JP2915486B2 - Indirect solid-phase antibody method - Google Patents

Description

The present invention relates to an indirect solid-phase antibody method. More specifically, the present invention relates to a method for quantifying an antigen-antibody reaction in a solution.

[Conventional technology]

Conventionally, as the quantification of the antigen-antibody reaction, a solid-phase enzyme antibody method, an immunoprecipitation method, a competitive immunization method, an inhibition test method and the like are known.

However, in the solid-phase antibody method, when the antigen is immobilized inside the reaction vessel, the antigenicity of the antigen often changes, and the antigen may be completely denatured. It was not possible to measure the reaction accurately.

In addition, in the immunoprecipitation method, there is a large possibility that the secondary antibody and the precipitant added to the reaction system and impurities contained therein will affect the reactivity and antigenicity of the antigen, and the antigen-antibody reaction under solution conditions Could not be measured accurately. In addition, there is a problem that it is not easy to measure the amount of the antigen or antibody in the precipitate with high sensitivity.

Similarly, in the competitive immunization method, the factor in the reaction solution affects the antigen-antibody reaction of the measurement system, so that it is impossible to measure the antigen-antibody reaction under solution conditions. The influence between the system and the measurement system could not be excluded, and accurate quantification of the antigen-antibody reaction could not be performed.

Therefore, it has not been possible to accurately quantify the antigen-antibody reaction under solution conditions even by using the above method. In particular, under the reaction solution conditions in which various factors are contained in the reaction solution, the influence of these factors must be considered, and it has been more difficult to measure the amount of the antibody. For example, in the enzyme-linked immunosorbent assay (ELISA), the signal intensity changes depending on both the factor that affects the antigen-antibody reaction and the factor that affects the color reaction by the enzyme. Measurement may not be possible.

[Problems to be solved by the invention]

Accordingly, an object of the present invention is to provide a method for accurately quantifying an antigen-antibody reaction in a solution state, and in particular, to provide a method for accurately quantifying an antigen-antibody reaction even in a solution containing various factors. The purpose is to do.

[Means for solving the problem]

Therefore, the present inventors have conducted intensive studies and found that the antigen-antibody reaction was stopped by diluting the reaction solution of the antigen-antibody reaction, and the effect of reaction system factors on the measurement was minimized to minimize the antigen-antibody reaction. It has been found that the present invention can be quantitatively determined, and the present invention has been completed. According to the method of the present invention, by measuring the amount of unreacted antibody after dilution, it is possible to completely remove the influence of factors contained in the reaction system on the measurement, and further, for example, the solid phase By using a condition in which the amount of the antibody is proportional to the signal intensity in the enzyme antibody method, the amount of the unreacted antibody can be accurately quantified.

That is, the present invention provides a method for quantifying an antigen-antibody reaction, comprising the steps of: reacting a known amount of an antibody with an antigen in a solution; diluting the solution to stop the antigen-antibody reaction; It is an object of the present invention to provide a quantification method including a step of minimizing an influence of a factor on a measurement and a step of quantifying an antibody which has not reacted with an antigen by a solid-phase antibody method.

Examples of combinations of antigens and antibodies used in the method of the present invention include recA protein antigen, ARM321 antibody, ARM4
Antibodies selected from the group consisting of 14 antibodies, ARM191 antibody and ARM193 antibody; recA430 antigen which is a mutant protein of recA protein; and antibodies selected from the group consisting of ARM321 antibody, ARM414 antibody, ARM191 antibody and ARM193 antibody.

The recA protein, ARM321 antibody, ARM414 antibody, ARM191 antibody and ARM193 antibody were obtained from The Journal of Biological Ch
emistry, 1985, 260 , pp15402-15405, recA430 is the
Journal of Biological Chemistry, 1989, 264 , pp21167−
21176. Among them, recA protein antigen, anti-RecA monoclonal antibody ARM321, anti-RecA monoclonal antibody ARM414, and anti-RecA monoclonal antibody ARM1
It is preferable to use an antibody selected from the group consisting of 91.

The solution for reacting the antigen and the antibody can be any solution, and any solution containing a factor that changes antigenicity can be selected. For example, when the recA protein is used as an antigen, the antigen-antibody reaction can be quantified in a solution containing ATP or DNA that may affect the antigenicity of the recA protein. In addition, any solution containing factors that change the pH, ionic strength, and the like can be selected.

As the reaction conditions, the reaction temperature can be arbitrarily selected, and the reaction time can be selected arbitrarily until the reaction between the antigen and the antibody reaches saturation.

