JPH03144367A - Indirect agglutination immunoassay method and device - Google Patents

Abstract

PURPOSE:To automate all processes of indirect agglutinating reaction by using magnetic particles or particles containing magnetic material as reagent to compose an immune measuring system by an indirect agglutination reaction. CONSTITUTION:This invention has a microplate supply device 1, a sample distributor 2, a reagent distributor 3, a sample/reagent agitator 4 and a forcible settlement promoting device 5 to settle magnetic particles or particles containing magnetic material by a magnetic force provided at a lower part of a microplate and subsequently, an tilt processor 6 for setting the device 5 tilted subsequently, a pattern reader 7 for reading a peeling condition of settled particle components as caused by tilting from the microplate and a microplate recovery device 8 for recovering microplates used. Then, the magnetic particles or the particles containing magnetic material are used as reagent to compose an immune measuring system by an indirect agglutinating reaction. After a reaction, components containing the particles are settled forcibly to the bottom of a container and then, the container is tilted to judge the presence of agglutinating reaction according to peeling condition of the settled particles from the container.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to an indirect agglutination immunoassay method and apparatus that utilizes antigen-antibody reactions using magnetic particles or particles containing magnetic substances.

(Prior art) As one of the immunoassay methods, the indirect agglutination reaction is a passive agglutination reaction or a reverse passive agglutination reaction in which the binding reaction caused by the antigen-antibody reaction is measured using particles to which antigens or antibodies are bound to increase the sensitivity. This reaction is called agglutination reaction and is widely used as a simple method for measuring large amounts of samples.

(Problems to be solved by the present invention) Immunoassay using indirect agglutination reaction is easier to operate than EIA (enzyme immunoassay), RIA (radioimmunoassay), etc. It has features such as not requiring any special equipment to determine the presence or absence.

However, there are drawbacks such as the need for skill to operate it and the difficulty of automation, and it is still in a state where it can only be partially automated.

In addition, as a pattern forming method for indirect agglutination, a predetermined reagent particle solution is added to a predetermined diluted sample solution in a microplate, stirred, and left to stand, and the presence or absence of an antigen-antibody reaction is determined from the sedimentation pattern of the particles (hereinafter referred to as (abbreviated as static method) and 7
A method in which particles are forcibly settled at the bottom of a letter-shaped microplate using centrifugal force, then the microplate is placed in an inclined position, and the presence or absence of an antigen-antibody reaction is determined from the separation of the particles from the bottom ( When determining the presence or absence of an antigen-antibody reaction from the sedimentation pattern by the former #% direct-standing method (hereinafter abbreviated as centrifugation method), it is important to avoid disturbance of the pattern due to vibration during standing, for example, induction of a slip phenomenon. The pattern area tends to decrease, making automation difficult. Another disadvantage of the standing method is that it takes a long time, about 0.5 to 3 hours, depending on the particles used, to complete pattern formation. In addition, with the latter centrifugation method, sedimentation is performed in a few minutes using a centrifuge, and then the microplate is tilted and the pattern can be read in a few minutes, and there is no need to worry about pattern disturbance due to vibrations during pattern formation. However, in practice, it is technically difficult to automatically perform immunoassay using a centrifuge, and these problems are the problems that the present invention aims to solve.

(Means for Solving the Problems) An object of the present invention is to provide a magnetic precipitation-enhanced indirect agglutination immunoassay method and apparatus that avoids the above-mentioned conventional drawbacks and automates the entire process of indirect agglutination reaction. It is.

The magnetic precipitation-enhanced indirect agglutination immunoassay method (hereinafter referred to as the "agglutination immunoassay method") of the present invention uses magnetic particles or particles containing a magnetic material as reagents to construct an immunoassay system based on an indirect agglutination reaction. This is an agglutination immunoassay method in which, after reaction, components containing the particles are forcibly settled at the bottom of a container by magnetic force, the container is then tilted, and the presence or absence of an immune reaction is determined from the separation of the precipitated particles from the container. In the agglutination immunoassay method of the present invention, an electromagnet or a permanent magnet may be used to forcibly precipitate magnetic particles or components containing magnetic particles using magnetic force.

The indirect agglutination immunoassay device of the present invention includes a means for receiving an immunoassay sample and an immunoassay reagent, and forcibly sedimenting a component containing a magnetic substance or particles containing a magnetic substance in the immunoassay reagent using a magnetic force; and means for placing the container in an inclined position. That is, in the present invention, the magnetic particles are forcibly settled on a sedimentation promotion table using a magnet, and are attracted to the bottom of the container, thereby achieving the same effect as if they were sedimented using a centrifugal method. This makes it possible to complete a measuring method and device that takes less than one-third of the time required by the conventional standing method and is not affected by vibrations or the like.

In the present invention, as the magnetic substance contained in the reagent, a ferromagnetic substance can be suitably used among magnetic substances in order to force the reagent to settle on the bottom surface. Further, specific examples of magnetic particles or particles containing a magnetic substance that can be used as a reagent in the present invention are as follows.

