JPH03154868A - Stabilizing method and stabilizing agent for settling antigen-antibody conjugate - Google Patents

Abstract

PURPOSE:To extremely easily execute the sepn. from the antigens or antibodies which do not react with an antigen-antibody conjugate by stabilizing the settling of the antigen-antibody conjugate with a vinyl acetate polymer. CONSTITUTION:Polyvinyl acetate or a copolymer of vinyl acetate and other monomer is brought into contact with the antigen-antibody conjugate to stabilize the settling of the antigen-antibody conjugate. The stabilizing agent for settling the antigen-antibody conjugate consists of the polyvinyl acetate or the copolymer of the vinyl acetate and the other monomer as its effective component. The polyvinyl acetate or the copolymer of the vinyl acetate and the other monomer is brought into contact with the antigen-antibody conjugate in an antigen- antibody reaction mixture to stabilize the settling of the antigen-antibody conjugate and thereafter, the sepn. operation is carried out at the time of separating the antigen-antibody conjugate and the non-conjugate from the antigen-antibody reaction mixture.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention provides a method for stabilizing an antigen-antibody conjugate precipitate, a stabilizer thereof, and a method for separating antigen-antibody conjugates and non-conjugates from an antigen-antibody reaction mixture. Regarding.

[Conventional technology]

In immunoassays, antigen-antibody reactions are used to quantify minute amounts of antigens or antibodies. A reaction is caused by adding an antibody specific to the antigen or an antigen or antibody labeled by an appropriate method to a solution containing a trace amount of the antigen or antibody. After the reaction is complete, the antigen-antibody complex (B) formed by the reaction and the unreacted antigen or antibody (II) are separated and quantified using a label to which B or F is simultaneously added. Alternatively, the concentration of the antibody is determined. Therefore, B/F separation is an essential technique in immunoassays, and the quality of this separation, along with the quality of the antibody used, has the greatest impact on the measurement results.

There are several methods of B/F separation with different basic principles. The main ones are (a) a liquid phase method in which either the antigen or the antigen-antibody complex is separated by insolubilizing the two by some method and generating a precipitate; (C) A method using electrophoresis. (C) A method using electrophoresis. In these (a) liquid phase method and (b) solid phase method, B/F
Conventionally, the techniques listed in (a) to (f) below have been used as specific means for separation.

(a) Ammonium sulfate precipitation method (a method in which proteins in a reaction solution are precipitated with high concentration ammonium sulfate, etc.) (b) Ethanol precipitation method (a method in which proteins in a reaction solution are denatured and precipitated with an organic solvent such as ethanol) ( C) Polyethylene glycol method (a method in which proteins in the reaction solution are insolubilized by polyethylene glycol) (d) Two-antibody method (a method in which a precipitate is generated using an antibody (second antibody) against the antibody in the reaction solution) (E) Dextran antibody method (in the dual antibody method (d) above, precipitation formation is promoted by the addition of dextran,
(f) Microparticle solid phase method (method of separating antibodies by immobilizing them on appropriate microparticles) [Problems to be solved by the invention] However, conventional B/F separation means have various problems. was there. That is, in the methods (a) to (c) above, most or all of the protein components present in the reaction solution are precipitated, and therefore cannot often be used for protein antigen measurement systems.

Therefore, it is not a common method. Also, (c)
The two-antibody method is still used as the most common method because it does not depend on the nature of the antigen.

However, in the case of the two-antibody method, the precipitate formed is generally loose and unstable, although it depends on the type of antibody used and the method. In that case, when the supernatant is removed from the reaction solution by suction, the precipitate flows out, causing a problem that the reproducibility of the results deteriorates. The dextran two-antibody method (e) is a method to improve this, but even with this method, sufficient stability of the precipitate cannot be obtained.

In the particulate solid-phase method described in (f), the precipitate is stable if a good particulate carrier is used, but nonspecific binding is large. Therefore, it is necessary to reduce the amount of solid phase fine particles used or to add a surfactant or the like. However, in the former case, the precipitate becomes almost unobservable with the naked eye, and the stability of the precipitate decreases. In the latter method, the shape of the precipitate is very poor and it tends to flow easily.

