JPH02141667A - Particle of blood corpuscle - Google Patents

Abstract

PURPOSE:To control the dispersing state of the particles of blood corpuscles magnetically and to make it possible to perform high speed analysis by sealing the magnetic bodies into the red blood corpuscles from cell films. CONSTITUTION:A red blood corpuscle is mixed with a magnetic body, and the mixture is dialyzed into a low surface-tension liquid. In this way, the magnetic body is sealed into the ghost of the red blood corpuscle by utilizing the film penetrating property of a cell. In the analysis wherein an object to be measured is an anti-A antibody in serum, an A antigen 2 is fixed to the inner surface of a reaction container 1. A solution incorporating the anti-A antibody 4 is inputted in the container 1. Thereafter, a red blood corpuscle particle 3 wherein the magnetic body is sealed into an A-type red blood corpuscle is inputted. The red blood corpuscle particle 3 is moved to the wall surface of the container 1 by the magnetic field of a magnet. The blood corpuscle particle 3 is bonded and aggregated to the A antigen 2 through the antibody 4. The presence or absence of the anti-A antibody in the serum is detected based on the aggregation pattern. In this method, the precipitation and reaction time can be shortened by the attracting force of the magnetic field.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to blood cell particles used for blood type determination and the like.

[Conventional technology and issues]

Most conventional blood determination methods are based on red blood cell agglutination reactions, and various automatic blood type determination devices that utilize such agglutination reactions have been developed. Specifically, Vox Sang,
, 8.235-241% McNel I in 19H
have reported that it is possible to determine blood type using the continuous flow method of Auto Analyzer. In addition, in 1970, the first Groupamatlc machine was created based on C. Matte's basic concept.
upasattc uses a test tube method, and the image of the tube bottom after centrifugation is optically read, similar to conventional inspection methods. Furthermore, the present applicant has also developed P, which has made it possible to perform ABO blood type, Rh blood type, irregular antibody screening, HBs antigen, syphilis testing, etc. using microplates.
Developed K-7100.

Blood tests are usually performed using front and back tests for ABO blood type, Rh blood type, and irregular antibody screening. The reagents used in these tests require not only antibody reagents but also blood cell reagents. Blood cell reagents are generally prepared by suspending predetermined red blood cells that have been analyzed for blood group antigens in a preservation solution. There are also attempts to effectively utilize membrane components with long-term stability in mind; for example, USP
There is also a method using hemolyzed stroma as described in No. 3958477.

By the way, separation and purification of blood type substances prior to blood determination is not always sufficient due to technical and economical aspects.
The biochemical entities of blood group substances are diverse, including membrane proteins, sugar chains such as glycoproteins and glycofats, and in some cases lipids, and some of these substances have not yet been identified. For this reason, current blood cell reagents include a variety of preserved blood cells. For this reason, the analysis format is limited to the one-flow type, the method using centrifugation, the method using a microplate, etc., as described above. In particular, in terms of shortening the analysis time, there was no effective means other than using centrifugation.

On the other hand, in the Coombs test used for screening for irregular antibodies, repeated centrifugal washing is done by boat fishing.
This caused delays in mass processing and automation.

The present invention was made to solve the above-mentioned conventional problems, and aims to provide blood cell particles whose dispersion state can be magnetically controlled and which can be applied to high-speed analysis without employing centrifugation. It is something to do.

[Means and effects for solving the problems] The present invention is a spherical particle characterized in that a magnetic substance is encapsulated inside at least the cell membrane of red blood cells.

Examples of the magnetic material include ultrafine particle magnetic material with a particle size of 100, such as Ferricide Sea 40 manufactured by Taiho Kogyo Co., Ltd., and magnetic latex manufactured by RHOME-POULENC.

Various methods can be used to encapsulate the magnetic material into red blood cells. Specifically, by mixing the red blood cells and the magnetic material to be encapsulated and dialyzing the mixture into a hypotonic solution, the magnetic material can be encapsulated in the red blood cell ghost by utilizing the membrane permeability of the cells. However, as the particle size of the magnetic material to be encapsulated increases, the introduction rate thereof decreases.

