JPS62180269A - Enzyme immunological measuring method for free thyroxine - Google Patents

Abstract

PURPOSE:To make substantial measurement of a free type thyroxine (FT4) in serum with about 2.7mul whole blood by making quantitative determination of the FT4 by an enzyme immunological method. CONSTITUTION:A 2-antibody solid phase method is preferable as the enzyme reaction which can be easily executed with relatively small hindrance. The free T4 (thyroxine) in the serum is quantitatively measured according to such method by bringing the measuring sample serum and the conjugate (T4-GOD) of T4 which is the labeled antigen and D-glucose oxidase (GOD) into contact reaction with the 1st antibody, then bringing the same into contact reaction with the 2nd antibody made into the solid phase, measuring the enzyme activity on the solid phase, and contrasting the same with a calibration curve preliminarily formed by using a standard T4 sample. A fluorescent method is usually high in sensitivity as a method for measuring the enzyme activity. The T4-GOD which is the labeled antigen is low affinity to the thyroxine-conjugated globulin (TBG) and serum albumin (HSA) and the measured value thereof is less affected by the TBG and HSA in the blood. Since said antigen has the high sensitivity, the substantial measurement of the FT4 is made possible with about 2.7mul whole blood.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a new method for measuring free thyroxine (hereinafter abbreviated as FT4) in serum using an enzyme immunoassay.

[Conventional technology]

As a method for determining FT4 in blood/m by enzyme immunoassay, there is a measurement method (C1ini
cal Chemistry730 16112 (198
4), same magazine 31 750 (1985)), Hose
Radish peroxidase (hereinafter referred to as HRP) wo4i1m-t7-,"s%Br @I←fi 2
8 666 (1982”) 1 was known.

[Problem that the invention seeks to solve]

Measurement of FT4 is effective for testing various thyroid functions such as neonatal thyroid dysfunction and postpartum thyroid dysfunction, but it is also effective for testing various thyroid functions such as neonatal thyroid dysfunction and postpartum thyroid dysfunction. It was strongly influenced by the proteins inside, and therefore it was necessary to obtain accurate measurements using enzyme immunoassay.

Recently, it has been discovered that the conjugate of thyroxine (hereinafter abbreviated as T4) and β-Gat (Ccnjugate) is less affected by TBG and H8A.
As mentioned above, a measurement method using β-Gat as a labeled enzyme was proposed. However, there has been a desire to develop a labeled enzyme to obtain a T4-enzyme conjugate that is even less affected by TBG, H13A, and the like.

[Failure to solve the problem]

The present inventor has been involved in the development and research of enzyme immunoassay methods for many years, and as he was conducting intensive research regarding this research, he discovered that D-
The present invention has been completed by discovering that when glucose oxidase (hereinafter abbreviated as GOD) is used as a labeling enzyme, it is possible to measure a considerable concentration range of TBG and H8A without being influenced by them.

In the enzyme immunoassay according to the present invention, the reaction method itself and the enzyme activity measurement itself can be carried out using various known methods [for example, Ishikawa, Tsutsugane, and Miyai, co-eds.: Enzyme immunoassay (2nd edition) (1982) However, as an enzymatic reaction, the two-antibody solid-phase method is preferred as it is simple and relatively unhindered (as a practicable method). According to this, free T4 in serum is obtained by contacting and reacting the measurement sample serum and a conjugate of T4 and GOD (hereinafter abbreviated as T4-GOD), which is a labeled antigen, with a first antibody, and then using a second antibody immobilized on a solid phase. The enzyme activity on the solid phase is measured and quantified by comparing it with a standard line prepared in advance using a T4 standard sample. Fluorescence is usually a highly sensitive method for measuring enzyme activity. For example, hydrogen peride produced by the glucose-glucose oxidase reaction,
Placed on a system of HRP and p-hydroxyphenylpropionic acid, excitation wavelength 320 nm.

The fluorescence intensity is measured at a fluorescence wavelength of 405 nm.

〔Example〕

Next, we will give examples and reference examples using the two-antibody solid phase method.
The method of the present invention will be explained in more detail, but the present invention is not limited thereto.

Reference Example 1 Preparation of buffer solution 1, 0.2 M (pH 8.6) barbital buffer solution (for testing) Parbital sodium 12.3708F was dissolved in approximately 500 g of water, adjusted to pH 8.6 with 12N hydrochloric acid, Add the pumped water containing bovine serum albumin α5F to a total volume of 50
Set it to 0p.

2, 101M (p) 17.0) Phosphate (contains food) buffer (for diluting FIRP) Potassium dihydrogen phosphate 1.3608? Dissolve 9 f of common salt in water to make a total amount of 1 t (A).

Dipotassium hydrogen phosphate 3.806F and salt 111F
Dissolve in water to obtain total t2t (B).

Combine the above A and B and adjust the pH to 7.0.

λ α25M(1)H5,O) Acetate buffer (for diluting glucose) Dissolve 17.01f of sodium acetate in 15a orttt of water.

Dissolve 14.31r/Ll of acetic acid in 1jlC of water, combine with the above sodium acetic acid solution, and adjust p)is, o to f3.

Reference example 2 Preparation of various solutions 1, HRP solution HRP (261Tl/my) (manufactured by TOYOBO) 1
Remove 0 curvature, dissolve 6.525ml of the above phosphate buffer (40QU/+ILl), and dilute 20 times before use (20-"
/yd) and use it.

2 α5% 3-(p-hydroxyphenyl)propionic acid (hereinafter abbreviated as HPPA) solution Dissolve 0.5 f of HPPA in IQOml of water.

