JPH0766000B2 - Labeled immunocarrier and method of using the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to the analysis of trace constituents in an analyte.

[Prior Art] Conventionally, various kinds of immunoglobulins, transferrin, β ₂ microglobulin, CRP, AFP, CEA, which are substances having a clear relationship between a trace component in a sample, for example, a disease and its concentration.
As a method for quantifying viruses and the like, radioimmunoassay (RIA), reverse passive agglutination reaction method (RPHA), complement fixation reaction method, enzyme immunoassay method (EIA) and the like are known. Of these, the RPHA method and complement fixation reaction method are methods in which each sample is diluted to the nth power of 2 (n = 1 to 2), and it is not suitable for multi-sample processing and there are large individual differences. It is said. Further, since red blood cells are used, there is a problem in stability. In addition, the RIA method uses an isotope that is difficult to handle and requires special facilities, so the equipment is expensive and cannot be handled easily.

In recent years, the EIA method, which is widely carried out, is rapidly spreading because it is easy to handle and expensive and does not require a device. However, the enzyme-labeled antibody used in this method is
When binding the enzyme to the antibody, a divalent reagent such as a glutaraldehyde or maleimide reagent, or a method of reducing the sugar chain of peroxidase using sodium periodate to bind to the antibody, or the method of binding the antibody or enzyme
Many methods for binding via SH groups have been announced, but in general, there are many difficult factors to prepare enzyme-labeled antibodies by these methods [enzyme antibody method, revised version, published by Interdisciplinary Planning Co., Ltd., 1985, see pages 21-23). On the other hand, in the homogeneous EIA method, agarose particles were used as a carrier, to which an antigen or highland and glucose-6-phosphate dehydrogenase (G-6-PDH) were immobilized, and a hexokinase-labeled antibody was added. A method for carrying out an antigen-antibody reaction has been reported, but this method has a very complicated reaction system [see Enzyme-linked immunosorbent assay, 2nd edition, published by Ikushosho, 1982, pp. 39-40].

[Problems to be Solved by the Invention] The enzyme-labeled antibody plays the most important role in the EIA method, and the results greatly vary depending on the performance of the enzyme-labeled antibody. The cause of this is the difficulty in producing the enzyme-labeled antibody. That is, the reaction between the crosslinking agent used for binding the antibody and the enzyme and the protein generally has low specificity, and as a result, the method has poor reproducibility. Generally, it is difficult to determine which part of the antibody or enzyme the cross-linking agent has bound to, and it is not uncommon for enzyme activity and / or antibody activity to decrease upon binding of enzyme and antibody. . Therefore, it is difficult to produce a long-term stable enzyme-labeled antibody that retains a high antibody titer and a high enzyme activity and is easy to produce by the conventional method of directly binding an antibody to an enzyme.

Therefore, the present invention provides a labeled immunocarrier that does not cause a decrease in antibody activity and enzyme activity without directly binding an antibody to an enzyme, and provides a simple assay method for a test substance using the same. To do.

[Means for Solving Problems] As a result of intensive studies in view of the above problems, the present inventors have made it possible to detect an antibody and an enzyme in carrier particles, thereby reducing both the activity of the enzyme and the antibody. Succeeded in developing a labeled immunocarrier having both high activity, and completed the present invention.

That is, the present invention is a water-insoluble fine particles, which can be suspended in water, and detect the antibody against the test substance and the test substance on a carrier of a size that cannot be visually confirmed at a low concentration. It is a labeled immunocarrier characterized by being bound with an enzyme which serves as an index for the purpose.

The material of the insoluble carrier particles is not particularly limited, and any carrier particles can be used, but fine particles of an organic polymer, such as polystyrene or styrene-butadiene copolymer, styrene-divinylbenzene or polyvinyltoluene. Etc., or carboxyl groups on them,
Known latex particles partially having a hydroxyl group or an amino group are used, and the particle size is about 100 μm or less, preferably 10 μm.
m or less is good.

