JPS62159046A - Method for measuring subunit of hormone - Google Patents

Abstract

PURPOSE:To exactly determine the subunits of respective hormones by a measuring system A which the monoclonal antibody specific to the beta subunit of the hormone to be measured is used as a labeling antibody and a measuring system B in which the monoclonal antibody having the specificness with the subunit to be measured is used for both of an immobilized antibody and labeling antibody. CONSTITUTION:The standard curve of the hormone themselves in the measuring systems A, B and the standard curve of the subunit to be measured by the measuring system B are formed. The quantity of the hormone itself in the specimen including the subunit to be measured is then measured with the system A and the absorbancy of the specimen is determined from the standard curve. The absorbancy b1 of the specimen is measured with the system B in succession and the absorbancy b2 of the system B corresponding to the quantity of the hormone itself determined by the system A is determined from the standard curve of the hormone itself by the system B. The quantity of the subunit is exactly determined from the absorbancy of the standard curve of the subunit in the system B corresponding to the difference between the absorbancies b1 and b2.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the use of hormones, particularly hormones selected from TSH (thyroid stimulating hormone), FSH (follicle stimulating hormone), LH (luteinizing hormone) and HCG (filioid gonadotropin). Concerning a method for measuring subunits.

Generally, these hormones have α and β + 7” −
It is said that the antigenicity of the α subunit is extremely similar to each of the four hormones. Measurement of its concentration in blood or body fluids is widely used for diagnosis of primary hypothyroidism, Graves' disease, fretinism, etc. However, it is said that in blood or body fluids, in addition to the hormone itself, which is composed of α and β subunits, free α and β subunits of each hormone also exist. Although various studies have been conducted on the dynamics of each of these subunit components in normal people or in patients with each of the above-mentioned symptoms, the dynamics of these subunit components has not yet been fully elucidated.

Conventionally, the concentrations of these free subunits of each hormone have been measured after separating each subunit from a large amount of serum using complicated procedures such as transfection with V:F5, but Wanibe et al. , 9 Journal of the Japanese Endocrine Society, vol.
55+1384~1394. (1979),
It is claimed that blood TSH subunit levels can be measured directly from serum samples using the R-artificial method. Also, corrida y, (
Kourides et al. have also reported that TSH subunits can be measured directly from the serum of thyroid disease patients using a similar R-artificial method (EndOOri, nolOgy94).
: 1411-1421 (1974)” and “J,C
11n, End.

cri-nol, Metab, 872-885. (1
975)''].However, since both of these RTA methods are competitive two-antibody methods, highly purified TSH-α
or require a β subunit and highly specific anti-TSH-α or β subunit antibodies. These α or β supplements are obtained from TSH extracted and purified from the human pituitary gland, which exists only in small amounts, and are extremely expensive and valuable. In addition, since anti-TS'H-α or β subunit antibodies use animal immune serum, that is, polyclonal antibodies, it is extremely difficult to purify them to improve specificity, and therefore they are valuable for immunization and purification. This necessitates the use of large amounts of TSH-α or β subunits. The same applies when measuring subunits of these hormones.

As the present inventors conducted research on methods for measuring the subunits of each hormone, which should be an effective means for analyzing the dynamics of hormone components in the blood, the inventors found that they found extremely effective methods for measuring the subunits of each hormone using specific monoclonal antibodies for each hormone. We found a measurement method.

Specific monoclonal antibodies for each hormone are known, for example, “CL Engineering CALCHEMISTRY”.
, Vol. 28. /Fll19. P.P., 1862-18
66.1982, H. Garrett, Wad.
a.

reported α-subunit-specific monoclonal antibodies and β-subunit-specific monoclonal antibodies for these hormones, and measurement of each hormone by a sandwich EIA method using these antibodies. According to this report, an α-subunit-specific monoclonal antibody was used as a solid phase and a β-subunit-specific monoclonal antibody was used as a labeled antibody, and the structural and immunochemical properties of the α-sag units of the above hormones were similar. Due to its nature, the solid-phase α-subunit monoclonal antibody may be an α-subunit monoclonal antibody of one hormone.

Furthermore, the present inventors have demonstrated that the epitope recognized by such hormone α-subunit-specific monoclonal antibodies or β-subunit-specific monoclonal antibodies, as well as the same α-subunit-specific (or β-subunit, 7)-specific monoclonal antibodies. It has been discovered that there are various monoclonal antibodies with different reactivity, that is, with different reactivity (see, for example, Japanese Patent Application No. 177813/1982).