3.0.5M glucose solution Glucose 22.52F was added to the acetate buffer 4ffi250
Dissolve in M.

Book sodium azide solution Dissolve 0.1 t of sodium azide in 100 d of water.

Reference Example 3 Preparation of T4-GOD Thyroxine methyl ester hydrochloride 3rn9 was added to 95% dimethylformamide 1M in a 101nl eggplant-shaped colben.
Glucose oxidase (105v/m9,
1d of an aqueous solution containing 109 (manufactured by TOYOBO) was added, stirred, and added dropwise with 1tiglutaraldehyde solution α21rttl, and reacted at room temperature for 2 hours. The resulting conjugate was dialyzed against 0.05 MIJ acid buffer i (pHa, O) overnight at 4°C and dialyzed in Tyopal HW55 (I x 5
(lcIn) column chromatography.

Reference Example 4 Preparation of anti-T4 serum A rabbit was immunized with T-hemigeltare-BSA as an antigen, and an antiserum was prepared (Clinical Chemistry 3)
Volume 1, page 430, 1985).

Reference Example 5 Preparation of immobilized second antibody 'n by affinity chromatography using Sephas gel bound with purified rabbit IG.
! The prepared anti-rabbit IgG goat Ig () was added to a 0.55% sodium phosphate solution (containing 1% sodium azide, pH 7,
One polyacetal bead (Fujirebio M) was immersed overnight at 4°C in a solution of 22 μm or more dissolved in 4), and then
．． Beads post-coated with 3% BSA solution are used.

Reference Example 6 Preparation of FT4 standard sample disk and specimen sample disk Blood cells prepared according to the conventional method (Clinical Chemistry Vol. 24, No. 2, p. 362, 1978) or Gant-free serum washed with physiological saline and ligand-free serum. , hematocrit). Part 4 m/
K T4 (free acid) diluted solution (400-6.25μ
Add 20 μt of each sample concentration of f/ytl and mix well. One drop of each of these solutions at various concentrations (zTμt
) and dried at room temperature overnight to obtain an FT4 standard sample disk. Instead of the diluted solution of T4 (free acid), one drop of specimen sample serum was placed on the same two papers as above, and the dried product was used as a specimen sample disk.

Example I Enzyme immunoassay for FT4 Anti-T4 serum (Reference example 4, X400000) 100μt
1 Dry sample disk (Reference example 6, T4 standard sample disk, 3 lines φ) 1 piece, T4-GODC Reference example 3, X250G
) 100 μt, 0.12 M barbital buffer [Reference Example 1-1) 250 μt and one immobilized second antibody bead (Reference Example 5) were mixed and left at 4° C. overnight.

After washing the collected beads three times with 1.5 liters of physiological saline,
0.5M glucose solution (#Example 2-3) SOQμt,
HRP solution (Reference Example 2-1) 50 μl and 0.5%
It is left at 37° C. for 1 hour with 50 μt of HPPA solution (Reference Example 2-2). The reaction solution was a 1:1 mixture of 2N aqueous sodium hydroxide solution and sodium acyde solution (Reference Example 2-4).
Add the mixed solution IQQμt and use a Hitachi fluorometer to measure 32
The fluorescence intensities at 0 nm (excitation wavelength) and 405 nm (fluorescence wavelength) were measured and quantified. The results are shown in Figure 1 (the line represents the test for FT4). The horizontal axis represents the amount of FT4 (nf/d4), and the vertical axis represents relative intensity. Bo is the strength without FT4, and B is the strength when FT4 is added.

Example 2 Effect of TBG In Example 1, the T4 standard sample concentration was set to 0.661'/d.
Add TBG to one reaction system using Z disk,
The reaction was carried out in the same manner as in Example 1, and the results are shown in FIG. 2 (indicated by * in the figure). For comparison, the influence of TBG on the measured values of FT4 using IT is shown on the same figure (marked with a square in the figure). ).

As is clear from the figure, in the measurement method using I-T4, the FT4 measurement value is rapidly affected by an increase in the TBG concentration, but the measurement value in the method of the present invention is significantly less affected.

Example 3 Effect of ISA The reaction was carried out in the same manner as in Example 2 using ISA instead of TBG. The results are shown in Figure 3. In the diagram ・
is the measurement result by the method of the present invention @ ” 12
5, and MU is based on the measurement results using -T4.

As is clear from the figure, the method of the present invention is almost insensitive to HIA.

〔Effect of the invention〕

T4-GOD, which is a labeled antigen, has extremely low affinity for TBG''? ISA, and therefore its measured value is rarely affected by TBG''? ISA in the blood.

Furthermore, since the sensitivity is very high, FT4 can be measured with just 2.7μ of whole blood. The detection limit of the method of the present invention is IIL05 nf/61 (1,35 ft/
tube).

[Brief explanation of drawings]

FIG. 1 is a calibration curve for T4, where the horizontal axis shows the amount of 4 in the sample and the vertical axis shows the relative value of the fluorescence intensity due to T4-GOD. Second
The figure shows the influence of TBG on the reaction between T and T-GOD and anti-T4 antibody.
The vertical axis indicates the relative value of fluorescence intensity by T4-COD. FIG. 3 shows the influence of ISA on the reaction between T4 and T4-GOD and anti-T4 antibody, where the horizontal axis shows the addition of ISA and the vertical axis shows the relative value of the fluorescence intensity due to T-GOD. B on the vertical axis of FIGS. 1 to 3 is the strength without the additive on the horizontal axis, and B is the strength in the presence of the additive.

Claims (1)

[Claims] A method for measuring free thyroxine in serum, characterized in that glucose oxidase is used as a labeling enzyme.