The antibody against the test substance is purified from the antiserum against the test substance according to a conventional method. For example, rabbit, cow,
Antiserum obtained regardless of the type of animal such as mouse, chicken, horse, pig, guinea pig or goat is fractionated with 18% sodium sulfate, or an antibody purified using an ion exchange resin having a diethylaminoethyl group Antibody-sensitized latex particles can be prepared by contacting with latex particles. A mouse monoclonal antibody produced by a cell fusion method may be used as the antibody.

On the other hand, as an enzyme to be bound to carrier particles together with an antibody,
Examples include peroxidase, glucose oxidase, hexokinase, alkaline phosphatase, β-galactosidase, urease or amylase, but not limited to these, the enzyme activity can be easily measured, the activity per molecule is high, and the enzyme is easy to handle. There is no particular problem.

Among such enzymes, those having a hydrophobic portion on the surface of the molecule can directly bind to the latex microparticles together with the antibody, but relatively hydrophilic enzyme cannot bind directly, and therefore, the present invention Then, a hydrophobic substituent is introduced into the enzyme itself to bind to the latex microparticles. Examples of the hydrophobic substituent include an alkyl chain, a phenyl chain, an amine having both of them, an aldehyde or a carboxylic acid. An enzyme having a sugar chain forms an aldehyde with an oxidizing agent such as sodium periodate according to a conventional method and introduces the above-mentioned substituent having an amino group. Further, the hydrophobic substituent having an aldehyde group can be introduced into the amino group of the enzyme. Hydrophobic substituents having a carboxyl group can be introduced at the amino group of the enzyme by using carbodiimide. As another substituent, 2,4-bis (o-methoxypolyethylenediglycol) -6-chloro-S-triazine (activated PEG ₂ ) can be introduced into the enzyme molecule. Besides, there is no particular limitation as long as it is a substituent that reacts with an enzyme to increase hydrophobicity, such as 2.4 dinitrofluorobenzene or tetranitrosulfonic acid that reacts with an amino group.

In this manner, the enzyme having the hydrophobic substituent introduced, the antibody and the latex particles are mixed at the same time, or the enzyme having the hydrophobic substituent and the latex microparticles are mixed and washed by centrifugation, and then mixed with the antibody. Alternatively, the antibody may be bound to the microparticle carrier first and then bound to the enzyme.
After these operations, the latex particles were washed with an appropriate buffer containing 0.9% sodium chloride aqueous solution, and then 1% to 10% bovine serum albumin, 0.5% skim milk, 1% BSA, 1%.
% Gelatin, 1% ovalbumin, 0.05% tween,
A labeled immune carrier can be prepared by blocking with 1% dextran, 1% γ-globulin, 1% serum or the like. As carrier particles in addition to latex particles,
For example, a labeled immune carrier can be prepared by binding an antibody and an enzyme to a bacterium such as erythrocyte, individually dispersed cocci or bacillus by a known chemical method. or,
The same applies when the antibody and the enzyme are bound to a colloid of bentonite, silica gel, an inorganic oxide such as alumina, or other mineral powder.

The thus-obtained labeled immunocarrier can be used in the same manner as the conventionally known enzyme-labeled antibody in which the enzyme and the antibody are directly bound to the diameter. The application of the use of labeled immunocarriers is thus another object of the invention. The application example is to provide a method for quantifying an analyte. The quantification method is, for example, spotting the test substance on a filter such as nitrocellulose, blocking it with BSA or skim milk, etc., and then developing the color with a labeled immunocarrier, or a dot immunoplot method, or after electrophoresis, the protein is It is a well-known immunological analysis method such as a protein blotting method for identifying a specific test substance by transferring it onto a cellulose membrane.

In the case of the sandwich method, the antibody to the test substance is immobilized on a stationary phase such as a microtiter plate or beads. This is blocked with 1-10% bovine serum albumin or the above blocking agent, and then washed with a phosphate buffer containing 0.9% sodium chloride aqueous solution. The test substance is brought into contact with the stationary phase, and after a certain period of time, it is washed with the above-mentioned buffer solution, a labeled immune carrier is added thereto, and the sample is left to stand. After a certain period of time, the sample is washed again with the above-mentioned buffer solution, a substrate for the enzyme bound to the chromogenic carrier microparticles is added thereto, and then the concentration of the test substance can be measured by colorimetric quantification.