The present invention was achieved using a combination of α or β subunit monoclonal antibodies having various reactivities, and is based on the following facts.

First, in a hormone measurement method such as EIA or EIA method, which involves reacting a sample containing a hormone to be measured, an immobilized antibody, and a labeled antibody, the α subunit specificity of the hormone to be measured as an immobilized antibody is determined. A measurement system using a monoclonal antibody and a monoclonal antibody specific for the β subunit of the hormone to be measured as a labeled antibody (or conversely, a monoclonal antibody specific for the β subunit as an immobilized antibody and α subunit, 71-monoclonal antibody as a labeled antibody) In a system using an antibody (hereinafter referred to as measurement system A), only the hormone itself can be measured, even if the sample to be measured contains not only the hormone itself but also its α or β subunits. This is because, for example, if an α-subunit-specific monoclonal antibody is used as the solid phase and a β-subunit-specific monoclonal antibody is used as the label, the solid phase will bind the hormone and its α subunit, but the β subunit will bind. Units are not combined. On the other hand, the labeled substance can bind the hormone to the β subunit, but not the α subunit.

As a result, the amount of label bound to the solid phase corresponds to the amount of hormone without free α and β Sag units.

In addition, both the immobilized antibody and the labeled body are monoclonal antibodies that have specificity for the subunit to be measured (however, the monoclonal antibodies used for the immobilized antibody and the labeled body must each recognize different epitopes). In the measurement system (hereinafter referred to as measurement system B), the solid phase adsorbs the hormone and the subunit to be measured for the same reason as above, and the label is bound to both, so the hormone + the subunit to be measured is The amount of units can be measured. However, the measured absorbance is a value for two substances with different molecular weights, so in reality,
The exact total amount cannot be determined from the absorbance value.

According to the present invention, using the measurement systems A and B described above, the subunits of each hormone can be accurately determined as follows.

First, a standard curve for the hormone itself in each of measurement systems A and B and a standard curve for the subunit to be measured in measurement system B are created.

Next, the amount of the hormone itself in the sample containing the subunit to be measured is measured using measurement system A, and determined from the standard curve based on the absorbance of the sample.

Subsequently, the absorbance b1 of the specimen is measured with measurement system B, and the absorbance b1 of measurement system B corresponding to the amount of the hormone itself determined with measurement system A is determined from the standard curve of the hormone itself obtained with measurement system B.

Thereafter, the amount of the subunit can be determined from the absorbance of the standard curve of the subunit to be measured in measurement system B, which corresponds to the absorbance of 1-absorbance 42=absorbance 3.

Monoclonal antibodies specific for the α or β subunits of various hormones used in the present invention can be obtained by conventional methods, such as fusing mouse myeloma cells with spleen cells of mice immunized with the hormones themselves or with the α or β subunits of each hormone. 7) Select a specific monoclonal antibody-producing hybridoma by examining its specificity from among the monoclonal antibody-producing hybridomas obtained by cloning. Alternatively, it can be obtained by preparing α or β subunit-specific monoclonal antibodies obtained by intraperitoneal proliferation in mice. Further, from among these, a combination of two α- or β-subunit-specific monoclonal antibodies with different recognition epitopes to be used in measurement system B was selected as described below.

By using such a monoclonal antibody, the present invention can be applied to conventional immunoassay methods such as EIA method, R
Technique A method, fluorescent antibody method Sonoike's method allows easy and effective measurement of α or β subunits of various hormones, and is an extremely effective method for analyzing the dynamics of each hormone component in the blood. can be provided.

As the carrier used for immobilizing these monoclonal antibodies, any carrier material commonly used in the art can be used, such as synthetic polymer materials such as silicone, polyacrylamide, polystyrene, Sephadex, agarose, agar, starch, etc. Examples include polysaccharides such as polysaccharides, proteins (polyamino acids), and glass.

In addition, as a labeling substance for obtaining a labeled substance, for example,
Enzymes such as bell-okindase, glycosidase, alkaline phosphatase, and galactosidase; radioisotopes such as 1 and 2; fluorescent dyes such as F-tech TC (fluorescein inthiocyanate) and rhodamine B, and the like can be used. Enzymes are preferred from the viewpoint of ease of handling.

Hereinafter, the present invention will be specifically explained with reference to Examples.

