JP2683944B2 - Indirect agglutination immunoassay method and device - Google Patents

Description

TECHNICAL FIELD The present invention relates to an indirect agglutination immunoassay method and apparatus using an antigen-antibody reaction using magnetic particles or particles containing a magnetic material.

(Prior art) As one of the immunoassay methods, an indirect agglutination reaction in which a binding reaction caused by an antigen-antibody reaction is measured with increased sensitivity using particles bound with an antigen or an antibody is a passive agglutination reaction or a reverse passive reaction. It is called agglutination reaction and has been widely put to practical use as a simple measurement method for a large amount of samples.

(Problems to be solved by the present invention) The immunoassay by indirect agglutination reaction is easier to operate than EIA (enzyme immunoassay) or RIA (radioimmunoassay), and the final antigen-antibody reaction It has the feature that it does not require any special equipment to judge the presence or absence.

However, there are drawbacks such as the skill required for the operation and the difficulty in automation, and the state is still beyond the scope of partial automation.

Further, as a pattern forming method of the indirect agglutination method, a predetermined reagent particle solution is added to a predetermined diluted sample solution in a U-shaped or V-shaped microplate, and the mixture is left standing after stirring. The method of determining the presence or absence (hereinafter abbreviated as a static method) and the method of forcibly settling particles on the bottom of a V-shaped microplate using centrifugal force, and then placing the microplate in an inclined state, There is a method of determining the presence or absence of an antigen-antibody reaction from the state of separation from the bottom of particles (hereinafter abbreviated as centrifugation method). When determining the presence or absence of an antigen-antibody reaction from the settling pattern by the former stationary method, the disturbance of the pattern due to the vibration during stationary, for example, the induction of a slip phenomenon, the reduction of the pattern area, etc., easily occurs and automation is difficult. It was Another disadvantage of the static method is that it takes about 0.5 to 3 hours to complete the pattern formation, depending on the particles used.
In the latter centrifugal method, sedimentation is performed in a few minutes using a centrifuge, the microplate is then tilted and the pattern can be read in a few minutes, and there is no need to worry about disturbance of the pattern due to vibration during pattern formation. Although there are advantages such as not being present, in practice, it is technically difficult to automatically perform an immunoassay using a centrifuge.

In addition, examples of the sample used for the measurement in both the above-mentioned conventional stationary method and centrifugation method include serum, urine, and other body fluids. In the conventional method, although the sample is used by diluting it, if the separation operation of blood cells and serum is omitted and whole blood (hereinafter abbreviated as whole blood) is used, it concentrates on one point on the bottom surface and forms a sedimentation pattern. Is known to have a great influence on the agglutination image and give an incorrect determination result. Therefore, a pretreatment operation for separating blood cells and serum is essential.

Furthermore, in other immunoassays such as enzyme immunoassay and radioimmunoassay, the procedure for separating blood cells and serum is
At present, it is used as a pretreatment operation to avoid nonspecific reactions.

(Means for Solving Problems) An object of the present invention is to avoid the above-mentioned drawbacks of the related art, and to perform a simple and automated whole process of indirect agglutination reaction. Is to provide.

The magnetic precipitation promoting type indirect agglutination immunoassay method of the present invention (hereinafter referred to as “aggregation immunoassay method”) comprises an indirect agglutination immunoassay system using magnetic particles or particles containing a magnetic substance as a reagent, After the reaction, the components containing the particles are forcibly settled on the bottom of the container by magnetic force, and then the container is tilted,
This is an agglutination immunoassay method for determining the presence or absence of an immune reaction based on the state of separation of precipitated particles from the container. In the agglutination immunoassay method of the present invention, an electromagnet or a permanent magnet may be used in order to forcibly settle the magnetic particles or the component containing magnetic particles by magnetic force.