Magnetic particles, gelatin particles containing ferromagnetic material (Unexamined Japanese Patent Publication No. 5
9-195161), particles coated with a magnetic material such as serum albumin, particles similarly coated with a base polymer,
These include particles containing a magnetic material in a synthetic polymer.

Further, as containers for receiving the specimens and reagents of the invention, U-shaped or 9-shaped containers made of plastic, such as polystyrene, ABS resin, or glass, can be used. Although the size of these containers does not matter, V-shaped microplates made of polystyrene can be suitably used in order to process large amounts of specimens, be easy to handle, and obtain clear images.

As described above, the agglutination immunoassay device of the present invention includes a microplate for receiving an immunoassay sample and an immunoassay reagent, and a means for forcefully sedimenting a component of the reagent containing a magnetic substance or particles containing a magnetic substance by magnetic force. , and means for tilting the microplate are essential,
When actually using this as a fully automatic aggregation measuring device, as shown in Figure 1, the microplate supply device 1
, a specimen dispensing device 2, a reagent dispensing device 3, a specimen and reagent stirring device 4, a forced sedimentation promoting device 5 that uses magnetic force to sediment magnetic particles or particles containing a magnetic material in the lower part of the microplate; It can be configured by combining a tilting processing device 6 that is placed in a tilted state, a pattern reading device 7 that reads the peeling state of the precipitated particle component from the microplate due to the tilt, and a microplate collection device 8 that collects used microplates. The configuration example shown in FIG. 1 is an embodiment of the measuring device of the present invention, and does not limit the present invention.

The present invention will be explained in more detail by showing examples below.

(Example) Preparation of CAFP-sensitized ferricolloid-containing gelatin particles] Anti-human AF was added to gelatin particles containing ferricolloid (average particle size of about 3 microns, see JP-A-59-195151).
Using P antibody (rabbit, DACO), the method of Barnard et al. (CIin, Chess, 27 (6)
832 (1981)), anti-human AFP sensitized ferricolloid-containing gelatin particles were prepared.

[Agglutination measurement]

Dilute the sample serum to 10 times with serum dilution solution, transfer it to a ■-shaped microplate, and add the above anti-human AFP to it.
Add 25 μl of a dispersion containing 0.09% of sensitized ferricolloid-containing gelatin particles, stir, and after 10 minutes, leave it standing on a sedimentation accelerating table using a magnet for 5 minutes, and then read the pattern without the influence of magnetic force. Hold the microplate at about 70° on the stand.
The microplate was tilted, and the presence or absence of an immune reaction was determined from the detachment of precipitated particles from the bottom of the well of the microplate. A case where sediment particles were observed to be peeled off from the bottom of the well was judged as negative, and a case where no separation was observed was judged as positive.

The above immunoassay method and Sero I Dia AF according to the present invention
Regarding the conventional standing method (conventional method) using Pmon (a reagent for measuring α-fetoprotein, manufactured by Fujirebio Co., Ltd.), a correlation diagram of each titer is shown in FIG.

(Effects of the Invention) In the immunoassay method and device for antigen-antibody reactions of the present invention, magnetic particles or particles containing magnetic particles are used as carriers, which are forcibly settled at the bottom of the container using magnetic force, and then Since the container is tilted and the presence or absence of an antigen-antibody reaction is determined from the separation status of the precipitated particles from the container, the same effect as the conventional centrifugation method can be achieved using magnetic particles and magnetic force, which is difficult with the conventional centrifugation method. A certain degree of full automation can be easily achieved, and measurements can be made in a much shorter time than conventional static methods and without being affected by vibrations or the like. Furthermore, there is a correlation between the agglutination immunoassay method of the present invention and the conventional static standing method, which substantially corresponds to the respective measurement results, and it can be used in place of the conventional static standing method.

[Brief explanation of the drawing]

FIG. 1 is a block diagram showing a configuration example of a fully automatic magnetic precipitation-enhanced indirect agglutination immunoassay device according to the present invention, and FIG. 2 shows an immunoassay method according to the present invention and a conventional static method (conventional method).
It is a correlation diagram of each potency for. DESCRIPTION OF SYMBOLS 1... Microplate supply device, 2... Sample dispensing device, 3... Reagent dispensing device, 4... Specimen and reagent stirring device, 5... Forced sedimentation promoting device, 6... Tilt processing device, 7... Pattern reading device, 8... Microplate recovery device.

Claims (2)

[Claims] (1) In an indirect agglutination immunoassay using magnetic particles or particles containing a magnetic substance, (a) a step of forcing the particles to settle at the bottom of the container by magnetic force, (b) a step of tilting the container, and (c) a step of causing the particles to settle at the bottom of the container. The measuring method comprises the step of determining the state of peeling from the bottom of the container. (2) a container for receiving an immunoassay sample and an immunoassay reagent; a means for forcibly settling components containing magnetic particles or magnetic particles of the immunoassay reagent; An indirect agglutination immunoassay device comprising: means for tilting.