norbutene antigen obtained by immunizing mammals with 1-Norbutene antigen at 2-week intervals for 6 months or more.
This is a quick and simple immunochemical measurement method for specifically measuring 1, -16α-G.

Regarding the antibodies used in the present invention, 1 To be more specific, the antibodies used in the RIA described above and the antibodies used in the present invention are significantly different in terms of antibody titer. It was difficult to adapt it to a standard measurement method. In other words, Reference Example 1 described below. The antibody titer determined by the microtiter method is 1 for the antibody used in RIA:
200 to 1:400 is sufficient), whereas the antibody titer suitable for the present invention is 1:1609 to 1:64.
00, an antibody titer of 4 to 32 times that required for RIA was required. One protein used in the present invention may be any protein having antigenicity, preferably bovine serum albumin (hereinafter referred to as 1BAA) or human serum albumin (hereinafter referred to as H8).
A) Tsuta Ovalbumin (hereinafter referred to as EA) 1
Bovine serum r-globulin (hereinafter referred to as BGG) 1 and rabbit serum albumin (hereinafter referred to as 5R8A) show good results.

Next, 4-16α-G antibody and Es-・16α-G-
Examples of carriers that sensitize protein complexes include carriers commonly used in immunochemical aggregation inhibition reactions, such as polystyrene latex particles, various species of animal blood cells, bacteria, and bentonite. Results of comparing the advantages and disadvantages of various conditions when producing a reagent for measuring Es-16α-G using a carrier and when measuring E and -16α-G in body fluids or excreted fluids. are shown in Table 1. According to this result, polystyrene latex particles are suitable for (1) manufacturing because of the small loft difference during manufacturing.

b) When measuring bird-16α-G as a subject - Even when blood is used, there is no visitor percentage or acceleration of the reaction, and the measurement can be performed in the same way as when using one urine sample.

C) The time required for measurement is the shortest, which is 12 to 3 minutes.

d) Example 3 described below. As shown in Figure 2, quantitative reactions can be carried out easily and accurately.

Production example 1 of anti-E, -16α-G antibody. t7) E, -16a-G-BSA 2m
g was dissolved in 1 mA' physiological saline, emulsified with the same amount of Freund's complete adipant, and injected intradermally into the foot and dorsal skin of adult rabbits. Administration was carried out at 2-week intervals for 2 months. After that, the interval was 1α month.The antiserum titer was E.
It was carried out by the microtiter method using -16α-G-R8A sensitized latex particles and reached 2,000 times in 3α months. Antiserum was collected 10 months after antigen administration. After absorbing the BSA antibody contained in the antibody, the antibody was purified by a salting-out method using ammonium sulfate.
α-G antibody and L7.

Production Example 3 Production of Ej-16α-G-R3A Sensitized Latex Suspension Production Example 1. NoE, -16a-G-R8A 2■ 0.
4 ml of 2-T-ruammonium buffer (pH 8,2)
The resulting mixture was added to 2 ml of a 5% concentration 0.90P latex suspension, reacted at 37°C for 1 hour, and then centrifuged at 4.00 Or, p, m for 20 minutes). To discard the sample, suspend the precipitate in 50 ml of the buffer solution and centrifuge it under the same conditions.After repeating this procedure twice, the precipitate was suspended at 1%.
Es-16 was suspended in 100 d of the buffer containing BSA.
A 0.14 suspension of α-G-R8A sensitized latex was obtained.

Production Example 4 Production method of E, -16α-G-R8A sensitized latex suspension Production Example 1. I, -16α-G-R3AI■ is 0.2
Dissolved in 4 ml of mol ammonium buffer (pH 8.2) and mixed with a 5% concentration particle size of 0.48. After adding to 2 ml of p latex suspension and reacting at 37°C for 1 hour,
．． 00Or, p.

-m, for 20 minutes and discard the supernatant).
Suspended in 10 rtrl of the buffer containing BSA,
, a 1% suspension of -16α-G-R8A sensitized latex was obtained.

Production Example 5 Production of anti-E, -16α-G antibody sensitized latex suspension Production Example 2. The anti-E, -16α-G antibody obtained in
% concentration in a 0.2 M ammonium buffer solution and added to 50 ml of a 296 suspension of latex particles with a particle size of 0.48H for 10 m/sealing) and reacted at 37° C. for 1 hour. After the reaction, 2 at 4.00 Or, p, m
After centrifugation for 0 minutes, the supernatant was discarded, and the precipitate was suspended in 100 ml of the buffer containing 5% BSA to obtain a 196 suspension of anti-bird-16α-G antibody-sensitized latex.

Production Example 6 Preparation of anti-E, +16α-G antibody sensitized latex suspension Production Example 2. The anti-E, -16α-G antibody obtained in
96 concentration in a 0.2M ammonium buffer to make 10 ml, and added to 50 ml of a 2% suspension of Latin 2F 1 particles with a particle size of 0.31P, and reacted at 50°C for 1 hour. did. After the reaction is completed, 10.000 r, p, m,
Centrifuge for 110 minutes) Discard the supernatant, and add the precipitate to 1% BS.
(suspended in 1,000 ml of the buffer containing A) anti-E,
A 0.1% suspension of -16α-G antibody-sensitized latex was obtained.

Test Example 1: Confirmation of antibody specificity by microtiter method. Antiserum 0.0, 0.25 R/1. diluted 1 times each with 0.025+/
) Production example 3. Obtained at iE, -16α-GwR8A
Sensitized latex suspension 0,025I+7! was added to examine the agglutination reaction of the antiserum in the presence of each steroid at each concentration.