After the antigen-antibody reaction, it is preferable to dilute the reaction solution using a diluent that does not dissociate the antigen-antibody bond.
Examples of such a diluent include PBS. PBS
Is a physiological saline solution containing a phosphate buffer. The dilution step is carried out so that the antigen-antibody reaction is effectively stopped, and the substance other than the antibody is below the detection limit of the solid phase antibody method. For example, when using an antibody selected from the group consisting of recA protein, ARM321 antibody, ARM414 antibody, and ARM191 antibody, it is preferable to dilute about 1500 times with PBS-Tween. Here, PBS-Tween refers to a solution in which 0.05% Tween is dissolved in PBS. Further, the dilution step is preferably performed at room temperature (about 25 ° C.) within 5 minutes.

Examples of the solid-phase antibody method for measuring the amount of the antibody in the reaction solution diluted as described above include an enzyme antibody method, a fluorescent antibody method, and a radioimmunoassay. Among them, the enzyme antibody method is preferred. Specifically, the antigen is immobilized in the wells of the microtiter plate, a solution obtained by diluting the reaction solution subjected to the antigen-antibody reaction is added to the well, and the unreacted antibody is allowed to react with the antigen immobilized in the well. The washed reaction solution is washed away, and a secondary antibody labeled with an enzyme is added to the well to react with the antibody reacted with the antigen immobilized in the well.Then, a substance serving as a substrate for the enzyme is added to the well, and the enzyme reaction is performed. The antigen-antibody complex can be quantified by the colorimetry of the substance. In the solid-phase antibody method, an antibody that has not reacted with an antigen is quantified. By subtracting this value from the amount of the antibody before the antigen-antibody reaction, the antigen-antibody reaction can be quantified.

〔The invention's effect〕

According to the method of the present invention, the antigen-antibody reaction in a solution state can be very easily and accurately quantified.

Furthermore, it has become possible to know changes in antigen-antibody reactions due to amino acid mutations and chemical modifications in proteins containing antigens. The quantification of the present invention is possible even if the antigenicity changes due to this amino acid mutation or chemical modification.

Hereinafter, examples will be given as embodiments of the present invention, but these are for explanation of the present invention and do not limit the scope of the present invention.

〔Example〕

The following examples were performed at room temperature (25 ° C.) unless otherwise specified.

The recA protein and DNA φ × 174 used in this example
Is described in J. Biol. Chem, 1981, 256 , p7557-764, ARM321 antibody, ARM414 antibody, ARM191 antibody, and ARM193 antibody are J. Bi
ol.Chem, 1985, 260 , PP15402-15404, recA430 is J. Bi
ol. Chem, 1989, 264 , pp. 21167-21176.

[Reference Example 1] Preparation of stationary phase 50 μl per well of a microtiter plate (96 wells)
Of recA protein (2 μg / ml) and left at 4 ° C. for 15 hours. Further, 200 μl of PBS (50 mM phosphate buffer, pH 7.
(2, 150 mM NaCl) three times, and the well walls were covered with recA protein and fixed. 100 μl for further blocking
5% calf serum albumin solution (1%)
After standing for about an hour, the wells were washed with 130 μl of PBS.

[Reference Example 2] Preparation of calibration curve In a stationary phase well prepared in Reference Example 2, PBS-Tween 20 (0.05% Tween 20 (manufactured by Bio-Rad) was added to PBS.
50 μl of an anti-recA protein antibody ARM321 antibody having a concentration of 0.025 nM dissolved in the above solution was added and left for 1 hour. The wells were washed three times with 130 μl of PBS-Tween. Next, 50 μl of an anti-mouse IgG antibody (manufactured by KPL) derived from a goat diluted with 500 labeled with peroxidase was added to the wells, left for 1 hour, washed three times with 130 μl of PBS-Tween, and then washed with 200 μl of PBS. Was. There, 100μ
l of o-phenylenediamine (1 mg / ml), 0.05 M citric acid, 0.05 M sodium citrate and 0.015% hydrogen peroxide solution were added and left for 30 minutes to perform a color reaction. The reaction was stopped by adding 32 μl of 4.5 M sulfuric acid. The color reaction was measured by measuring the absorbance at 490 nm. Perform the above operation on ARM321
Antibody concentrations of 0.038 nM, 0.05 nM, 0.063 nM, 0.094 nM, 0.1
25nM, 0.156nM, 0.188nM, 0.219nM, 0.250nM, 0.281n
A calibration curve was prepared by changing the M and 0.313 nM and repeating.

