JPH03191864A - Indirect agglutination immunoassay method and device - Google Patents

Abstract

PURPOSE:To make it possible to automate the entire process of indirect agglutination simply by using magnetic particles or particles containing magnetic material as a carrier, and forcibly settling the carrier to the bottom part of a container by using magnetic force. CONSTITUTION:Magnetic particles or particles containing magnetic material are used as reagent, and an immunity measuring system comprising indirect agglutination is constituted. After the reaction, the component containing the particles is forcibly settled to the bottom of a container with magnetic force. Then, the container is inclined, and the presence or absence of the immunity reaction is judged based on the releasing state of the settled particles from the container. When the component containing the magnetic particles or the particles containing the magnetic material are forcibly settled with the magnetic force, an electromagnet or a permanent magnet can be used. The magnetic particles are forcibly settled on a settling accelerating stage using the magnet and attracted to the bottom surface of the container. The same action as the settlement using a centrifugal method can be obtained. Thus, the measurement can be carried out in a short time with no influence of variation or the like.

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 U-shaped or V-shaped microplate, stirred and left to stand, and the antigen-antibody reaction is detected from the sedimentation pattern of the particles. The method for determining the presence or absence of particles (hereinafter abbreviated as the standing method) involves forcibly settling particles at the bottom of a ■-shaped microplate using centrifugal force, and then placing the microplate in an inclined state. There is a method (hereinafter abbreviated as centrifugation method) in which the presence or absence of an antigen-antibody reaction is determined based on the state of separation of particles from the bottom. When determining the presence or absence of an antigen-antibody reaction from the sedimentation pattern obtained by the former standing method, the pattern is likely to be disturbed due to vibration during the standing, such as inducing a slip phenomenon and reducing the pattern area, making automation difficult. Ta. Another disadvantage of the static method is that the amount of time required to complete pattern formation varies depending on the particles used.
It takes a long time of about 0.5 to 3 hours. 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.

In addition, examples of specimens used for measurement in both the above-mentioned conventional static method and centrifugation method include serum, urine, and other body fluids. In conventional methods, the sample is diluted before use, but when whole blood (hereinafter referred to as whole blood) is used without the separation of blood cells and serum, blood components concentrate at one point on the bottom and form a sedimentation pattern. is known to have a large effect on the agglutination image and give incorrect judgment results.

Therefore, a pretreatment operation for separating blood cells and serum is essential.

Furthermore, in other immunoassay methods such as enzyme immunoassay and radioimmunoassay, the separation of blood cells and serum is currently used as a pretreatment procedure to avoid non-specific reactions. .

(Means for Solving the Problems) An object of the present invention is to avoid the above-mentioned drawbacks of the conventional methods, and to provide a method and apparatus for indirect agglutination immunoassay using magnetic precipitation promotion, which is simple and automates the entire process of indirect agglutination reaction. The goal is to provide the following.

The magnetic precipitation-promoted indirect agglutination immunoassay method (hereinafter referred to as "Agglutination immunoassay method J") of the present invention comprises an immunoassay system based on an indirect agglutination reaction using magnetic particles or particles containing a magnetic substance as a reagent, This is an agglutination immunoassay method in which the component containing the particles is forcibly settled at the bottom of the container after the reaction by magnetic force, the container is then tilted, and the presence or absence of an immune reaction is determined from the detachment of the precipitated particles from the container. In the agglutination immunoassay method of the invention, an electromagnet or a permanent magnet may be used to forcibly settle magnetic particles or a component containing particles containing a magnetic substance by 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 synthetic polymer,
These include particles containing a magnetic material in a synthetic polymer.

As specimens used in the measurement of the present invention, serum, urine, body fluids, etc. used in conventional static methods and centrifugation methods, as well as whole blood can be used. When whole blood is used, after the indirect agglutination reaction, only the magnetic particles or particle components containing the magnetic material are forcibly settled, so the blood cell components spread throughout the container and the magnetic particles or magnetic material are concentrated at one point on the bottom. selectively concentrate only on components containing particles containing . Therefore, even if whole blood is used, the influence of magnetic particles or components containing magnetic particles on pattern formation can be virtually ignored. In addition, if the entire container is colored red due to blood cell components and is difficult to judge, the top of the plate can be covered with a filter to facilitate the judgment. As the filter, one colored in red can be used, and it can be selected from glass, film, cellophane, etc.