The indirect agglutination immunoassay apparatus of the present invention comprises means for forcibly sedimenting a magnetic substance or a component containing particles containing a magnetic substance of the immunoassay reagent for receiving an immunoassay sample and an immunoassay reagent by magnetic force, And means for placing the container in a tilted state. That is, in the present invention, it is possible to obtain the same effect as if the magnetic particles were forcibly settled on the settling promotion table using a magnet and pulled to the bottom of the container, and settled by the centrifugal method. As a result, it is possible to complete a measuring method and apparatus that are less than one-third of the time required for conventional stationary methods and that are not affected by vibration or the like.

In the present invention, as the magnetic material contained in the reagent, a ferromagnetic material can be preferably used among the magnetic materials in order to forcefully settle on the bottom surface. Specific examples of magnetic particles or particles containing a magnetic material that can be used as a reagent in the present invention are as follows.

Magnetic particles, gelatin particles containing ferromagnetic material
59-195161), particles coated with a magnetic substance such as serum albumin, particles similarly coated with a synthetic polymer, and particles containing a magnetic substance in a synthetic polymer.

As the sample used for the measurement of the present invention, whole blood such as serum, urine, and body fluid used in the conventional static method and centrifugation method can be used. When whole blood is used, since only magnetic particles or particle components containing magnetic substances are forcibly settled after the indirect agglutination reaction, blood cell components are spread over the entire container, and magnetic particles or magnetic substances are present at one point on the bottom surface. Only the component containing the particles containing is selectively concentrated. Therefore, even if whole blood is used, its effect on the pattern formation of magnetic particles or components containing magnetic particles can be virtually ignored. If the container is colored red due to blood cell components and it is difficult to judge,
The plate can be covered with a filter to facilitate the determination. As the filter, a reddish one can be used, and it can be selected from glass, film, cellophane and the like.

As the container for receiving the sample and reagent of the invention, a U-shaped or V-shaped container made of plastic, for example, polystyrene resin, ABS resin or glass can be used. The size of these containers is not limited, but V-shaped microplates made of polystyrene can be preferably used because a large amount of specimens can be processed, handling is easy, and a clear image can be obtained.

In the agglutination immunoassay device of the present invention, as described above,
A microplate for receiving an immunoassay sample and an immunoassay reagent, a means for forcibly sedimenting magnetic particles or components containing particles containing a magnetic material of the reagent by magnetic force, and means for tilting the microplate Is essential, but when actually using it as a fully automatic agglutination measuring device, as shown in FIG. 1, a microplate supplying device 1, a sample dispensing device 2, a reagent dispensing device 3, Stirrer for sample and reagent 4, Forced sedimentation promoting device 5 for magnetically settling magnetic particles or particles containing a magnetic substance in the lower part of the microplate, Gradient treatment device 6 for placing the particles in an inclined state, sedimented particle component Pattern reading device 7 for reading the peeling condition of the microplate from the microplate and the microplate collecting device 8 for collecting the used microplate
Can be configured in combination. The configuration example shown in FIG. 1 is an example of the measuring apparatus of the present invention and does not limit the present invention.

The present invention will be described in more detail with reference to the following examples.

(Example) Example 1 Preparation of anti-human α-fetoprotein (AFP) -sensitized ferri colloid-containing gelatin particles Gelatin particles containing ferri colloid (average particle size of about 3 microns, see JP-A-59-195151) were used as anti-human. Using AFP antibody (rabbit, DACO), Barnard's method (Clin.Che
m., 27 (6) 832 (1981), anti-human AFP-sensitized ferri colloid-containing gelatin particles were prepared.

Example 2 Measurement of AFP in Serum Sample serum containing each concentration of AFP was diluted 10-fold with a serum-diluting solution, placed on a V-shaped microplate, and the above-described anti-human prepared in Example 1 was added thereto. AFP-sensitized ferri colloid-containing gelatin particles containing 0.09% dispersion 25μ was added, stirred, and allowed to stand for 10 minutes on a sedimentation accelerating table using a magnet for 5 minutes, after which the pattern reading table was not affected by magnetic force. The microplate was tilted at about 70 ° and the presence or absence of an immune reaction was determined from the state of separation of sedimented particles from the bottom of the well of the microplate. From the bottom of the well, those in which the separation of the sedimented particles was observed were determined as negative, and those in which they were not observed were determined as positive.