As described above, conventional B/F separation means have not been able to sufficiently satisfy both the stabilization of the B precipitate and the reduction of non-specific binding.

That is, in order to stabilize the precipitate, a certain amount of precipitate is required as a prerequisite. If the amount of precipitate is small, the strength of the precipitate will decrease, and even a small amount of precipitate flowing out will have a large effect on the results. The greater the amount of precipitate, the greater the intensity of the precipitate, and even the release of a small amount of precipitate has less relative influence on the results. However, since the stability of precipitate depends not only on the amount of precipitate but also on the properties of the precipitate itself, generally the precipitate does not become stronger in proportion to the amount of precipitate. On the other hand, non-specific binding increases in proportion to the amount of precipitation.

Non-specific binding is a reaction in which a labeled antigen or antibody binds to a precipitate through a reaction that is not a specific antigen-antibody reaction.

The most important feature of immunoassays is that they can selectively and sensitively detect antigens in serum, etc. by using highly specific antibodies.

However, if non-specific binding is large, this accuracy will deteriorate. Particularly in immunoradiometric assays using the sandwich method, this non-specific binding must be minimized in order to increase detection sensitivity.

Therefore, an object of the present invention is to provide a method for stabilizing a precipitate of antigen-antibody conjugates generated in an immunoassay without causing almost any non-specific binding, the stabilizing agent, and an efficient B/P separation method. There is a particular thing.

[Means to solve the problem]

As a result of extensive research to solve the above problems, the present inventors have found that using polyvinyl acetate or a copolymer of vinyl acetate and other monomers allows antigen-antibody binding with almost no non-specific binding. The present invention was completed by discovering that the precipitate of substances can be stabilized and B/F separation can be performed efficiently and easily.

That is, the present invention provides a method for stabilizing a precipitate of an antigen-antibody conjugate, which is characterized by contacting an antigen-antibody conjugate with polyvinyl acetate or a copolymer of vinyl acetate and another monomer, An antigen-antibody conjugate precipitate stabilizer containing a copolymer of and other monomers as an active ingredient,
In addition, in separating antigen-antibody bound substances and non-bound substances from the antigen-antibody reaction mixture, polyvinyl acetate or a copolymer of vinyl acetate and other monomers is brought into contact with the antigen-antibody bound substance in the antigen-antibody reaction mixture. This is a method for separating antigen-antibody bound substances (B) and non-bound substances (F), which is characterized in that a separation operation is performed after the antigen-antibody bound substance precipitate is stabilized.

The polyvinyl acetate used to stabilize the precipitate of the antigen-antibody conjugate according to the present invention is not particularly limited, and vinyl acetate is radically polymerized by a conventional method such as emulsion polymerization, suspension polymerization, solution polymerization, etc. can be mentioned. Examples of copolymers of vinyl acetate and other polymers include vinyl acetate-ethylene copolymers, vinyl acetate-vinyl versatate copolymers, and the like. Further, the degree of polymerization of polyvinyl acetate or a copolymer of vinyl acetate and other monomers (hereinafter simply referred to as "vinyl acetate polymer") is not particularly limited, but is preferably 300 or more.

The stabilizer for the antigen-antibody conjugate precipitate of the present invention is not particularly limited as long as it contains the vinyl acetate polymer described above, but is preferably in the form of an emulsion. This emulsion contains various buffers, dextran, a second antibody, and components normally included in commercially available vinyl acetate resin emulsions, such as polymerization initiators, plasticizers, surfactants, and antifreeze agents. You can leave it there. Such emulsions contain 0.01 to 20% w/v of vinyl acetate polymer (
(hereinafter simply expressed as "%"), particularly preferably 0.01 to 1%.

To carry out the B/P separation method of the present invention, after the antigen-antibody reaction in a normal immunoassay, a vinyl acetate polymer is added to the antigen-antibody reaction mixture, and then a normal B/P separation method is carried out.
F separation operation may be performed.