Second, the highest dilution factor of the antiserum at which agglutination was observed was shown.

Table 2: Reaction between E, -16α-G-RsA sensitized latex and anti-E, -16α-G antibody in the present invention
-16α-G) anti-E, -16α-G
This shows that the antibody hardly reacts with steroids other than E and -16α-G.

Test/Test Example 2゜Steroid addition recovery test E using male urine -16α-
G and 2. 0.025m/1 and 0.025mA of the buffer (pH 8,2) were prepared by diluting samples prepared by adding male urine to 10.2M ammonium buffer (pH 8,2). , and further production example 1. I, -16α
- G-R8A i p9/IIIt buffer solution 0.0
25m/ was mixed on a slide, and to this was prepared Example 5. anti-bird-16α-G antibody sensitized latex suspension of 0.025
m/ was added and mixed, and after 93 minutes, the presence or absence of aggregation was determined with the naked eye. This is shown in Table 3. In addition, one of the conventional methods, 7s, is a sensitized latex suspension of antibodies obtained by binding -16α-G-B8A (or -R8A) by the mixed acid anhydride method and immunizing a supplemented mammal. E, -16α-G-R8A
Table 4 shows the measurements of the same sample.

Both the present invention and the conventional method have a measurement sensitivity of I, -16α-G.
The criterion for 1 was as follows, assuming 1p9/m/.

-2 clear aggregation image (1, -16α-G is below measurement sensitivity) +: complete aggregation inhibition image (E is above measurement sensitivity, -16α
-G is included in the sample) Table 3 Table 4 In the method of the present invention, only 1 added E and -16α-G are accurately measured. .

In the method of the present invention, E, -16α-G-R8A sensitized 2-gex and anti-1ii, -16α-G antiserum or anti-Mo1
6α-G antibody sensitized latex 16α-G-R8A
, or a measurement reagent using a combination of E, -16α-G-R8A sensitized latex and anti-E, -16a-G antibody sensitized latex, etc. 1 The measurement sensitivity can be adjusted to any value. . For example, E, -16α-G-
Sensitivity can be adjusted by changing the amount of R8A or anti-bird-16α-G antibody sensitized to latex, or by changing the concentration of anti-E, -16α-G antiserum or the concentration of 16α-G-R8A solution. It is.

Example 1 Measurement of urinary avian-16α-G in pregnant women Late pregnancy method Three samples were each diluted 25.50.100.200.3001 times with a 0.2 molar ammonium buffer solution to 0.025 mj. The buffer 0.025
1 production example 2 per ml. anti-E, -16α-G antiserum 0.0
25 ml) to which Production Example 4.0E, -16α-
After adding 0.025 m of G-R8A sensitized latex suspension and reacting on the slide for 3 minutes, the agglutination image was visually judged. In this example, the sensitivity of the Company 1 measurement reagent was set to 0.2P.
It was adjusted so that aggregation could be inhibited up to 4-16α-4G at a concentration of 9 g/l. The judgment criteria are Test Example 2. The results of the measurements were as shown in Table 5, line by line.

Table 5 Implementation @2゜Measurement of bird-16α-G in blood of pregnant women 6 samples of peripheral blood (blood T) of pregnant women were mixed with 0.2M ammonium buffer solution at 5% each 10 S20, 30
．． 0.05*/
and 1 Production Example 4.0E, -16α-G-R8A sensitized latex suspension 0.025111/, and Production Example 5. 4-16α-G antibody sensitized latex suspension 0.025+
*/ was thoroughly mixed on a slide, and after reacting for 13 minutes, the agglutination image was determined with the naked eye. The sensitivity of the determination reagent in this example is 0.
001)19/'' (1n9/+a/).The measurement results were as shown in Table 6.

Table 6 Example 3 Measurement of -16α-G in blood of pregnant women Production example 6. Add 0.1- of pregnant women's serum to 1.0- of the anti-E, + 16α-G antibody sensitized latex reagent prepared in above, mix well, and place in a cell with an electric path width of 0.4 cm. 1. Next, manufacture.
Example 1. 4-16α-G-R8A O, 1ml manufactured by
The intensity of the scattered light 1 or the absorbance is measured over time while irradiating light with a constant wavelength in the range of 600 mp to 1200 mF, and the rate of change in the scattering intensity -% or the change in absorbance per certain period of time is measured. Find the rate.

Similarly to the above method, a calibration curve was prepared using a 1-concentration 16α-G solution with a known concentration.
The amount was measured.

Here is an example of the results: measurement temperature 37℃ 1 measurement wavelength 6
The amount of 4-16α-G in pregnant women's blood determined from the calibration curve (Figure 1) determined by the change in absorbance at 50 mH and 1.
Shown in the table. Note) Table 7 shows the conventional radioimmunological analysis method (R Engineering A) method (accepted by Mitsuteru) for comparison. .1977) are also shown.

Table 7

[Brief explanation of the drawing]

Figure 1 shows the concentration of E, -16α-, obtained by the method of the present invention.
It represents the calibration curve prepared using G solution, and the λ vertical axis represents the change in absorbance at a wavelength of 650 mp per minute (Δ0,
D,) 1 The horizontal axis shows F, -16a-G amount (ng/mO. Patent applicant Eiken Chemical Co., Ltd. Figure 1 25 50 75 1°oE, -16ff-G
Amount (ng/*/)

Claims (1)

[Claims] An antibody obtained by immunizing a mammal with an estriol-16α-glucuronide-protein complex in which estriol-16α-glucuronide and a protein are bound together by a Mannich reaction; Estriol in body fluids or excreta using the
Immunochemical assay characterized by measuring tea-glucuronide