JPS58149700A - Composite containing peroxidase, its preparation and reagent - Google Patents

Abstract

PURPOSE:To prepare an enzyme composite useful as a immunochemical determination reagent having high determination sensitivity, by bonding a peroxidase and an immunochemically active substance with a compound having succinimide group and maleimide group. CONSTITUTION:A peroxidase (B-NH2) and a compound of formula II (n is integer of 0-5; R is chemical bond or 6-membered cyclic hydrocarbon residue) are made to react with each other in a buffer solution having about 6-8pH at about 10-50 deg.C for about 10min-24hr to obtain the compound of formula III (B is peroxidase residue; n and R are same as defined above), which is made to react with an immunochemically active substance (A-SH) such as immunoglobulin, albumin, etc. in a buffer solution of about 6pH at about 0-40 deg.C for about 1-48hr to obtain the composite material of the peroxidase and the immunochemically active substance and represented by the formulaI(A is residue of the immunochemically active substance; n, R and B are same as defined above).

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a reagent for immunochemical analysis, particularly for enzyme immunoassay (hereinafter abbreviated as BIA).

EIA is radioimmunoassay C, hereinafter abbreviated as RIA. )
Similarly, it is an excellent clinical testing method that can measure extremely small amounts of substances, and is frequently used in the diagnosis, understanding, and management of diseases. However, RIA has drawbacks due to the use of radioactive isotopes, which limits its widespread use. On the other hand, BIA uses enzymes for labeling and has the characteristic that it does not require a special laboratory, so it has developed rapidly in recent years. “Enzyme immunoassay” as a BIA method [edited by Eiji Ishikawa, Igaku Shoin (
1978)], which are divided into competitive methods and non-competitive methods. In particular, the non-competitive method, ie the Sanderutsch method, and similar methods are frequently used.

As a labeling enzyme used in BIA, it is desirable that it be stable, enable highly sensitive measurements, and not be damaged during the labeling reaction. So far, peroxidase, β-D-lactosidase, alkaline phosphatase, glucose oxidase, etc. have been used, but among the enzymes mentioned above, peroxidase is an extremely stable enzyme with a molecular weight of about 40,000 and has high enzymatic activity. Most frequently used.

When using peroxidase for EIA, it is necessary to bind peroxidase and an immunochemically active substance in advance. The commonly used methods are shown below, but each has drawbacks and improvements have been desired.

Glugulaldehyde crosslinking method: This is a method in which peroxidase and an immunochemically active substance, such as a protein, are crosslinked with glutaraldehyde. A one-step method in which peroxidase and protein are added simultaneously [Immunochemistry (Immu
[Immunochemistry, Vol. 8 (l. 971), vol. [see page 1175], but in the former case, the obtained enzyme complex is extremely heterogeneous and contains a polymer. In the latter case, there is relatively little polymerization, but the yield for enzyme complex formation is 10
% or less, resulting in a low yield.

Periodic acid cross-linking method; method developed by Nakane et al.
tochemistry and cytochemi
22 (1974), page 1084]
The sugar moiety of peroxidase is oxidized with periodate to generate an aldehyde group, which is combined with an immunochemically active substance such as an amino acid of a protein to create a Schiff base, and further reduced to form a stable enzyme complex. Prepare.

Although this method has a high enzyme utilization rate, it has the disadvantage of producing a polymer.

The enzyme complex obtained by the glutaraldehyde crosslinking method or the periodic acid crosslinking method is Sephadex G-200 (Fallemacia Fine Chemicals (Nueten) III).
Even if purified by gel chromatography such as It was deemed appropriate. In addition, these methods suffer from a decrease in the sensitivity of the measurement, since the immunochemically active substance, such as the protein, bound to the pe-oxidase is damaged to varying degrees. .

The inventors of the present invention conducted extensive studies on a method for creating a peroxidase complex that allows for highly sensitive measurements, and found that the above-mentioned drawbacks can be overcome by combining peroxidase and an immunochemically active substance with a compound having a succinimide group and a maleimide group. It was discovered that an extremely excellent enzyme complex could be obtained, and based on this, further research was conducted, and as a result, the present invention was completed.