Antigen-antibody reactions to which the method of the present invention can be applied include, for example, the two-antibody method and the fine particle solid phase method. Here, the objects to be measured include various components contained in solutions such as serum, urine, and saliva.

The amount of vinyl acetate polymer added to the antigen-antibody reaction mixture is 5 to 5% relative to the amount of antigen-antibody conjugate in the reaction mixture.
Approximately 0 times the weight is preferable. If the amount is less than 5 times by weight, the stabilizing effect on the antigen-antibody conjugate precipitate will not be sufficient, and if it exceeds 50 times by weight, non-specific binding may occur, which is not preferable.

The addition of vinyl acetate polymer stabilizes the antigen-antibody conjugate precipitate in the antigen-antibody reaction mixture, and the subsequent B/F
Separation operation becomes extremely easy. Therefore, 8/F separation can be easily performed by operations such as centrifugation, suction, and decantation.

Highly accurate immunoassay can be performed by measuring the amount of labeled substance in the supernatant or precipitate of B/F separation operation. Here, various enzymes, coloring substances, fluorescent substances, radioactive isotopes, etc. can be used as labels.

[Effect]

The vinyl acetate polymer used in the present invention has a particle specific gravity close to that of the antigen-antibody conjugate precipitate and is highly stable in itself. It is thought to stabilize the precipitate. Furthermore, since vinyl acetate polymer has a hydrophilic surface and is chemically inert itself, it does not cause physical adsorption or chemical bonding of proteins, so nonspecific binding is presumed to be extremely low.

〔Example〕

Next, the present invention will be further explained with reference to Examples.

Implementation/Example 1 β-2 microglobulin radioimmunoassay (1) Reagent buffer solution used: 1% BSA, 0.1% Tween 20, 50
mM phosphate buffer solution (p) l? , 4) β-2 microglobulin standard solution β-2 microglobulin antigen 0.0.1°0.3,
1. 3. 10. 30mg/i! dissolved in a buffer solution at a concentration of

Iodinated (IIJ) β-2 microglobulin solution β-2 microglobulin 15 kB labeled with radioactive iodine 125 to have a specific radioactivity of 10 kBq/μg
q/ml! dissolved in a buffer solution.

Anti-β-2 microglobulin monoclonal antibody solution Anti-β-2 microglobulin monoclonal antibody, 0.
6% normal mouse serum dissolved in buffer solution.

Second antibody solution containing precipitation stabilizer 4% second antibody antiserum, 3% dextran, 0.2% polyvinyl acetate emulsion in 50mM phosphate buffer solution (p
Contained in H7,4).

(Use diluted commercially available vinyl acetate resin emulsion solution [approximately 40% concentration]) (2) Assay method 1: Transfer 25 μl of the test specimen or standard solution to a test tube.

2. 50μ of iodinated (12J) β-2 microglobulin solution! Add to each tube.

3. 50μ of anti-β-2 microglobulin monoclonal antibody solution! Add to each tube.

4. Leave at room temperature for 1 hour.

5. Add 500 μl of a second antibody solution containing a precipitate stabilizer.

6. Leave at room temperature for 30 minutes.

7. Perform centrifugation (2000g, 30 minutes, 20°C).

8. Separate the precipitate and the upper groove by suction.

9. Measure the radioactivity of the precipitate using a scintillation counter.

10. Create a standard curve with the radioactivity of the standard solution on the vertical axis and the concentration on the horizontal axis (Figure 1), and read the unknown sample concentration from the curve.

(3) Assay Results The standard solution was measured in triplicate, and the unknown specimen was measured in nine replicates. The results are shown in Table 1. Moreover, the standard curve is shown in FIG.

Note that CV in the table indicates the coefficient of variation.

Table 1 Below Table 1 and Figure 1 show that, according to the method of the present invention, the precipitate is stable, B/F separation by suction is easy, good Enoki and quasi-curves are obtained, and the coefficient of variation is extremely small, making it reproducible. The quality is also very good.