The above-mentioned immunoassay method according to the present invention and Cerodia AFPm
Regarding the conventional stationary method (conventional method) using ono (a reagent for measuring α-fetoprotein, manufactured by Fujirebio Inc.),
The correlation diagram of each titer is shown in FIG.

Example 3 Measurement of AFP in Serum Sample serum containing AFP was diluted 10-fold with a serum-diluting solution, and 50 μm thereof was placed in the first hole of a V-shaped microplate, and 2
25 μl of the diluting solution was added to the 8th to 8th holes, 25 μm was further taken from the 1st hole, and 2n was successively diluted from the 2nd hole to the 8th hole. A dispersion liquid containing 0.14% of the above-mentioned anti-human AFP-sensitized ferricolloid-containing gelatin particles prepared in Example 1 25
Add μ, stir for 3 minutes, and leave it on a sedimentation promotion table using a magnet for 3 minutes. Then, tilt the microplate at about 45 ° with a pattern reading table without the influence of magnetic force, and settle particles from the bottom of the well of the microplate. The presence or absence of an immune reaction was determined in the same manner as in Example 2 from the peeling state of No. Table 1 shows the results.

Example 4 Measurement of AFP in whole blood An equal amount of whole blood of a healthy person was added to diluted serum diluted 5 times with a serum-diluting solution of a sample serum containing AFP, and this was taken in the first hole of a V-shaped microplate. The same measurement as in Example 3 was performed and the results were obtained. Table 1 shows the results.

(Effects of the Invention) In the immunoassay method and apparatus for antigen-antibody reaction of the present invention, magnetic particles or particles containing a magnetic material are used as a carrier, and these are magnetically forced to settle to the bottom of the container. Since the container is tilted and the presence or absence of an antigen-antibody reaction is determined from the state of separation from the sedimented particle container, the same effect as the conventional centrifugation method can be achieved with magnetic particles and magnetic force, which is difficult with the conventional centrifugation method. Some full automation can also be easily achieved. Furthermore, it is possible to perform measurement using whole blood in a much shorter time than the conventional static method, without being affected by vibration at all, and using the whole blood, which is completely impossible by the centrifugation method. Furthermore, the agglutination immunoassay method of the present invention and the conventional static method have a correlation substantially corresponding to each measurement result, and can be used in place of the conventional static method.

In addition, the method of the present invention is also capable of measurement using whole blood such as serum, urine, and body fluid, which are samples of conventional immunoassays, and provides a simpler method.

[Brief description of the drawings]

FIG. 1 is a block diagram showing an example of the configuration of a fully automatic magnetic precipitation promoting type indirect agglutination immunoassay device according to the present invention, and FIG. 2 is an immunoassay method according to the present invention and a conventional stationary method (conventional method). FIG. 3 is a correlation diagram of each titer of A. 1 ... Microplate supply device, 2 ... Sample dispensing device, 3 ... Reagent dispensing device, 4 ... Sample and reagent stirring device, 5 ... Forced sedimentation promoting device, 6 ... Inclination processing device, 7
...... Pattern reader, 8 ・ ・ ・ Microplate recovery device.

Claims (3)

(57) [Claims] 1. In an indirect agglutination immunoassay method using magnetic particles or particles containing a magnetic material, (a) forcibly settling the particles to the bottom of the container by magnetic force (b) inclining the container (c) The measuring method, which comprises a step of judging the state of separation of particles from the bottom of the container. 2. The method according to claim 1, wherein whole blood is used as the sample. 3. A container for receiving a sample for immunoassay and a reagent for immunoassay, means for forcibly sedimenting magnetic particles or a component containing particles containing magnetic material of the reagent for immunoassay by magnetic force, An indirect agglutination immunoassay device comprising: a means for tilting the container.