In the formula C, A is the residue of the immunochemically active substance (A-8H), B is the residue of Beryl reoxidase (B-NH2), and n is 0.
R represents an integer from 5 to 5, and R represents a chemical bond or a 6-membered cyclic hydrocarbon residue. ] A complex of peroxidase and an immunochemically active substance fl), (2)
Peroxidase (B-NH2) [wherein n is an integer of 0 to 5, and R represents a chemical bond or a 6-membered cyclic hydrocarbon residue. [In the formula, B represents a peroxidase residue.

n and R have the same meanings as above. ] is obtained, and then an immunochemically active substance (A-) is obtained.
8H) [wherein A represents a residue of an immunochemically active substance].

B, n and R have the same meanings as above. ] A complex of peroxidase and an immunochemically active substance (
1) Production method and (3) general formula [wherein A is the residue of the immunochemically active substance (A-8H), B is the residue of peroxidase (B-NH2), and n is 0 to 5 an integer of
R represents a chemical bond or a 6-membered cyclic hydrocarbon residue, respectively. This is an immunochemical measurement reagent containing a complex (1) of peroxidase and an immunochemically active substance represented by the following formula.

The immunochemically active substance represented by A-8H must have a thiol group in its molecule. Even when there is no thiol group in the molecule, a thiol group may be introduced into the immunochemically active substance using S-acetylmercaptosuccinic anhydride or the like. When the molecule has a disulfide bond, a thiol group may be generated using a reducing agent such as 2-mercaptoethylamine.

Immunochemically active substances in the present invention include antigens, haptens, and antibodies. Specifically, for example, immunoglobulin, albumin, fibrinogen (fibrin and its degradation products), α-fetoprotein, and C-reactive protein.

β2-microglobulin, myoglobulin, carcinoembryonic antigen, hepatitis virus antigen, human chorionic gonadotropin (
(hereinafter abbreviated as hCG), human placental lactogen,
proteins such as insulin, hormones, administered drugs, etc.
Also included are antibodies thereof.

Peroxidases of various origins can be used, examples include horseradish,
Pineapple, figs, and sweet potato.

Examples include peroxidases obtained from broad beans and corn, especially horseradish peroxidase extracted from horseradish.
4sh peroxjdase) is preferred.

In the above formula, the 6-membered cyclic hydrocarbon residue represented by R may be either saturated or unsaturated. Examples of saturated 6-membered cyclic hydrocarbon residues include cyclohexylene, and examples of unsaturated 6-membered cyclic hydrocarbon residues include phenylene.

Cyclohexylene is particularly preferred as the 6-membered cyclic hydrocarbon residue.

The compound [111] used in the method of the present invention has been published, for example, in The Journal of Biochemistry (T
he Journal of Biochemistry
y) Vol. 79, p. 288 (1976), European
Journal of Biochemistry (Europe)
anJournal of Biochernistr.
y) Volume 101, page 395 (1979), Japanese Patent Application Publication No. 1973-
It can be produced by the methods described in JP-A-85168, JP-A-52-85164, etc., or according to these methods. For example, the general formula [wherein X represents a hydroxyl group or a halogen atom].

n and R have the same meanings as above. ] Maleimide compound a) represented by the general formula [wherein Y represents a hydrogen atom or an alkali metal atom].

It can be produced by reacting the succinimide compound M represented by ] in the presence of a dehydrating agent or a deoxidizing agent. In the above general formula, examples of the halogen atom include chlorine and bromine, and examples of the alkali metal atom include sodium and potassium. Examples of dehydrating agents used in the reaction include sulfuric acid and dicyclohexylcarbodiimide, and examples of deoxidizing agents include pyridine and triethylamine fxt.

The compound side can be produced, for example, by the method described in JP-A-52-85164 or in accordance with the method. For example, it can be obtained by dehydrating and ring-closing a compound represented by the general formula (wherein n and R have the same meanings as defined above). The dehydration ring closure can be carried out by using a dehydrating agent such as acetic anhydride or acetic anhydride and acetic acid anhydride, and by heating gently.

Furthermore, as another method, Helvetian force, Kimi force, Acta (H
elvetica Chimica Acta) No. 58
Vol. (1975), p. 531, or according to the method described therein.

For example, N-alkoxycarbonylmaleimide represented by the general formula (Z represents an alkyl group) and the general formula % (%) (in which n and R have the same meanings as above). ] is reacted with an amino acid represented by the general formula [where n and R have the same meanings as above. ] is obtained. Next, a succinimide compound represented by general 2M is added and reacted in the presence of the same dehydrating agent or deoxidizing agent as described above.

In the compound represented by the above general formula (VI), the alkyl represented by Z includes methyl and ethyl.

