JP2516793B2 - Non-specific adsorption inhibitor and non-specific adsorption prevention method - Google Patents

Description

TECHNICAL FIELD The present invention relates to a non-specific adsorption inhibitor and a non-specific adsorption prevention method used for immunological measurement in the field of medicine and the like.

(Prior art) In recent years, immunological techniques such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and western blotting have been used in clinical examination, immunology, biochemistry, molecular biology, and other fields. Became heavily used.

In these measurements, the target substances are immobilized on a solid phase such as a plate or nitrocellulose membrane, and then an isotope or enzyme-labeled antibody (probe) that specifically binds to these substances is added to determine the radiation level and enzyme activity. By measuring, the target substance is fixed or quantified. At this time, it is important that the probe reacts only with the target substance, and it should not be nonspecifically adsorbed on the solid phase.

Therefore, after immobilizing the target substance on the solid phase, a blocking agent (non-specific adsorption inhibitor) must be added to block the adsorption on the solid phase. For example, in ELISA, a blocking agent is added after immobilizing an antigen on a plate, and then a probe is added. The blocking agent functions to prevent antibodies that are not specific for the antigen from adsorbing to the plate, or prevent the probe from adsorbing non-specifically to the plate.

Usually, bovine serum albumin (BS
A) Solution is used. Generally, BSA must be dissolved in a concentration of 1% or more, and in order to completely prevent non-specific adsorption, it must be dissolved and used in a concentration of 2% or more, preferably about 5%. Although BSA is a relatively available protein,
This is one of the factors that raises the analysis cost due to the time required for dissolution and the high price.

However, logically, any substance that does not interact with the target substance or probe and that adsorbs well to the solid phase can be used as a blocking agent. The development of was desired.

In 1984, Johnson et al. Reported that skim milk powder (SMP) was effective instead of BSA in the Southern blotting method, and that 5% SMP solution also effectively blocked in ELISA (Gene Analytical). -Technology; Gene Anal.Technol., 1 , 3-8, 1984).
Also, Miskimins et al. Have shown that a 5% SMP solution can be an effective blocking agent (Proceeding of National Academy of Sciences: Proc.
Natl. Acad. Sci., 82 , 6741-6744, 1985), and Ohta also described the effectiveness of SMP in an introduction of the cloning method using the expression vector λgt11 (Cell engineering, 5 , 264-27).
0,1986).

(Problems to be Solved by the Invention) In these examples, SMP is dissolved in Tris buffer and used as a blocking agent, but if it is left as it is, cloudiness will be severe and the commercial value as a research reagent will be significantly impaired. Also,
When mass-produced and distributed as a product, it is far more advantageous in terms of cost to distribute the product at room temperature than to distribute it at low temperature. The SMP solution must be sterilized in an autoclave or the like for circulation at room temperature, but if SMP is simply dissolved in water or Tris or a phosphate buffer,
It has been found that heat causes a marked browning, an increase in turbidity, or even the formation of a precipitate, which reduces the commercial value.

INDUSTRIAL APPLICABILITY The present invention has a transparent feeling and does not turn brown even when sterilized.
Moreover, it is an object of the present invention to provide a blocking agent having an excellent blocking effect.

(Means for Solving the Problems) The present invention relates to a non-specific adsorption inhibitor that is inexpensive and can be stored for a long period at room temperature in an immunological assay, and uses milk protein as an active ingredient, It is characterized by being combined with a buffer solution containing at least one selected from organic acids and organic acid salts as a main component.

Milk protein used in the present invention is one of casein, whey protein concentrate (WPC), skim milk powder, or skim milk,
Alternatively, two or more kinds can be used in combination. When WPC is blended, it is desirable that the content be 50% or less in the milk-solid content. If the content is 50% or more, precipitation may occur after the heat treatment. In addition, these milk proteins may be used after being limitedly degraded with a protease, but if the degree of decomposition is too high, it becomes unstable at the time of sterilization. Therefore, it should be limited to partial hydrolysis.

The buffer solution used in the present invention contains, as a main component, at least one selected from organic acids and organic acid salts such as citric acid, maleic acid, malic acid, succinic acid, and malonic acid.

Preferably, the pH of the buffer is between 5.3 and 7.0. Adjustment of the pH is, for example, an alkaline solution, such as sodium hydroxide,
It can be carried out with potassium hydroxide or the like, an acid solution such as hydrochloric acid, sulfuric acid, acetic acid or the like, or one or a combination of the above organic acids and their bases. When the pH is 5.3 or less, the active ingredient is difficult to dissolve, so the pH is adjusted to 5.3 or more, particularly preferably 5.5 or more.
The upper limit of pH is preferably 7.0, and particularly preferably 6.5 or less. When the pH exceeds 7, not only the buffering ability of the above-mentioned organic acid is lost, but also browning becomes severe when sterilized.

The ratio of milk solid content concentration (%) / organic acid concentration (mM) is preferably 0.1 or less, and particularly preferably 0.02 or less. At 0.1 or more, for example 0.2, there is a transparent feeling after sterilization, but browning cannot be prevented, and at 2.0, for example, the transparent feeling disappears.

After preparing the active ingredient under the conditions as described above, it can be sterilized. Sterilization conditions may be normal autoclave conditions, such as 121 ° C. for 10 minutes, and conditions used during normal long-life beverage production, such as 140 to 150.
C. and 2-4 seconds are sufficient.

Furthermore, the method for preventing non-specific adsorption of the present invention comprises milk protein as an active ingredient and is selected from organic acids and organic acid salts.
It is characterized by using a solution consisting of a buffer solution containing at least one species as a main component.