The combination of the chromogenic substrate and the enzyme is, for example, hydrogen peroxide, 4-aminoantipyrine, phenol or hydrogen peroxide in the case of peroxidase, p-nitrophenyl phosphate or phenyl phosphorus in the case of alkaline phosphatase. Acid, 4-aminoantipyrine, sodium periodate, glucose oxidase, glucose, peroxidase, 4-aminoantipyrine, phenol, β-galactosidase, β
-D-galactoside, p-nitrophenyl-β-galactoside and the like can be mentioned.

The analytes that can be measured in this manner include various immunoglobulins in serum, ceruloplasmin, transferrin, antitrypsin, β ₂ -microglobulin, CRP, AFP,
CEA, HBs and the like can be mentioned.

Hereinafter, the present invention will be described with reference to examples.

Example 1 (1) Preparation of labeled immunocarriers Enzyme modification Peroxidase (POD) [Boehringa grade I
I] 5 mg was dissolved in 1 ml of 0.3 M carbonate buffer (pH 8.1) to give 1%
Add 0.2 ml of 2,4-dinitrofluorobenzene (2,4-DFB) and stir slowly with a stirrer at room temperature. After 1 hour, 1 ml of 0.06 M sodium periodate is added, and the mixture is reacted at room temperature for 30 minutes. To this, 1 ml of 0.16M ethylene glycol is added and stirred for 1 hour. Add this solution to 0.01M carbonate buffer (pH
It dialyzed by 9.5) and the 2,4-DFB-ized POD was obtained.

Antibody An anti-HBs monoclonal antibody obtained from mouse membrane water (ascitic fluid was fractionated with 18% sodium sulfate and purified using diethylaminoethyl cellulose) was used.

Binding to carrier particles 2,4-DFB-modified POD (2 mg / ml) 1 ml, anti-HBs monoclonal antibody (1 mg / ml) 1 ml and latex particles [Sekisui Chemical Co., Ltd., 0.0
95 μm] (0.2% v / v) 1 ml. After leaving it at 4 ° C for one day and night, it was centrifugally washed with a phosphate buffer containing 0.9% sodium chloride aqueous solution and blocked with a glycine phosphate buffer containing 1% BSA to prepare a labeled immunocarrier.

(2) Confirmation of labeled immune carrier HBs antigen 1μg / ml 50μ on a microtiter plate
1 was added and the mixture was left at 4 ° C. for one day. Blocking was performed with a phosphate buffer containing 1% BSA. Aspirate the supernatant,
After adding 50 μl of 0.002% labeled immunocarrier, the mixture was allowed to stand at 37 ° C. for 1 hour and then washed with a phosphate buffer containing 0.05% Tween 20. Subsequent to this, add 200 μl of POD measuring reagent below the substrate [4-aminoantipyrine 0.5 mM, phenol 10 mM, hydrogen peroxide 0.3 mM, Tris-hydrochloric acid buffer (pH 7.5) 50 mM] and measure the absorbance after standing at 37 ° C for 15 minutes. did. The results showed that the HBs antigen plate had A ₅₀₅ = 0.213, and the plate coated with BSA had A ₅₀₅ = 0.213.
_{It was 505} = 0.008, and it was confirmed that the antibody and the enzyme were bound to the latex particles.

(3) Quantification of HBs antigen 5 μg of anti-HBs monoclonal antibody obtained from mouse ascites
Add 50 μl / ml to the microtiter plate,
After allowing to stand at 4 ° C for one day and night, it was sucked, blocked with a phosphate buffer containing 1% BSA at 37 ° C for 2 hours, and washed with a phosphate buffer. HBs antigen, 0, 50, 100,
50 μl of 200, 500 and 1000 ng / ml samples were added and left at 37 ° C. for 1 hour. After washing with a phosphate buffer, 50 μl of the labeled immune carrier prepared in (1) was added and left at 37 ° C. for 1 hour, and then washed with a phosphate buffer containing 0.05% Tween 20.
After adding 200 μl of the POD measurement reagent used in (2) and storing at 37 ° C., the absorbance after 30 minutes was measured. This result, as shown in FIG. 1, was able to quantify the HBs antigen.