On the other hand, since the precipitate in the case where the precipitate stabilizer of the present invention was not used was unstable, the precipitate was broken by the water flow generated during suction. Furthermore, since the adhesiveness between the precipitate and the test tube was weak, the precipitate itself flowed. Furthermore, during suction, the precipitate was also aspirated together with the supernatant, resulting in large variations in measured values, extremely low accuracy, and extremely poor reproducibility.

Example 2 Human FSII immunoradiometric assay using solid phase microparticle method (1) Reagent used Standard solution Human PSH 0, 2, 5, 12, 5, 50, 200 ml 1U
/ml! contained in normal horse serum.

Iodine (l″-lylated anti-FS) I antibody solution with radioactive iodine 125 and specific radioactivity of 450 kBq/μg
Anti-FSII monoclonal antibody labeled as
en2Q, contained in 50mM phosphate buffer solution p16.4.

Anti-FSH antibody-conjugated solid phase microparticle liquid Aggregated bovine T globulin bound to an anti-FSH monoclonal antibody different from that used for labeling in an amount of 2 μg per assay is suspended in distilled water.

Precipitation stabilizer polyvinyl acetate emulsion mixed in water at a concentration of 0.1%.

(Use diluted commercially available vinyl acetate resin emulsion solution [approximately 40% concentration]) (2) Assay result ■, unknown sample or 4! Take quasi-solution 1007J1 into a test tube.

2. Add 100 μl of anti-FSH antibody-bound solid phase microparticle solution.

3. Add 100 μl of iodinated anti-FS) I antibody solution.

4. Leave at room temperature for 1 hour.

5. 2mj of precipitation stabilizer! Dispense.

6. Perform centrifugation (2000g for 30 minutes, 20t).

7. Separate the precipitate and supernatant by suction.

8. Measure the radioactivity of the remaining precipitate.

9. Create a standard curve in which the radioactivity of the standard solution is plotted on the vertical axis and the concentration on the horizontal axis (Figure 2), and read the concentration of the unknown sample.

(3) Assay Results Table 2 shows the results of triplicate measurements of the standard solution and triplicate measurements of two unknown samples. The standard curve is shown in Figure 2.

Table 2 Margin below From Table 2 and FIG. 2, the method of the present invention shows good results.

A standard curve was obtained and the results were reproducible.

The precipitate was also stable, with no decomposition or flow observed.

Furthermore, non-specific binding was also extremely small.

Comparative Example In Example 2, microsepharose, polystyrene latex, or polyethylene glycol was used instead of polyacetate/vinyl as a precipitate stabilizer, and the same assay was conducted. As a result, non-specific binding is extremely high;
The accuracy was poor.

〔Effect of the invention〕

According to the present invention, since the antigen-antibody conjugate precipitate is stabilized by the vinyl acetate polymer, B/P separation becomes extremely easy. Furthermore, since the vinyl acetate polymer does not increase non-specific binding in any way, the immunoassay using the B/F separation method of the present invention has extremely high accuracy.

[Brief explanation of the drawing]

Figure 1 is a drawing showing the standard curve for radioimmunoassay of β-2 microglobulin, and Figure 2 is a diagram showing the standard curve for β-2 microglobulin radioimmunoassay.
1 is a drawing showing a standard curve in an immunoradiometric assay of S H. that's all

Claims (1)

[Scope of Claims] 1. A method for stabilizing a precipitate of an antigen-antibody conjugate, which comprises bringing the antigen-antibody conjugate into contact with polyvinyl acetate or a copolymer of vinyl acetate and another monomer. 2. A stabilizer for antigen-antibody conjugate precipitate containing polyvinyl acetate or a copolymer of vinyl acetate and other monomers as an active ingredient. 3. In separating antigen-antibody bound substances and non-bound substances from the antigen-antibody reaction mixture, the antigen-antibody bound substances in the antigen-antibody reaction mixture are brought into contact with polyvinyl acetate or a copolymer of vinyl acetate and other monomers. A method for separating antigen-antibody bound substances and non-bound substances, which comprises stabilizing the antigen-antibody bound substance precipitate and then carrying out a separation operation.