To react a compound (Ill) with peroxidase,
Both were dissolved in a buffer solution with a pH of about 6 to 8 at a pH of about 10 to 50%.
The reaction is carried out at a temperature of .degree. C. for about 10 minutes to 24 hours. As the buffer solution, for example, pH 7
0.05 in 0.1 M phosphate buffer, pH 6.8
Examples include MIJ acid buffer.

Compound (1) is thus obtained. The obtained compound (1) can be purified, for example, by gel chromatography. The carrier used in the gel chromatography is, for example, Sephadex G-25 (Pharmacia Fine Chemicals (Sweden) IIJ).

Examples include Pyoge VP-2 (manufactured by Bio-Wood Laboratories, Inc. (USA)).

To react compound (I) with an immunochemically active substance,
This can be carried out by reacting both in a buffer at a temperature of about 0°C to 40°C for about 1 to 48 hours. As the buffer solution, 1. For example, 5 m at pH 6.0
0.1 containing M ethylenediaminetetraacetic acid sodium salt
Examples include M iJ phosphate buffer.

Compound (I) thus obtained can be purified, for example, by gel chromatography.

As the carrier used in the gel chromatography,
Examples include Ultrogel AcA44 (manufactured by LKB (Sweden)) and Sephacryl S-200C (manufactured by Pharmacia Fine Chemikasi (Sweden)).

Substances to be measured when performing immunochemical measurements using the complex (T) of the present invention include substances that can be used in clinical diagnosis, such as human immunoglobulin G and human immunoglobulin M contained in body fluids. , human immunoglobulin A, human immunoglobulin E, human fulbumin, human fibrinogen (fibrin and their degradation products)
, α-fetoprotein, C-reactive protein, β2-microglobulin, myoglobin, carcinoembryonic antigen, hepatitis virus antigen, human chorionic gonadotropin, human placental lactokene. proteins such as insulin, hormones,
Examples include administered drugs.

The complex (T) of the present invention can be used as a reagent in immunochemical analysis.

For example, in complex +11, A is a residue of an antibody, which can be used in an enzyme immunoassay using the Sand-Deutsch method.

As an example of an EIA reagent containing the complex (1) of the present invention,
Listed below are kits using the Sanderutsch method.

(1) Antibody supported on a carrier (2) Peroxidase-labeled antibody fragment (Fab') obtained by the method of the present invention.

(8) Standard product of the analyte (4) Reagents (2) to (3) above and a buffer used for diluting the test sample (a buffer that can be used for diluting the reagent and the test sample) (Examples include phosphate buffer or glycine buffer with pH 6 to 9).

(5) After incubation, the buffer used to wash the carrier (any buffer that can be used to wash the carrier may be used, examples of which include phosphate buffer or glycine buffer) ) (6) Reagents necessary for peroxidase activity measurement.

For example, in the case of a fluorescence method, P-hydroxydiphenylacetic acid and hydrogen peroxide are used as enzyme substrates, and in the case of a colorimetric method,
0-phenylenediamine and hydrogen peroxide. Examples include a buffer (preferably a phosphate buffer) used for dissolving the enzyme substrate and an enzyme reaction termination solution.

The above kit can be used, for example, by the method described below.

Standard product of the substance to be measured or test solution approximately lθ ~ 200μl
Add reagent (4) to dilute it, add a certain amount of reagent (1), then 10 to 800 μl of reagent (2), and then
React at ~40°C. After reacting for about 1 to 24 hours, add the reagent (
5) and measure the activity of v-oxidase bound on the carrier. That is, after about 10 to 1000 μl of the substrate solution is added and reacted at about 20 to 40° C. for about 0.2 to 24 hours, the enzyme reaction is stopped and the absorbance or fluorescence intensity in the reaction solution is measured.

By using the reagent for immunochemical analysis of the present invention, high-sensitivity measurements can be performed with simple operations in ordinary laboratories.

(:BIA is extremely useful as a clinical diagnostic method, but the enzyme complex of the present invention is also extremely effective in the field of immunohistochemistry because it causes less group I-specific reactions.

The binding reagent used in the method of the present invention has a succinimide group and a maleimide group, reacts with the amino group present in PeM oxidase to introduce a maleimide group into the enzyme, and further binds to immunochemically active substances such as A complex of an enzyme and an immunochemically active substance can be easily prepared by reacting with the 1,000-ol group present in an immunoglobulin fragment (Fab'). The composite of the present invention (according to the production method No. 11, the yield is extremely excellent at about 70% or more, and hardly any polymerized product of the composite is observed. When the enzyme complex of the present invention is subjected to EIA using a non-competitive method, the measurement sensitivity is lower than that obtained using the enzyme complex obtained using the method described above. The improvement is about 10 times or more.Furthermore, even if a high concentration of the enzyme complex in the complex of the present invention is used, nonspecific adsorption to the solid phase does not increase, so the time required for measurement can be shortened.

