JPH04323559A - Measuring method for immunity - Google Patents

Abstract

PURPOSE:To make effective use of magnetic particulates so as to enhance the efficiency of reflection by disposing a magnet in such a way as being movable along the direction of the depth of a reaction container proportionately to the amount of a reacted liquid in the container. CONSTITUTION:A sample and at least a liquid of antibody-solidifying magnetic particulates are separately infused into a reaction container 10 and the particulates reacted with an antibody in the sample are attracted by a magnet 11 and any unnecessary liquid containing an antigen which is not reacted with the particulates is removed. In that case, the depth of the liquid is different during the reaction from during B/F separation. The magnet 11 is disposed in such a way as being movable along the pipe wall of the container 10 proportionately to the amount of the reacted liquid in the container 10 and thereby the magnet 11 can be disposed in its optimum position and the magnetic particulates can be effectively used and the efficiency of reflection can be enhanced.

Description

[Detailed description of the invention]

[Purpose of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immunoassay method for measuring the amount of antigen in a sample using magnetic fine particles.

0002

2. Description of the Related Art A conventional RIA (radioimmunoassay) method using a radioactive element is used for quantitative analysis of the amount of a specific antigen in a sample (specimen). However, since this RIA method uses radioactive elements, it requires the installation of special equipment and the operation by a qualified operator, and is troublesome, such as requiring careful disposal of waste.

[0003] For this reason, instead of the RIA method, an EIA (enzyme immunoassay) method, which performs analysis using an enzymatic reaction, has been used. Recently, an analysis method using antibody-immobilized fine particles such as a magnetic material has become popular for the purpose of speeding up the EIA method.

FIG. 13 explains the principle of such an EIA method. First, a solution of magnetic particles (hereinafter referred to as MP) 1 on which a first antibody (first reagent) 2 is immobilized is prepared. By dispensing a specimen (sample) 4 containing the antigen 3 to be measured into this, a first antigen-antibody reaction occurs, and a portion of the antigen 3 is bound to the first antibody 2. The unbound antigen 3' exists in a free state. Therefore, by adsorbing MP1 using a magnet, free antigen 3' is removed in an aggregated state, and so-called B/F separation is performed. Next, a second antigen-antibody reaction occurs by dispensing the second antibody (second reagent) 6 labeled with the enzyme 5, and the antigen 3 to be measured binds to MP1 and a part of the enzyme-labeled antibody 6. It is then made into a sandwich. The unbound enzyme-labeled antibody 6' exists in a free state. Therefore, similar to the above, B/F separation is performed using a magnet to remove free enzyme-labeled antibody 6'.

Next, by dispensing the substrate solution (third reagent) 7 into this, a third reaction, so-called enzyme reaction, occurs and a reaction product 8 is produced. Since the enzyme 5 is bound to the antigen 3, the amount of the antigen 3 proportional to the amount of the enzyme 5 can be measured by photometrically measuring the third reaction state by spectrophotometry.

FIG. 12 illustrates an immunoassay method based on such a principle. First, a reaction vessel 10, for example a rectangular glass vessel, is prepared and a sample 4 is dispensed into it. The first reaction then occurs by dispensing the MP1 solution therein. Note that sample 4 and MP1 solution may be dispensed in any order. Antigen 3 in sample 4 by the first reaction
Since a part of the antigen 3' binds to the first antibody 2, a magnet 11 is used to prevent the antigen 3' remaining in a free state from influencing the next reaction.
In a state in which MP1 is adsorbed and aggregated by bringing it close to the antigen 3', unnecessary liquid containing free antigen 3' is removed by suction with a nozzle to perform B/F separation.

Next, an excess cleaning solution (generally a buffer solution) is discharged into the reaction container 10, and after stirring and dispersing the solution, the magnet 11
is used again to perform B/F separation, and the remaining unreacted (excess) antigen 3' is removed by suction. Next, the second antibody (
After dispensing the second reagent) 6, the washing liquid is discharged and stirred. Hereinafter, such a reaction is repeated according to the principle shown in FIG.

[0008] In such an immunoassay method using the EIA method, during the reaction, excess antigen or enzyme-labeled antibody contained in the reaction solution is removed by B/B so as not to affect the next reaction.
It is important to remove it by F separation. Generally E.I.
In case A, the amount of reaction liquid prepared in the reaction vessel 10 is 50 to 200 μl, whereas the amount of washing liquid used for B/F separation is prepared to be twice to several times the amount of reaction liquid. is normal.