Further, as the container for receiving the specimen and reagent of the invention, a U-shaped or V-shaped container made of plastics, such as polystyrene resin, 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 a large amount of specimen, be easy to handle, and obtain clear images.

As described above, in the agglutination immunoassay device of the present invention, a microplate that receives an immunoassay sample and an immunoassay reagent, and a component containing magnetic particles or magnetic particles of the reagent are forcibly sedimented by magnetic force. A means for tilting the microplate and a means for tilting the microplate are essential, but when actually using this as a fully automatic agglutination measuring device, as shown in FIG. 1. Sample dispensing bag W2, reagent dispensing device 3, sample and reagent stirring device 4, forced sedimentation promoting device 5 that uses magnetic force to sediment magnetic particles or particles containing magnetic material in the lower part of the microplate.
, a tilting processing device 6 that subsequently places the same in a tilted state;
It can be constructed by combining a pattern reading device 7 that reads the state of separation of settled particle components from a microplate due to inclination, and a microplate collection device 8 that collects used microplates. The configuration example shown in FIG.
This is an example of the measuring device of the present invention, and is not intended to limit the present invention.

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

(Example) ! ML-1 Preparation of gelatin particles containing ferricolloid sensitized with anti-human α-fetoprotein (AFP) Anti-human AF
Using the P antibody (rabbit, DACO), the method of Barnard et al. (CIin, Chem, +27 (6)
832 (1981)), anti-human AFP sensitized ferricolloid-containing gelatin particles were prepared.

Base-I Measurement of AFP in Serum Sample serum containing AFP of each concentration was diluted 10 times with serum dilution solution, placed in a single-shaped microplate, and the above prepared in Example 1 was added to the sample serum. A dispersion containing 0.09% of anti-human AFP-sensitized ferricolloid-containing gelatin particles 25
Add μl, stir, and after 10 minutes, leave it for 5 minutes on a sedimentation promotion stand using a magnet. Then, tilt the microplate about 70° on a pattern reading stand that is not affected by magnetic force, and The presence or absence of an immune reaction was determined from the detachment of the precipitated particles. 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 according to the present invention and Celodia AF
Conventional standing method using Pmono (α-fetoprotein measurement reagent, manufactured by Fujirebio Co., Ltd.) (conventional method)
Figure 2 shows a correlation diagram of the respective titers.

Su JiJ Juice - 1 Measurement of AFP in serum Dilute the sample serum containing AFP 10 times with serum dilution solution, add 50 μm of this to the first hole of a single-shaped microplate, and place it in 8 wells from the 2nd hole. Add 25 μl of the dilution solution up to the first hole, and then take 25 μl from the first hole and dilute it 2n sequentially from the second hole to the eighth hole.

To this was added 25μ2 of the dispersion containing 0.14% of gelatin particles containing anti-AFP-induced ferricolloids prepared in Example 1, stirred for 3 minutes, and left to stand for 3 minutes on a sedimentation accelerator using a magnet. After that, the microplate was tilted at about 45° on a pattern reading stand that is not affected by magnetic force, and the presence or absence of an immune reaction was determined in the same manner as in Example 2 from the detachment of precipitated particles from the bottom of the wells of the microplate. The results are shown in Table 1.

Measurement of AFP in whole blood A sample serum containing AFP was diluted 5 times with a serum dilution solution.An equal amount of whole blood from a healthy individual was added to the diluted serum, and this was placed in a V-shaped microplate.
A hole was taken, and the same measurements as in Example 3 were performed to obtain the results.

The results are shown in Table 1.

(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. Some full automation can also be easily achieved. Furthermore, it is much shorter than the conventional static method and is completely unaffected by vibrations, and it is possible to perform measurements using whole blood, which is completely impossible with the centrifugation method.

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 the method can be used in place of the conventional static standing method.

Furthermore, the method of the present invention allows measurement using whole blood in addition to conventional immunoassay specimens such as serum, urine, and body fluids, thereby providing a simpler method.

[Brief explanation of drawings]

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 (3)

[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) The method according to claim (1), which uses whole blood as the specimen. (3) a container for receiving an immunoassay sample and an immunoassay reagent; a means for forcibly settling magnetic particles or a component containing magnetic particles of the immunoassay reagent; An indirect agglutination immunoassay device comprising: means for tilting.