As mentioned above, the reason why non-specific adsorption is reduced by using the enzyme complex of the present invention and measurement sensitivity is improved is that approximately one molecule of immunochemically active substance binds to one molecule of be-oxidase. This is also thought to be due to the fact that the amount of polymer produced in the composite is extremely small and the prepared composite is extremely uniform.

The present invention will be explained in more detail with reference to Reference Examples and Examples below.

Reference Example 1 Periodic acid cross-linking method Nobune et al.'s method [The Journal of Histochemistry and Cytochemistry (The Jou
RNA of Histochemistry a
ndCytochemistry) Volume 22 (1974
2006), p. 1084]. Dissolve 7 da of horseradish peroxidase in 1 ml of 0.3 M sodium bicarbonate solution (pH 8.1) and add 0.1 ml of 1% 1
-Fluoro-2,4-dinitrobenzene was processed and reacted for 1 hour at room temperature. Then 0.06 M Na l041
After adding 111 and stirring at room temperature for 30 minutes, 0.16
1 liter of M ethylene glycol aqueous solution was added and allowed to stand at room temperature for 1 hour. 0.61M carbonate) IIum buffer (p
Dialysis was performed overnight against H9,5).

Kato et al.'s method [The Journal op Immunology (
The Journal of Immunolog
Rabbit anti-goat rgG (Fab' fragment) 5 obtained by
'IF was added to 0.1M phosphate buffer (pH 6,0) containing 24mM ethyl remaleimide to block thiol groups (at room temperature for 80 minutes), purified by gel chromatography using a Sephadex G-150 column, and purified with collodion. After concentrating using the membrane method, it was mixed with the previously prepared aredehyde peroxidase and reacted at room temperature for 8 hours.
'q of NaHB4 was added and reacted overnight at 4°C. 0.
0.01M phosphate buffer containing 15M NaCl (pH
After overnight dialysis at 4 °C against AcA44 (LKJt (;x.

Column (1,5 x 451) filled with
gel chromatography using 0.1MIJ
Elution was carried out with acid buffer (pH 6.5). 28 of the eluate
Absorbance at 0 and 408 nm and enzyme activity were measured. The enzyme activity of the eluate was 0.19
0.1MIJ acid buffer containing 6 bovine serum albumin (
After diluting 500 times with pI (y, o), this 10μ
Guilbalt et al.'s method for l [Analytical book Chemistry] +7-(4nalyt
ical Chemistry) Volume 40 (196
8 years), page 1256]. That is, 10 μl of the diluted eluate contained 0.196 bovine serum albumin, 0 μl dissolved in xM phosphate buffer (pH 7, O),
Add 0.25 ml of enyl acetic acid (5% p-hydroxyf) and mix.
'C for 5 minutes. Next, 0.05 ml of 0.01% hydrogen peroxide was added and reacted at 80°C for 20 minutes. 0.1M glycine buffer (pH 10,8) 2.5
ml was added to stop the enzyme reaction, and the fluorescence intensity at 405 nm was measured using excitation light of 320 nm, setting the fluorescence intensity of lμf/me2 of kinin as 100. The results are shown in Figure 1. In Figure 1, -0- is the absorbance at 280 nm, ÷ is the absorbance at 408 nm,
+ indicates peroxidase activity (as fluorescence intensity), respectively.

Example 1 (1) Introduction of maleimide group 6TIIg horseradish peroxidase (manufactured by Boerin Mannheim (West Germany)) was dissolved in 1 ml of 01M phosphate buffer (pH 7.0), and 50 μl of n-, - Coupling reagent MMC dissolved in dimethyl and a small amount of himamide
(Compound with general formula fnl+nite, n=1, R=cyclohexylene) Add 4.8# and heat at 30°C to 6
The reaction was allowed to proceed with stirring for 0 minutes. The generated precipitate was removed by centrifugation, and the supernatant was passed through a Sephadex G-25 column (equipped with 1.0×45) and eluted with IMIJ acid buffer. A protein-containing fraction was collected and concentrated using a collodion membrane.