The above operation was repeated using the ARM414 antibody, the ARM191 antibody, and the ARM193 antibody.

The results are shown in FIG. From these results, the intensity of the signal (absorbance at 490 nm) of the enzyme antibody method is proportional to the amount of anti-recA protein antibody. Unreacted anti-recA protein antibody can be accurately quantified up to 0.2 nM by the enzyme antibody method. We can see that we can do it. Therefore, in this embodiment, after the dilution operation,
Unreacted antibody concentration was adjusted to 0.13 nM.

[Reference Example 3] Concentration of anti-rec protein antibody (IgG) and signal intensity of enzyme antibody method 21 μl of reaction solution A (31 mM Tris buffer, pH 7.5, 13 mM)
Magnesium chloride, 2.8 mM dithiothreitol, 88 μg / m
l In calf serum albumin, 2% glycerin)
ARM32, one of the M anti-recA protein monoclonal IgG
One antibody was allowed to react with recA protein, which is an antigen at a concentration of 0.2 μM, 0.5 μM, 1 μM, and 2 μM, at 37 ° C. for 10 minutes. 4 μl of the reaction solution was taken out, and the antigen-antibody reaction was stopped by adding 1,500-fold volume (6 ml) of PBS-Tween. After dilution at room temperature, within 50 minutes after dilution, 50 μl of the diluted solution was already transferred to the wall of the well. Was placed in the well of the microtiter plate of Reference Example 1 fixed in Example 1 and left for 1 hour. The wells were washed three times with 130 μl of PBS-Tween. Next, 50μ
l of peroxidase-labeled goat-derived anti-mouse IgG antibody (manufactured by KPL) derived from goat diluted 1/500, allowed to stand for 1 hour, then three times with 130 µl of PBS-Tween, and then 200 µl of P-Tween.
Washed with BS. 100 μl of o-phenylenediamine (1 mg / ml), 0.05 M citric acid, 0.05 M sodium citrate, and 0.015% hydrogen peroxide solution were added thereto, and the mixture was allowed to stand for 30 minutes to develop color. Then, 32 μl of 4.5 M sulfuric acid was added. The reaction was stopped. 49
The absorbance at 0 nm was measured, and the amount of unreacted antibody was determined from the absorbance using the calibration curve of Reference Example 2.

The above operation was repeated for the ARM414 antibody and the ARM191 antibody.

The results are shown in FIG. For the above antibodies, it is shown that the amount of unreacted antibody decreases as the concentration of recA protein increases. About 0.5 μM of the recA protein eliminated unreacted antibodies.

[Reference Example 4] Stability of antigen-antibody complex against 1,500-fold dilution 0.2 μM antibody (ARM414 antibody, ARM321 antibody, ARM191 antibody, ARM193 antibody) and 1 μM recA protein were used in Reference Example 3
After the reaction was performed under the same conditions as described above, the mixture was diluted with a 1,500-fold volume of PBS-Tween, and the amount of the antibody not bound to the antigen was quantified by an enzyme-linked immunosorbent assay by changing the time of standing at room temperature.

The results are shown in FIG. In FIG. 3, ● represents a complete system containing an antigen and an antibody; Δ represents a control excluding the antibody; and ○ represents a control excluding the antigen (recA protein). From these results, ARM414 antibody, ARM321 antibody, ARM191
As for the antibody, the complex was stably present for 60 minutes after dilution, but it can be seen that all of the ARM193 antibody gave a signal immediately after dilution. This is because the ARM193 antibody and recA
Binding to the protein did not withstand this dilution, indicating that the method of the invention cannot be used with this antibody.

[Reference Example 5] Effect of stopping treatment by 1,500-fold dilution of antigen-antibody reaction 4 μl of 1 μM recA protein was added to 6 ml of PBS-Tween.
In addition, immediately after, 4 μl of a 0.2 μM antibody (ARM414 antibody, ARM321 antibody, ARM191 antibody) solution was added, and the time at which the solution was allowed to stand at room temperature was changed, and the amount of the antibody that did not bind to the antigen was quantified by the enzyme antibody method.

The results are shown in FIG. In FIG. 4, ● represents a complete system containing an antigen and an antibody; Δ represents a control excluding the antibody; and ○ represents a control excluding the antigen (recA protein). This result indicates that, after moderate dilution, the antigen-antibody reaction does not occur or occurs very slowly even if the antigen and antibody coexist.
Therefore, it is understood that the antigen-antibody reaction can be effectively stopped by this dilution.

