JPS5816146B2 - In-gel sedimentation reaction measurement method - Google Patents

Description

[Detailed description of the invention] The binding of antigen and antibody takes place in an extremely short time.

This conjugate has the most suitable concentration ratio of antigen and antibody.
When in this state, they aggregate to form sediment.

Antigens and antibodies are diffused into a gel such as agar gel that allows protein molecules to freely pass through, and the precipitate (line) that appears at the position of the "optimal ratio" is observed, and the antigen or antibody is measured and analyzed. This is commonly used as an immunological method.

Examples include the immunoelectrophoresis method, the offtelony method, and the one-way immunodiffusion method.

Of these, the offtelony method is special. It has advantages such as no equipment required, simple operation, short reaction time, and ability to identify the antigen (or antibody) from the shape of the sedimentation line.

However, when analyzing an unknown concentration of antigen (or antibody), this method dilutes the antigen solution (or antibody solution) in several stages in order to prevent negative coagulation caused by too much or too little antigen (or antibody). It takes a lot of effort to carry out all the tasks at the same time.
Alternatively, they have drawbacks such as the inability to quantify.

The present invention is an in-gel precipitation reaction method that is characterized by compensating for the above two drawbacks without losing the advantages of the offtelony method.

The most commonly practiced offtelony method involves drilling holes in a gel layer centered at the center of a regular hexagon and at each of the six vertices, as shown in Figure 1.

Next, antibodies are injected into hole A in the center of these hexagons, and test antigens or standard antigens containing known antigens are injected into the six surrounding holes, and they are reacted for a certain period of time under constant humidity conditions to perform measurement and analysis. It is.

Therefore, this method uses the central hole (antibody-receiving hole) and the surrounding holes (
The distance between the antigen-receiving pores is constant.

The offtelony method is carried out by arranging each pore in a regular hexagonal shape and changing the pore arrangement as appropriate depending on the purpose of the analysis, but in either case, the distance between the antigen-receptive pore and the antibody-receptor pore is constant. there were.

However, the present invention is characterized in that the antigen-antibody receptor holes are regularly arranged at different intervals.

For example, as shown in Fig. 2, a central hole A and holes S and -S are made at different intervals around the center hole A in a gel layer such as agar, and the antibody solution is poured into the center hole A (antibody receiving hole) in the surrounding holes S+-. 8a(
Inject the test antigen solution into the antigen receiving hole).

After placing this gel layer in a humidity chamber and allowing it to react for a certain period of time,
Observe which antigen-receiving pore the appearance of the sedimentation line between the antigen-receiving pore and the antibody-receiving pore reaches, and use that position as a quantitative value.

Therefore, the principle of the present invention is to adjust the distance that the antigen (or antibody) diffuses within the gel, and measure the concentration of the antigen (or antibody) based on whether a sedimentation line appears after a certain time within a given distance. become.

This is different in principle from the method of measuring the position where a sedimentation line occurs, as seen in conventional quantitative in-gel precipitation reaction methods such as quantitative immunoelectrophoresis and immunodiffusion.

In the present invention, the most quantitative combination of the spacing between the antigen-receiving hole and the antibody-receiving hole can be determined experimentally.

For example, as shown in Figure 2, the distance from the edge of the antigen receptor hole to the edge of the antibody receptor hole is 2.0 mm, 3.2 mm, and 4.2 mm.
The purpose of clinical diagnosis can be achieved by measuring α-fetoprotein in serum using an antiserum against this protein using agar gel layers set at 5.0 mm and 5.6 mm. Ta.

The results are shown in FIG.

In addition, the present invention allows antigen (
or antibodies).

When measuring a high concentration of antigen (or antibody) in a test liquid using the present invention, it is possible to prevent false negatives due to excess antigen to some extent.

For example, as shown in Table 1, using the conventional offtelony method (see Figure 1) and the present invention, α in patient serum can be detected.
- When comparing the measurement of fetoprotein, in the offtelony method, the sedimentation line already disappears at a concentration of α-fetoprotein of 75 μg/− or higher, indicating a false negative state.

However, according to the present invention, the concentration of α-fetoprotein is 1
The appearance of false negatives can be prevented until reaching 50 μg/−.

In addition, for the measurement of C-reactive protein in patient serum using the present invention, a layered comparison of the measurement sensitivity with the conventional method and the one-way immunodiffusion method is shown in Table 2. It was also demonstrated that there was no difference at all.

Example 1 A 1.2% agar gel layer (height 1.5 mm) was prepared on a slide glass, holes with a diameter of 3rn7rt were punched as shown in Fig. 3, and anti-α-fetoprotein serum was poured into the antibody receiving hole A. Inject 5-7 μl of α-fetoprotein into antigen-receiving hole st, and add standard serum containing 60 μg/− of α-fetoprotein into antigen-receiving hole st.
5 to 7 μl of the same patient serum was injected into each, and the sedimentation line that appeared after 4 hours of reaction at room temperature in a humidity chamber merged with the standard serum sedimentation line (single sedimentation line), and further 81 to S4. For example, if a sedimentation line appears up to S3, a measurement value of 3+ is obtained.

Example 2 A 142% agar gel layer (height 1.5 mm) was prepared on a glass slide, holes were made as shown in Fig. 4, and anti-C-reactive protein serum was injected into the antibody receiving groove A. o, i ml
5 to 7 μl of the same patient serum was injected into each of the antigen receiving holes 81 to S5, and after reacting for 4 hours at room temperature in a humidity chamber, measurements were performed in the same manner as in Example 1.

It was measured by the method of Example 1.

The measurement value of one-way immunodispersibility was determined by the method described in Example 2.

[Brief explanation of drawings]

FIG. 1 is an explanatory diagram showing the conventional implementation method, FIGS. 2 and 3 are explanatory diagrams showing the position of the antibody receptor hole relative to the antigen receptor hole in the present invention, and FIG. 4 is an explanatory diagram showing the position of the antibody receptor hole relative to the antigen receptor hole in the present invention. FIG. 5 is an explanatory diagram showing the positions of antibodies, and FIG. 5 is an explanatory diagram showing the relationship between the measured values of α-fetoprotein in serum by the method of the present invention and the measured values by the one-way immunodiffusion method.

Claims (1)

[Claims] 1. The antigen-receiving holes and antibody-receiving holes are drilled in a gel layer that allows protein molecules to freely diffuse, and the intervals between the antigen-receiving holes and the antibody-receiving holes are arranged regularly so that each hole is different, and an antigen solution or an antibody solution is injected into each hole. , a method for measuring sedimentation reaction in a gel, which is characterized by observing a sedimentation line that occurs after a certain period of reaction.