Example 2 (1) Preparation of labeled immune carrier Enzyme modification Peroxidase (POD) [Boehringa, Grade I] 5 mg was dissolved in 0.3 M carbonate buffer (pH 8.1) 1 ml to prepare 1
% 0.24-dinitrofluorobenzene (2,4-DFB) (0.2 ml) was added, and the mixture was slowly stirred with a stirrer at room temperature for 1 hour.
Then, 0.16 M ethylene glycol 1 ml was added and stirred for 1 hour. The POD is dialyzed against 0.01 M carbonate buffer (pH 9.5) to obtain 2,4-DFB-modified periodate-treated POD. 3.5 ml of this mixture and 2 ml of 10 mM octylamine were mixed, stirred at 25 ° C for 3 hours, and then dialyzed against phosphate buffer to obtain octylamine-modified POD.
Was produced.

Antibodies Anti-CRP antiserum (goat) prepared by a conventional method was fractionated with 18% sodium sulfate, the 0-18% fraction was dialyzed against 0.9% sodium chloride solution, and then immunized / globulin with a diethylaminoethylcellulose ion exchange column. Fraction was obtained.

Binding to carrier particles 50 μl of octylamine POD (0.5 mg / ml), 500 μl of anti-CRP antibody (1 mg / ml) and latex particles [Sekisui Chemical Co., Ltd.,
0.095 μm] (0.2% v / v) 500 μl were mixed and left at 37 ° C. for 2 hours, then centrifugally washed with phosphate buffer containing 0.9% sodium chloride aqueous solution, and with glycine phosphate buffer containing 1% BSA. Blocking was performed to prepare a labeled immune carrier.

(2) Confirmation of labeled immune carrier 50 μC of CRP (5 μg / ml) on a microtiter plate
Add 1 l and leave at 4 ° C for 1 day to fix CRP, then add 1% B
Blocking was performed with a phosphate buffer containing SA. 50 μl of the labeled immunocarrier prepared in (1) was added to this plate and the reaction was carried out for 30 minutes. After washing with a phosphate buffer containing 0.05% Tween 20, 200 μl of the POD measuring reagent used in Example 1- (2) was added, and the absorbance after standing at 37 ° C. for 15 minutes was measured by. The results show that the CRP coated plate is A _505.
= 0.104, the plate coated only with BSA had A ₅₀₅ = 0.013, and it could be confirmed that the antibody and enzyme were bound to the latex particles.

(3) Quantification of CRP 5 μg / ml of the anti-CRP antibody prepared in (1) was added to the microtiter plate in an amount of 50 μl and left at 4 ° C. for 1 day and then
Blocking was performed with a phosphate buffer containing 1% BSA. CRP, 0, 20, 40, 60, 80, 100ng / m on this plate
50 μl of each sample was added to each well and allowed to stand at 37 ° C. for 1 hour, followed by washing with a phosphate buffer, and then 50 μl of 0.003% of the labeled immune carrier prepared in (1) was added and left at 37 ° C. for 1 hour. Wash with phosphate buffer containing 0.05% Tween 20,
200 μl of the POD measurement reagent used in Example 1- (2) was added
The absorbance was measured after 30 minutes at 37 ° C. As a result, as shown in FIG. 1, CRP could be quantified.

[Brief description of drawings]

FIG. 1 shows the relationship between the dilution and the absorbance of the serum sample in Example 1 and FIG.

Claims (3)

[Claims] 1. A labeled immunocarrier, wherein an antibody corresponding to an analyte and an enzyme having a hydrophobic substituent introduced therein are bound to a water-insoluble microparticle carrier. 2. An enzyme having a hydrophobic substituent introduced therein and an antibody corresponding to the analyte are simultaneously bound to the microparticle carrier,
Alternatively, a method for producing a labeled immune carrier, characterized in that one of an enzyme having a hydrophobic substituent introduced and an antibody corresponding to a test substance is bound first and the other is bound to a microparticle carrier later. 3. A method for immunologically quantifying a trace component in a specimen, wherein an antibody corresponding to the specimen and an enzyme having a hydrophobic substituent introduced are bound together to a water-insoluble microparticle carrier. A quantification method comprising using the labeled immune carrier.