[0009]

[Problems to be Solved by the Invention] However, in conventional immunoassay methods, it is difficult to effectively utilize magnetic fine particles, so there is a problem of poor reaction efficiency. This is because the depth of the liquid is different during the reaction and during the B/F separation, so it is difficult to arrange the magnet at the optimum position. For example, if the magnet is placed on the side with a smaller amount of reaction liquid, the magnetic particles will not aggregate properly during washing, whereas if the magnet is placed on the side with a larger amount of reaction liquid, the magnetic particles will be collected during the next reaction after B/F separation. will be insufficiently dispersed. This is because, as described above, since the amount of reaction liquid (particularly the amount of reagent liquid) is smaller than the amount of washing liquid, magnetic fine particles remain on the wall surface of the reaction container. In either case, it becomes difficult to utilize magnetic fine particles effectively. As a result, reaction efficiency deteriorates, which tends to result in defective measurement data.

The present invention was made in response to the above-mentioned problems, and it is an object of the present invention to provide an immunoassay method that effectively utilizes magnetic fine particles to improve reaction efficiency. be.

[Configuration of the invention]

0012

[Means for Solving the Problems] In order to achieve the above object, the present invention dispenses a sample and at least an antibody-immobilized magnetic particle liquid into a reaction container, and the antibody-immobilized magnetic particles reacted with the antigen in the sample. In an immunoassay method in which an unnecessary liquid containing an unreacted antigen is removed while fine particles are adsorbed by a magnet, the magnet can be moved along the depth direction of the tube wall of the reaction vessel according to the amount of reaction liquid in the reaction vessel. It is characterized by being placed in.

[0013]

[Function] The magnet is arranged so that it can be moved along the depth direction of the tube wall of the reaction vessel according to the amount of reaction liquid in the reaction vessel, so when the amount of reaction liquid is small, the magnet position is moved deeper and the amount of reaction liquid is lowered. When there is a large amount of water, B/F separation can be performed by freely moving the position of the magnet so that it becomes shallower. This makes it possible to effectively utilize the magnetic particles, thereby improving reaction efficiency.

[0014]

EXAMPLES Examples of the immunoassay method of the present invention will be described below with reference to the drawings.

Example 1 As shown in FIG. 1, the reaction vessel 10 has an inner diameter of 5 mm x 6 mm,
A rectangular glass tube with a length of 40 mm was prepared and the following reaction was carried out.

First, 10μ of magnetic fine particle (hereinafter referred to as MP) suspension solution (manufactured by Advanced Magnetic Co., Ltd.) was added.
1 into the reaction container 10, then add 100 to 400 μl of 0.1M phosphate buffer (PH 6.5) and stir to homogenize. Then, as shown in FIG. 2, place the magnet 11 close to the reaction container 10. Then, MP was aggregated by adsorption. When the amount of liquid in the reaction vessel 10 was varied and the center position h in the depth direction of the magnet 11 at which MP was absorbed most quickly was measured in each case, the results shown in Table 1 below were obtained.

[0017]

[Table 1]

In each case, MP was adsorbed onto the wall of the reaction vessel 10 within about 30 seconds. As is clear from Table 1, it was confirmed that by changing the position of the magnet 11 in the depth direction according to the amount of liquid, the MP of the liquid could be efficiently adsorbed.

Example 2 Under the same conditions as Example 1, 10 μl of MP solution and 400 μl
0.1M phosphate buffer was dispensed into the reaction container 10 and mixed. Next, the magnet 11 was brought close to the reaction vessel 10 and its position in the depth direction was changed, and MP was adsorbed for about 30 seconds in each case. In this state, the absorbance of the supernatant of the mixture was set to 660n.
The measurement was performed at a wavelength of m, and the aggregation rate of MP was determined based on this result. That is, after measuring the absorbance A1 (100%) at 660 nm of the mixed liquid in the reaction container 10 as shown in FIGS.
Measure the absorbance A2 at 60 nm, (A1 - A2)
% was taken as the aggregation rate. In this case, the relationship between the magnet position and the aggregation rate was as shown in Table 2.

[0020]

[Table 2]

As is clear from Table 2, the optimum position of the magnet 11 in this case is 7 mm, where the aggregation rate is maximum. From Table 2, it was confirmed that when the magnet 11 deviates from the optimum position, the MP aggregation rate decreases and effective use of MP is hindered.

Example 3 Under the same conditions as in Example 2, 400 μl of a mixed solution of MP and phosphate buffer was prepared, and the magnet 10 was placed at a height of 7 mm from the bottom of the reaction vessel 10 to collect MP. I did it. Next, the reaction was divided into two systems, A and B, and continued.