In the maleimidated peroxidase thus prepared, the number of maleimide groups introduced per molecule of peroxidase was 1.0 to 1.2 (calculated assuming the molecular weight of peroxidase to be 40.0 OOE1% = 22.75.゜(2) Maleimidated peroxidase and rabbit anti-Yaki I
Rabbit anti-goat I conjugated to gGFab' fragment
From gG serum [manufactured by Miles Laboratories (USA)], the method of Kato et al. [The Journal of Immunology]
Fab fragments were prepared according to the method described in J. Ogy, Vol. 116 (1976), p. 1554]. That is, rabbit anti-goat IgG serum was dialyzed by the sodium sulfate method, and DE
Ig by column chromatography of AE-Setv loin
After making the G fraction, it was digested with pepsin to obtain F(ab
) 2, further reduced with 2-mercaptoethylamine, and purified by gel chromatography on a Sephadex G-25 column to obtain a rabbit anti-goat IgG Fab' fragment.

Example 1-(Manimidated peroxidase 1.5 cape prepared in 11) was added to 0.1M phosphate buffer (pH 6,0) 0
．． Rabbit anti-goat IgGFa obtained previously was dissolved in 15 g/
Add 0.1M phosphate buffer (pH 6,0) containing 5mM ethylenediaminetetraacetic acid sodium salt in which 1.7wv of the b' fragment had been dissolved, and incubate at 4°C for 2 hours.
The reaction was allowed to proceed for 0 hours. After reaction, Ultrogel A c A
Gel chromatography was performed using a column packed with 44 (1,5X j5cM) and eluted with 0.1M phosphate buffer (pH 6,5). Measure the absorbance of the eluate at 280 and 4Q31m and the enzyme activity.

It was confirmed by the following method that a complex of peroxidase and rabbit TgGFab fragment was produced.

First, enzyme activity was measured using the method of Gilpart et al. [Analytical Chemistry]
istry), Vol. 40 (1968), p. 1256]. That is, each fraction of the eluate was 0.1
0.1 M phosphate buffer containing % bovine serum albumin (p
It was diluted 1800 times with H7,0).

005 containing 01% bovine serum albumin in this 10μl
M sodium acetate buffer (pH 5,0) dissolved in 0
, 0.25 yxl of 5% p-hydroxyphenylacetic acid was added, mixed, and incubated at 30°C for 5 minutes. Next, add 0.05 ml of 0.01% hydrogen peroxide and incubate at 30°.
The reaction was carried out at C for 20 minutes. p, 1 M glycine buffer (
The enzyme reaction was stopped by adding 2.5 ml of kinin (pH 10.8), and the fluorescence intensity at 405 nm with excitation light at 820 nm was measured, setting the fluorescence intensity of 1 μI/ml kinin to 100. The results are shown in Figure 2. In Figure 2, min represents the absorbance at 280 nm, and f represents the absorbance at 40:3.
Absorbance at nm is indicated, and + indicates peroxidase activity (as fluorescence intensity), respectively.

Comparing with the elution pattern obtained in Reference Example 1 (Fig. 1),
No polymerized enzyme complex eluted around fraction 29 was observed, indicating that the complex between peroxidase and rabbit anti-goat IgG Fab' fragment around fraction 85 was produced extremely well.

+81EIA The following were used as reagents for EIA.

■ Human IgG K-made silicone rubber piece ■ Complex of peroxidase obtained in Example 1 (2) and rabbit anti-goat IgG Fab fragment ■ Standard goat anti-human IgG ■ 0, IMNaC used for washing and dilution of ■ and ■
I, 1mM MgCI2, 0.1% bovine serum albumin, pH 7.0 containing 0.002% merthiolate.
．． OIM phosphoric acid solution ■ Reagent required for peroxidase activity measurement 05% p-
Acetic acid buffer containing hydroxyphenylacetic acid at pH 5.0, 0.01% hydrogen peroxide solution and reaction stop solution (pH 1
0.8 0.1M glycine buffer) Preparation of antibody-binding solid phase, clean silicone strip (inner diameter 3mm, length 4n+, silicone hs manufactured by Fuji Polymer Industries Co., Ltd.)
, SH type) was added to a 0.25 M phosphate buffer (PH47.5) containing 100 μf Al of human IgG for 30 min at room temperature.
After soaking for 1 minute, it was left at 4°C overnight. 0.25M phosphate buffer (PH7.5) and buffer A (0.1M
001M with NaCl, 1mM MgCl2, 0.1% Bovine Serum Albumin, 0.002% MeI Rethiolate
Wash with phosphate buffer (, p) 47.0)) and buffer A
I kept it cold inside.