Preparation Example Preparation of monoclonal antibody First, 50 Mg of TSH, TSH-α or β subunit (both Calbiochem standard products) was added to 3 m
The antigen for basic immunization was prepared by dissolving the antigen in 0.9% physiological saline and adding 3 ml of FCA l from Deng FC○ as an adjuvant to form a water-in-oil droplet. In addition, a booster antigen prepared by dissolving 50 Mg of TSH or TSH-β subunit in 1.3 ml of 0.9% physiological saline was used.

Mouse spleen cells obtained by immunizing 74-5 week old BALB/C mice according to a general immunization schedule are as follows:
Ordinary methods, such as "Hyglidoma method and monoclonal antibodies J, supervised by Takeshi Watanabe, P, 20-P, 29 R Co., Ltd.
Mouse myeloma cells (P3-X63-Ag8-Ul) were prepared by the method described in &D Planning Co., Ltd., 1982.
By fusion with TSH-α and β-subunit-specific monoclonal antibodies, 11 types of hygliomas were obtained.

Each monoclonal antibody was obtained by growing each in the peritoneal cavity of Maune.

The antigen specificity of the monoclonal antibodies produced by the 11 types of hybridomas obtained above was measured as follows.

First, one-label TSE (.

12511t1m TSH-α subunit and 125-labeled TSEI-β subunit were chloramine-treated.
Prepared by method T.

The immunoglobulin content of the culture supernatant of each hybridoma as a sample was adjusted to 20 Mg/mJ10
0μm and each tracer approximately 20000cpm/100
The sample adjusted to μm and 100 μm were mixed in a test tube and incubated at 37°C for 1 hour. Next, add 20% of commercially available anti-Maunui immunoglobulin goat serum (manufactured by Kappel).
Add 50 μm of the diluted product, and then incubate at 37°C for 30 minutes.
Incubate for a minute. Subsequently, 200 μm of commercially available Staphylococcus aureus Cowan I strain Absoap G (manufactured by Kaketsuken) was added and incubated at 37°C for 30 minutes. Then, 0.15M phosphate buffered saline (pH 7,
After washing with 2), Cpm in the sediment was counted with a gamma counter to measure the antigenicity of each hybridoma.

We also identified the immunoglobulin subclasses of mouse α, γ1, r2a1, 2J! r, γ3, μ, and each hybridoma culture supernatant were measured by the Ophthalony method using each antiserum against the λ chain.

The results are shown in Table 1, and it can be seen that monoclonal antibodies specific for either TSH-α or β sag unit were obtained.

Next, in order to find a combination of α-α or β-β monoclonal antibodies with different recognition epitopes for use in measurement system B, the following recognition epitope confirmation test was conducted.

Recognition Epitope Confirmation Test The differences in epitopes recognized by the five types of TSH-α subunit-specific monoclonal antibodies and the six types of TSH-β subunit-specific monoclonal antibodies obtained above were determined as follows.

Namely, TSH) racer (32,000 (J)
m, 200 μm), and each of the above monoclonal antibodies was adjusted to a concentration of 50 μg7-, d to 200 μj.
- was added and reacted at 37°C for 2 hours, and the above monoclonal antibody fixed on polystyrene beads was further added and reacted at 37°C for 12 hours. After washing, the cpm of the solid phase was measured, and those with a high value, ie, those that did not inhibit the reaction, were designated as different combinations of recognized epitopes.

The results are shown in Tables 2 and 3.
Subunits) Place in a test tube together with a polystyrene piece on which the special Jl monoclonal antibody is immobilized and react at 37°C for 2 hours, then add 5%) U4-:/ (Tween)2
After washing the beads three times with 0-PBS, they were transferred to another test tube.6 In addition, 25mf of a pH 4.8 solution containing 0.1M citric acid and 0.2M sodium phosphate, 25μ of 5% H20
! and 15 mg of O-phelenediamine dihydrochloride were added, reacted in the dark at 37°C for 11 hours, and further heated for 6N.

The reaction was stopped by adding 125 μm of H2SO4, and O.D.

The relationship between the absorbance measured at 492 nm and the concentration of TSH was plotted to obtain a standard curve shown as measurement system A (indicated as A system) in FIG.

Next, in measurement system B using the TSH-β subunit monoclonal antibody of 57 instead of the TSH-α subunit monoclonal antibody of Todoroki 1 as the immobilized antibody, another TSH Standard curve (
(shown as B series in FIG. 1) was obtained.

In addition, h) Each TSH-βS Goonit standard solution was prepared in the same manner as the above TSH standard solution using TSH-β subunit standard product (manufactured by Calbiochem, 1 oomg/vial lyophilized product), and in measurement system B, A pTsH-β subunit standard curve was obtained by the same procedure as above (second
figure).