[0023] As shown in FIG. 7, the A line was left as it was for 1 minute. For the B system, the height position is changed stepwise to 7 mm, 5 mm, 3 mm, and 1 as shown in Figures 8 to 11 every 15 seconds.
Changed to mm. After 1 minute for each system, the supernatant was removed, and 100 μl of buffer was newly added to each reaction container 10 (those in FIGS. 7 and 11). Subsequently, each mixed solution was stirred using a rotary stirrer, and then diluted 20 times with 50 μl of suspension solution, and the absorbance at 660 nm was measured. By repeating the above reaction 10 times, the results shown in Table 3 were obtained.

[0024]

[Table 3]

Note that the standard absorbance at 660 nm was 550.

As is clear from Table 3, if the position of the magnet 11 is changed over time to move the MP as in the B system, the average absorbance becomes close to the standard value, and as a result, the dispersion value and the variation rate decrease. It means being held small. As a result, when moving from cleaning for B/F separation to the next reaction step, by moving the position of the magnet 11 lower and moving the MP to the bottom of the reaction vessel 10, the MP in the liquid can be removed.
It was confirmed that the redispersion value of can be improved. Note that in system A, as described above, when the amount of reagent is small compared to the washing liquid, MP remains on the wall of the reaction vessel 10 and is not dispersed, making it impossible to obtain desired results.

As described above, according to the embodiment of the present invention, the magnet is moved along the depth direction according to the amount of reaction liquid in the reaction vessel, so that the aggregation rate of magnetic fine particles during B/F dispersion is reduced. loss can be prevented. Further, the dispersibility of the magnetic fine particles after B/F dispersion can be improved. This makes it possible to effectively utilize the magnetic particles, thereby improving reaction efficiency and improving the reliability of measurement data.

[0028] The arrangement of the magnets can be arbitrarily set; for example, a plurality of magnets may be arranged facing the reaction vessel, and each magnet may be placed at a higher or lower position depending on the reaction process. can do. Alternatively, the magnet may be moved vertically along the reaction vessel. Alternatively, by arranging multiple magnets along the reaction vessel while changing their vertical positions little by little, and moving the reaction vessel along the arranged magnets, the magnetic particles inside are guided to the desired position. It is possible to do so.

[0029]

As described above, according to the present invention, the magnets are arranged so as to be movable along the depth direction of the reaction vessel according to the amount of reaction liquid in the reaction vessel. It can be used effectively and reaction efficiency can be improved.

[Brief explanation of the drawing]

FIG. 1 is a perspective view showing a first embodiment of the immunoassay method of the present invention.

FIG. 2 is a side view showing a first embodiment of the immunoassay method of the present invention.

FIG. 3 is a top view showing a second embodiment of the invention.

FIG. 4 is a side view showing a second embodiment of the invention.

FIG. 5 is a top view showing a second embodiment of the invention.

FIG. 6 is a side view showing a second embodiment of the invention.

FIG. 7 is a side view showing a third embodiment of the present invention.

FIG. 8 is a side view showing a third embodiment of the present invention.

FIG. 9 is a side view showing a third embodiment of the present invention.

FIG. 10 is a side view showing a third embodiment of the present invention.

FIG. 11 is a side view showing a third embodiment of the present invention.

FIG. 12 is a process diagram illustrating an immunoassay method.

FIG. 13 is an explanatory diagram of the principle of EIA method.

[Explanation of symbols]

10 Reaction container 11 Magnet

Claims (3)

[Claims] 1. A sample and at least an antibody-immobilized magnetic particle liquid are dispensed into a reaction container, and the antibody-immobilized magnetic particles that have reacted with the antigen in the sample are adsorbed by a magnet, and an unnecessary liquid containing the unreacted antigen is removed. 1. An immunoassay method in which a magnet is disposed so as to be movable along the depth direction of a tube wall of the reaction vessel according to the amount of reaction liquid in the reaction vessel. 2. Further dispense an enzyme-labeled antibody solution into the reaction container, react the enzyme-labeled antibody with the antigen, and sandwich the antigen with the antibody-immobilized magnetic particles. Then, use a magnet to bind the antibody-immobilized magnetic particles. 2. The immunoassay method according to claim 1, wherein an unnecessary liquid containing the enzyme-labeled antibody that does not react in an adsorbed state is removed. 3. The immunoassay method according to claim 1, wherein the magnetic particles are moved to a target position by moving a tube wall using a magnet with the magnetic particles adsorbed thereon.