Measurement: goat anti-human) IgG (I
gG fraction) C. Miles Laborado IJ-'s (
(manufactured in the United States) and the previously prepared IgG-sensitized silicone rubber pieces were reacted at 87°C for 6 hours, and then at 4°C overnight. After washing the silicone rubber piece twice with buffer A, the peroxider-v-rabbit anti-goat IgG (Fab' fragment) complex solution prepared in Example 1 (2) (Fig. 2, fractions 37 to 39 was collected) After adding 0.15 ng/d) (indicated by -0- in Figure 8) and reacting at 87°C for 6 hours, the silicone rubber piece was diluted with buffer solution A. After washing twice with A, the enzyme activity bound to the solid phase was measured according to the method of Gilpart et al. shown in Example 1 (2). The results are expressed as peroxidase-rabbit/goat Ig obtained by the periodic acid cross-linking method obtained in Reference Example 1.
G (Fab fragment) complex solution + 11 Fig. 1 Fractions 87-89 were collected as fractions with less heavy inclusions and diluted with buffer A (11020 n/s + j
X in Figure 8--e-te zRT. ).

Fractions 28-80 as large fractions of polymer contamination
was collected and diluted with buffer A (SOOng/j) (
Indicated by + in FIG. ). ] using the same assay procedure (the enzyme activity of each complex solution is the same). As shown in Figure 3, the system using the enzyme complex obtained in Example 1 (2) of the present invention has minimal non-specific adsorption to the solid phase, which is about 10 times higher than other systems. It was sensitivity.

Example 2. Measurement of human IgG (1) Peroxidase and rabbit anti-human IgG (F
ab' fragment) Maleimidated peroxidase 1 obtained in Example 1 (1)
．． 5 Wv and rabbit anti-human) IgG (Pab' fragment l11.7W) obtained from rabbit anti-human) IgG serum [Miles Labora, manufactured by IJ-S (USA)] in the same manner as in Example 1 (2). was combined in the same manner as in Example 1 (2), and peroxidase-rabbit anti-human) IgG (Fab
' Obtained a fragment complex.

(2) EIA The following were used as reagents for EIA.

■ Rabbit anti-human IgG (IgG fraction) sensitized silicone rubber piece ■ Complex of peroxidase obtained in Example 2 (1) and rabbit anti-human IgG Fab' fragment ■ Standard human IgG ■ For washing and dilution of ■ and ■ 0, 1 MN used
aCI, 1mMgcI2. 0.01M phosphate buffer at pH 7.0 containing O, t% bovine serum albumin, 0.002% merthiolate■ Reagents required for peroxidase activity measurement 0.5%p
- Acetate buffer at pH 5.0 containing hydroxyphenylacetic acid, 0.01% hydrogen peroxide solution and reaction stop solution (p
H10,8 0.1 M glycine buffer) Preparation of antibody-binding solid phase: Clean silicone rubber piece (inner diameter (3
tm, length 4m, manufactured by Fuji Polymer Industries Co., Ltd. (C + ) coyNn8 + SH type) was used as rabbit anti-human IgG.
(IgG fraction) containing 1ooμg//Ml.
The sample was immersed in a 25 MIJ acidic acid solution (pH 7.5) for 30 minutes at room temperature, and then left overnight at 4°C. Wash with 0.25MIJ acid buffer (PH7,5) and buffer A,
Stored inside in a cool place.

For measurement, human IgG (manufactured by Miles Laboratories, USA) in 0.15 m/buffer A and the previously prepared rabbit anti-human IgG sensitized silicone rubber pieces were incubated at 37°C for 6 hours, then at 4°C. The mixture was allowed to react overnight.

After washing the silicone rubber piece twice with buffer A, Example 2 (1
peroxidase-rabbit anti-human IgG prepared with
After adding 10.15 ml of Fab'fragme7)3 complex solution and reacting at 87°C for 6 hours, the silicone rubber piece was washed twice with buffer A, and the enzyme activity bound to the solid phase was determined. It was measured according to the method of Gilbert et al. shown in 1(2). The results are shown in Figure 4. In Figure 4, ÷ is M
The results obtained when using a complex bound by MC are shown. The results are shown using both a very small fraction and a fraction relatively contaminated with polymer. As is clear from FIG. 4, when the complex of the present invention is used, non-specific adsorption to the solid phase is extremely small.
The sensitivity was 10 to 20 times higher than when using a peroxidase-rabbit anti-human IgG (Fab' fragment) complex by periodic acid crosslinking.