(Effects of the Invention) According to the present invention, it is possible to provide a non-specific adsorption inhibitor that has a transparent feeling, is inexpensive, and does not brown even when sterilized, in place of the BSA that has been conventionally used. . In addition, an extremely excellent blocking effect can be obtained in the immunological measurement. Furthermore, it has the advantage that it can be distributed at room temperature and is easy to handle.

(Example) Example 1 Nonspecific Adsorption of Enzyme-Labeled Antibody to 96-Well ELISA Plate Each well of a 96-well ELISA plate (manufactured by Sumitomo Bakelite Co., Ltd.) was dispensed with 400μ of nonspecific adsorption inhibitor, Blocking was performed by leaving it at room temperature for 1 hour. Then
Peroxidase-labeled anti-human IgG (manufactured by Tago) was diluted 1,000-fold with a non-specific adsorption inhibitor, and 50μ was added to each well.
We dispensed each. After incubating at room temperature for 1 hour, the plate was washed with phosphate buffer (PBS-Tween) containing 0.05% Tween 20 for 6 days.
After washing twice, ABTS (2,2'-azinobis- (3-ethylbenzothioazoline-6-sulfonic acid)) was added as a substrate. The absorbance at 405 nm after 10 minutes was measured by Titer Tech Multiscan (manufactured by Flow Co.). Both are 3
Conducted in a row.

As a non-specific adsorption inhibitor, dissolve 2% BSA in PBS,
Diluted with deionized water to the specified concentration and 0.1M citric acid after adjusting the pH to 6.2 with sodium hydroxide, then SMP 1%
The solution was dissolved so that it became so that it was treated with an autoclave at 121 ° C. for 10 minutes, and diluted with deionized water to a predetermined concentration.
The results are shown in Table 1.

Very high nonspecific adsorption was observed without using the nonspecific adsorption inhibitor, but with BSA, nonspecific adsorption could be suppressed within the measurement error range at a concentration of 1.0% or more.

On the other hand, with SMP, SMP 0.0625%, citric acid 6.25 mM
Even when it was diluted to 1, non-specific adsorption could be completely suppressed.

Example 2 Implementation of ELISA 96-well E of anti-human κ and anti-human λ IgG (manufactured by Tago) mixed solution 50 μ diluted 1,000 times with 0.05 M sodium hydrogen carbonate
Dispense into a LISA plate (Sumitomo Bakelite Co., Ltd.) 4
It was left still overnight at ℃. The next morning, the antibody solution was discarded, each well was filled with a nonspecific adsorption inhibitor, and left for 1 hour. PBS-Twe
After washing twice with en, 50 μm of human serum IgG (Midori Cross) at 0 to 1000 ng / ml was added and left still for 1 hour. After washing 6 times, ABTS was added and the absorbance at 405 nm was measured.

As a non-specific adsorption inhibitor, 2% BSA / PBS and 1% SMP / 0.1M citric acid-Na (pH 6.2) shown in Example 1 were treated with an autoclave and then diluted 10 times with deionized water. I used it.

The results are shown in Fig. 1, but no difference was observed between the two.

Example 3 Browning degree after sterilization After dissolving SMP in various buffer solutions (pH 6.1) so as to be 1%, the mixture was treated in an autoclave at 121 ° C. for 10 minutes, and the degree of turbidity, presence of precipitate, and browning The degree was judged visually.

The results, as shown in Table 2, were transparent, did not cause precipitation, and did not cause browning when citric acid, maleic acid, succinic acid, malic acid, or malonic acid was used.

Example 4 Types of milk proteins and blocking effect The following were prepared as non-specific adsorption inhibitors.

1) 1% BSA / PBS (pH7.2) 2) 1% nonfat dry milk / 50 mM citric acid + 50 mM maleic acid (pH
6.3) 3) 0.5% casein / 100 mM maleic acid (pH 6.1) 4) 0.7% casein + 0.3% WPC / 100 mM citric acid (pH 6.2) 5) 0.5% skim milk powder + 0.5% casein / 50 mM maleic acid +5
Regarding 0 mM malonic acid (pH 6.1) 2) to 5), it was indirectly sterilized at 140 ° C. for 2 seconds.

The test of the blocking effect was carried out in the same manner as in Example 1, except that the peroxidase-labeled anti-human IgG was replaced by
Peroxidase-labeled anti-mouse IgG (manufactured by Tago) was used. Further, the absorbance at 405 nm was measured after adding ABTS and leaving it at room temperature for 15 minutes. The results are shown in Table 3.

With milk protein, the background was low and the blocking effect was superior to that of BSA. In milk protein
4) containing WPC was a little bad, but it does not pose any problem in practical use.

[Brief description of drawings]

FIG. 1 shows the case where 2% BSA / PBS was used as the non-specific adsorption inhibitor according to Example 2 of the present invention (FIG. A), and 1% S.
It is a graph which shows the standard curve by ELISA of human IgG when MP / 0.1M citrate-Na (pH6.2) is used (FIG. B).

Claims (5)

(57) [Claims] 1. Non-specific adsorption prevention in an immunological assay, which comprises a milk protein as an active ingredient and a buffer solution containing at least one selected from organic acids and organic acid salts as a main component. Agent. 2. When producing a sterilizing and / or sterilizing non-specific adsorption inhibitor containing milk protein as an active ingredient, it is combined with a buffer solution containing at least one selected from organic acids and organic acid salts. A method for producing a non-specific adsorption inhibitor in an immunological assay, which comprises: 3. The method according to claim 2, wherein the pH of the buffer solution is 5.3 to 7.0. 4. The method according to claim 2, wherein the ratio of milk solid content concentration (%) / organic acid concentration (mM) is 0.1 or less. 5. A non-specific method for immunological measurement, which comprises using a solution consisting of milk protein as an active ingredient and a buffer solution containing at least one selected from organic acids and organic acid salts as main components. Adsorption prevention method.