Subsequently, using the above TSH-free serum, TSH 5μU/m
1 and 'I'SH-β, unit lng/m4
Prepare a sample solution containing O, D, 492 nm using measurement system A.
When I measured the absorbance at
J-, which was consistent with the sample concentration. Next, in measurement B, the absorbance of the specimen was measured at 61 tl, and the result was O, D.

492nm=1.25. Measurement system B Te (7) TS
The absorbance l3-2 corresponding to 5 μU/m 1 of TSH in the H standard curve is <0.25, as is clear from FIG. It becomes 00.

This absorbance originates from the TSH-β subunit, and when the fiTsH-β subunit concentration is determined from the TSH-β subunit standard line in FIG. 2 from this absorbance of 43, it is exactly 1. Ong/ml, which correctly matched the initial sample concentration.

TSH-α subunit standard it human TS at various concentrations
H-α subunit standard product (manufactured by Calbiochem, too
mg/><Ial lyophilized product) and TSH of Example 1
Prepared using free serum.

Measurement systems A and B were prepared in the same manner as in Example 1, except that Clone &5 (labeled antibody) and Clone Haruka 3 (immobilized antibody), which are both TSH-α subunit-specific monoclonal antibodies, were used as measurement system B. TSE (standard curve (Figure 3) and TSE in measurement system B (-α subuni, 7)
A standard curve (Figure 4) was obtained.

As specimens, T S H10ttU/mJL O and TS
A solution containing 2 ng/mA of H-α supunits I was subjected to TSE (
-α Subuni 7+- Prepared using the standard product and the TSH-free serum of Example 1.

When the absorbance of the sample was measured using measurement system A in the same manner as in Example 1, it was found that ○, D, 492 nm = 0.6, and the pTsH concentration was determined from this absorbance according to FIG. 3.
The content was 0 AU/ml, which coincided with the actual content. Also, T
The absorbance good 2 from the standard curve in measurement system B corresponding to a SH concentration of 10 μU/mJ is ○, D, 492 nm = 0.25
Met.

Next, the absorbance of the sample in measurement system B measured in the same manner is O, D, 492 nm = 1.7, 43, that is, 4l-42 = 1.7-0.25 = 1.45. Ta.

When looking at the TSH-α subunit concentration corresponding to this absorbance 63=1.45 using FIG. 4, it was 2 ng/mf, which matched the actual concentration in the sample.

In addition, when measuring the TSEI-α subunit from an actual sample, in order to avoid cross-reactivity with Ike's hormone (as mentioned above, due to the similar antigenicity of the α-subunit of each phor7in), Therefore, HCO
, LH and FSH are preferably removed. Furthermore, in the case of α-subunits, not only the α-subunits of one hormone but the amounts of α-subunits of all hormones are measured.

[Brief explanation of drawings]

FIG. 1 shows the TSH standard curve in measurement systems A and B for measuring TSH-β subunit, and FIG. 2 shows the TSH-β subunit standard curve in measurement system B. Figure 3 shows the TSH standard curve in measurement systems A and B for measuring the TSE (-α subunit), and Figure 4 shows the TSH-α subunit standard curve in measurement system B. TSH Mida () IU/ml) Figure 2 Figure 3

Claims (1)

[Claims] (1) Using an α subunit-specific monoclonal antibody and a β subunit-specific monoclonal antibody of a hormone selected from TSH, FSH, LH and HCG, a)
Measurement system consisting of an immobilized antibody (or labeled antibody) of an α-subunit-specific monoclonal antibody and a labeled antibody (or immobilized antibody) of a β-subunit-specific monoclonal antibody A and B) Same subunit as the subunit to be measured Establishment of an immobilized antibody-labeled antibody measurement system B using two types of monoclonal antibodies that recognize different epitopes, which are specific monoclonal antibodies; Create a standard curve for the subunit to be measured using measurement system B. Measure the sample containing the subunit to be measured using measurement system A, and determine the amount of the hormone itself based on the absorbance of the sample. Measure the sample containing the subunit to be measured with measurement system B to obtain the absorbance b_1 of the sample; Obtain the absorbance b_2 of measurement system B corresponding to the amount of the hormone itself measured with measurement system A using measurement system B. Determine from the standard curve of the hormone itself obtained in
3. Determining the amount of the subunit from the corresponding absorbance of the standard curve of the subunit to be measured in measurement system B.