Example 8. Measurement of hCG (1) Production of antibody Approximately 10,0 OOIU/cape of hcGIIv purified from human urine by a known method was dissolved in physiological saline 1111, and Freund's complete adjuvant [Freund'Sco
complete adjuvant, biochemistry of immunity,
Tachibana et al., Kyoritsu Shuppan Co., Ltd. (1967)] 11m is added and mixed well to form an emulsion, which is injected into both thigh muscles and several locations on the back and lower part of the rabbit. Perform the above operations 8
Blood was collected 1 week after the final immunization, and anti-hCG
Serum was obtained.

hcG5W containing 0.5M NaCl, 1M NaH
Dissolved in COB 8s+/N/1,000HC in advance
The mixture was added to Bromcyan-activated Sepharose 4B (manufactured by Remacia Fine Chemicals Co., Ltd.) washed with Phyl I and stirred overnight at 5°C. After the reaction, the same 0.5MNa
Thoroughly wash with 0.1 M NaHCOB containing Cl, then add 10 m+/ of 0.5 M ethanolamine adjusted to pH 8 with HCl and react for 1 hour at room temperature. Acetate buffer (pH 4,0'), (2) IM NaCl
0.1 MM phosphate buffer pH 8.0) and (
8) Washed sequentially with 0.02M borate buffer (pH 8, o) containing 0.15M NaCl and packed into a column.

8 ml of the anti-hCG serum obtained previously was subjected to salting-out precipitation using 1.5 g of anhydrous sodium sulfate, and the obtained γ-globulin fraction was applied to the hCG-binding Sepharose 4B column (
0.9x4n).

The column was washed with 0.02 M borate buffer (p) (s, o) containing 0.15 M NaCl, then 0.02 M borate buffer (p) (s, o) containing 0.
Rabbit anti-hCG antibody was obtained by elution with 17M glycine-hydrochloric acid buffer (pH 2,8).

(2) Complex of peroxidase and rabbit anti-hCG (Fab' fragment) Maleimidated peroxidase 1 obtained in Example 1 (1)
．． 5 W and rabbit anti-hcG prepared in Example 8 (1)
The rabbit anti-hc antibody was obtained according to the method of Example 1 (2).
G(Fab')-)gmen day 1.8 Iv and Example 1 (
A peroxidase-rabbit anti-hCG (Fab' fragment complex) was obtained by conjugation using the method shown in 2).

+81EIA The following were used as reagents for EIA.

■ Anti-hCG antibody sensitized polystyrene pole ■ Example 3
Peroxidase obtained in (2) and rabbit anti-hCGF
Complex with ab' fragment ■ Standard hCG ■ M# solution used for washing and dilution of ■ and ■ ■ Reagents necessary for peroxidase activity measurement 002% hydrogen peroxide and 0.15% 0-phenylenediamine containing PH4,8
0.1 M citric acid-Nina phosphate) IJum buffer and reaction stop solution (IN-HCl) Preparation of antibody binding solid phase: Polystyrene balls (diameter 6.4
g, Precision Plastics Ball Company (Pr.
ecision Plastics Ba1l C
o.

(Made in the United States)] 800 pieces of the anti-h
CG antibody 2.51v was added to 0.01 M phosphate buffer (PH
7,7) A solution dissolved in 501I+/ was added and immersed at room temperature for 1 hour, and then left overnight at 4°C. 0.05M phosphate buffer (pH 7.0) containing 0.1% bovine serum albumin
) and then stored in a cool place until use.

Measurement: hCG and previously prepared rabbit anti-hC in buffer A.
The G-sensitized polystyrene pole was reacted overnight at room temperature.

After washing the polystyrene balls with buffer iA, perform 8 ml.
Peroxidase-rabbit anti-hcG prepared in (2)
(Fab' fragment) After adding complex solution 0.1517 and reacting at 37°C for 6 hours, the polystyrene ball was washed with buffer A, and this was mixed with 0.02% hydrogen peroxide and 0.15% - 0.1 M containing phenylenediamine
Citric acid-disodium phosphate buffer (pH 4,8)
0.5 g/-(After reacting at 80°C for 1 hour, 2 ml of 1N hydrochloric acid was added to stop the reaction, and 492
The absorbance at nm was measured to obtain a standard curve. The results are shown in Figure 5, and the non-specific adsorption to the solid phase is extremely small.
High sensitivity measurement was possible.