[Example 1] In the reaction solution A of Reference Example 3, 1 μM of the recA protein and 0.2
After reacting with μM ARM321 antibody in the presence of ATP (Yamasa Shoyu Co., Ltd.) concentrations of 0, 0.1, 0.3, 1.3, and 2.6 mM at 37 ° C. for a different reaction time, unreacted by the enzyme antibody method. Was measured.

The results are shown in FIG. ATP concentration: 0 mM, ● 0.1 mM,
◆ 0.3mM, ▲ 1.3mM, ▼ 2.6mM. ○ indicates a control excluding recA protein (in the absence of ATP), and ▽ indicates rec
A control excluding A protein (ATP 2.6 mM) is shown. From these results, recA protein is converted to A by the presence of ATP.
It turns out that it loses the antigenicity with respect to RM321 antibody.

Example 2 In the reaction solution A of Reference Example 3, 1 μM of the recA protein and 0.2
μM ARM414 antibody was used in the presence of ATPγS (manufactured by Boehringer Mannheim) (1.3 mM) and single-stranded DNA φ × 174 (50 μM), in the presence of ATPγS (1.3 mM), and single-stranded DNA φ174 (15 mM).
(0 μM) at 37 ° C. for various reaction times, and the amount of unreacted antibody was measured by the enzyme antibody method.

The results are shown in FIG. ▼ indicates ATPγS and single-stranded DNA φ × 17
4, in the presence of ATPγS, ●: single-stranded DNA φ × 174
Indicates the presence of 〇 indicates a control containing only the recA protein (in the absence of ATP and DNA), and ☆ indicates a control containing only the antibody excluding the recA protein (in the absence of ATP and DNA).

These results show that the presence of ATPγS and single-stranded DNA φ × 174 causes recA protein to lose antigenicity to ARM414 antibody.

Example 3 The method of Example 2 was repeated using recA430 as an antigen and ARM414 as an antibody.

[Brief description of the drawings]

FIG. 1 is a calibration curve of an antibody against the recA protein antigen. FIG. 1A is a calibration curve of ARM321, and FIG. 1B is
FIG. 1C is a calibration curve of ARM191, and FIG. 1D is a calibration curve of ARM193. FIG. 2 shows the absorbance at 490 nm when the amount of recA protein antigen was changed for a given amount of antibody. FIG. 2A shows
FIG. 2B shows the case where ARM321 was used for the antibody, and FIG. 2C shows the case where ARM191 was used for the antibody. FIG. 3 shows the stability of the recA protein antigen / antibody complex to a 1,500-fold dilution. FIG. 3A shows ARM321 for the antibody, FIG. 3B shows ARM414 for the antibody, and FIG.
Fig. 3 shows a case where ARM191 was used for the antibody, and Fig. 3D shows a case where ARM193 was used for the antibody. FIG. 4 shows the effect of stopping treatment of the recA protein antigen-antibody reaction by a 1,500-fold dilution. FIG. 4A shows
FIG. 4B shows the case where ARM321 was used for the antibody, and FIG. 4C shows the case where ARM191 was used for the antibody. FIG. 5 quantifies the change in the antigenicity of the recA protein by ATP. FIG. 6 quantifies the change in the antigenicity of the recA protein by ATPγS and single-stranded DNA φ × 174.

Claims (6)

(57) [Claims] 1. A method for quantifying an antigen-antibody reaction, comprising: reacting a known amount of an antibody with an antigen in a solution; diluting the solution to stop the antigen-antibody reaction; A method for minimizing the effect of the enzyme on the measurement, and a method for quantifying an antibody that has not reacted with the antigen by a solid-phase antibody method. 2. The method according to claim 1, wherein the antigen-antibody reaction in a solution containing a factor that changes antigenicity is determined. 3. The method according to claim 1, wherein the antibody is a monoclonal antibody.
The described method. 4. The method according to claim 2, wherein the antibody is IgG. 5. The method according to claim 1, wherein the antigen is a polymer. 6. The method according to claim 1, wherein the antigen is a recA protein or a mutant protein, the antibody is selected from the group consisting of anti-RecA monoclonal antibody ARM321, anti-RecA monoclonal antibody ARM414, and anti-RecA monoclonal antibody ARM191, and the diluent is PBS-Tween. Item 7. The method according to Item 1.