Example 4. Binding horseradish oxidase by m-maleimidobenzoyl-N-hydroxysacunnimide CMB S [Boehringer Mannheim (
611y (manufactured in West Germany) was dissolved in 0.1M phosphate buffer (pH
7,0) and 50 μl of N,N'-dimethylfo!
Binding reagent MBS dissolved in remamide [general formula full
(compounds in which n=o, R-phenylene) 4.8
Wg was added and reacted at 25°C for 30 minutes with stirring. The reaction product was then passed through a column of Sephadex G-25 (1, Ox 45m) and 0.05M acetate buffer (p
It eluted with H5,0).

A protein-containing fraction was collected and concentrated using a collodion membrane. In the maleimidated peroxidase thus obtained, the number of maleimide groups introduced per molecule of peroxidase was 068 to 0.78.

Kato et al.'s method [The Journal of Immunology,
116 (1976), p. 1554] rabbit anti-goat IgG (Fab' fragment) 1. '
5mM ethylenediaminetetraacetic acid dissolved in tyv)
0.1 M IJ acid buffer (pH 6) containing IJ salt
.
H6,0) was added to 0.15 liters and reacted at 4°C for 20 hours. After the reaction, gel chromatography was performed using a column (1.5 x 45 cIl) packed with Ultrogel AcA44, and 0.1 M phosphate buffer (pH 6.5) was applied.
Elute with peroxidase-rabbit anti-goat gG (
Fab' fragment) complex was obtained.

When this complex was assayed according to the EIA procedure shown in Example 1 (3), it was found that non-specific adsorption to the solid phase was extremely small and it provided high sensitivity.

The following reagents were used in EIA.

■ Silicone rubber piece made from human IgGrE ■ Complex of peroxidase obtained in Example 4 and rabbit anti-goat IgG Fab' fragment ■ Standard goat anti-human IgG ■ Buffer used for washing and dilution of ■ and ■ ■ For peroxidase activity measurement Necessary Reagents: Acetate buffer at pH 5.0 containing 0.5% p-hydroxyphenylacetic acid;
0.01% hydrogen peroxide solution and reaction stop solution (pH 10J
0.1 M glycine buffer)

[Brief explanation of drawings]

Figure 1 shows the elution pattern in gel chromatography of the reaction product of the peroxidase obtained in Reference Example 1 and the rabbit anti-goat IgG Fab' fragment, and Figure 2 shows the elution pattern of the reaction product of the peroxidase obtained in Example 1 and the rabbit anti-goat IgG Fab' fragment. Figure 3 shows the elution pattern of the fragmented reaction product in gel chromatography. Goat anti-human) IgG standard curve,
Figure 4 shows the standard curve of human IgG obtained in Example 2 or using the complex of peroxidase and rabbit anti-human IgG Fab' fragment by the periodic acid cross-linking method, and Figure 5 shows the standard curve of human IgG obtained in Example 8. The standard curves of hCG obtained using the peroxidase and rabbit anti-hCG Fab' fragments obtained are shown, respectively. Broom 1 Number of brooms, fraction 4 (human IgG ng/broom)

Claims (3)

[Claims] (1) General formula [wherein A is the residue of the immunochemically active substance (A-8H), B is the residue of peroxidase (B-NH,), n is an integer from 0 to 5, R represent a chemical bond or a 6-membered cyclic hydrocarbon residue, respectively. ] A complex of peroxidase and an immunochemically active substance. (2) A general formula or a 6-membered cyclic hydrocarbon residue is shown in be-Nt oxidase (B-NHt), respectively. ] A compound represented by the general formula [where B represents a peroxidase residue] is reacted. n and R have the same meanings as above. ] was obtained, and then an immunochemically active substance (A-8H
) is reacted with - [wherein A represents a residue of an immunochemically active substance]. B, n and R have the same meanings as above. ] A method for producing a complex of peroxidase and an immunochemically active substance. (3) General formula [wherein A is the remainder Jl of the immunochemically active substance (A-8H)]
-, B is the residue of peroxidase (B -NHg),
n represents an integer from 0 to 5, and R represents a chemical bond or a 6-membered cyclic hydrocarbon residue. ] An immunochemical measurement reagent containing a complex of peroxidase and an immunochemically active substance represented by the following.