In such a case, by not only making the red blood cells hypotonic but also using high voltage pulses, it becomes possible to encapsulate a magnetic substance with a relatively large particle size inside the red blood cells.

The blood cell particles of the present invention are extremely useful when carrying out ABO blood type back test and irregular antibody screening.

For example, in an analysis where the measurement target is anti-A antibody in serum, A
After immobilizing the antigen 2 and placing the solution containing the anti-A antibody in the reaction container 1, the blood cell particles of the present invention in which a magnetic material is encapsulated are placed in type A red blood cells, and the blood cell particles are blown by the magnetic field of a magnet (not shown). Immunization is carried out on the wall of the reaction vessel l, and the blood cell particles 3 are bound to the A antigen 2 via the anti-A antibody 4 to be measured and aggregated, and the presence or absence of the anti-A antibody in the serum is detected from the agglutination pattern. It can be used for scientific n1 determination method. In this case, the blood cell particles can settle and react in a short period of time due to the attraction force caused by the magnetic field of the magnetic substance enclosed therein, and the n1 constant time can be shortened.

In addition, when performing the Coombs test used to screen for irregular antibodies in blood transfusion tests, see Figure 2 (
As shown in A), a solution 13 containing antibodies and blood cells 12 containing type O red blood cells encapsulated with a magnetic substance is placed in a reaction container 11, and after binding the antibodies to the blood cells 12, as shown in FIG. Then, a magnet 14 is placed on the side of the reaction container 11 from the outside, and its magnetic field collects the blood cell particles 12 to which antibodies have been bound onto the inner surface of the reaction container 11. Subsequently, as shown in FIG. By inhaling and discharging the solution 13 in the reaction vessel 11, the antibodies bound to the blood cell particles 12 collected on the inner surface of the reaction vessel 11 can be easily washed away, eliminating the need for centrifugation.

Furthermore, a substance (for example, red blood cells) that specifically reacts with or competes with the substance to be measured is immobilized on the inner surface of the reaction vessel, and the substance to be measured and the substance that specifically reacts with or competes with the substance are immobilized in the reaction vessel. After adding a solution containing competing magnetic particles, a magnetic field is applied to the magnetic particles to form a distribution pattern of the magnetic particles, and the state of binding between type O red blood cells fixed on the inner surface of the container and the substance to be measured is measured. In the solid phase treatment in the immunoassay method, as shown in FIG. A solution 24 containing blood cell particles 23 of the present invention encapsulating a magnetic substance is placed, and the blood cells 23 are attracted to the inner surface of the reaction container 21 by the magnetic field of a magnet 25 placed below the reaction container 21, as shown in FIG. This makes it possible to immobilize red blood cells at a higher speed than in the conventional method of leaving them to stand.

〔Example〕

Examples of the present invention will be described in detail below.

Preparation of B-type blood cell solid-phase plate> NUNC U-shaped bottom microplate 3 shown in Figure 4
0.01M PBS Spl+7.
Wheat germ lectin (WGA) adjusted to 0 and 10tg/-
) [Seikagaku Corporation, manufacturing number 92107] too
uI was added and treated at room temperature for 30 minutes. Subsequently, WGA was prepared by washing 5 times with 300 μp each of 0.01 M PBS, pH 7.0, and drying at room temperature.
A treated plate was obtained. Subsequently, as shown in FIG. 4, the support table 32 is moved so that the magnet (diameter 8 mm, thickness 3III+1, residual magnetic flux density 2000 Gauss) 34 thereon approaches the bottom of each well 32 of the microplate 31. In this manner, a magnetic field was applied in the vertical direction of the bottom surface of each well 32. Next, magnetic particles with a particle size of 100 (manufactured by Taiho Kogyo Co., Ltd.) were encapsulated in a hypotonic solution.
The concentration of hemocytes was adjusted to 0.3% with physiological saline, 25 μg/well was added, and the mixture was left for 10 to 30 seconds.

Contains 0.1% bovine serum albumin (BSA) at the end [
A type B blood cell solid-phase plate was prepared by washing three times with 1,01 M PBS 5 pH 7,0 using 200 layers/well.

Detection of Anti-B Antibody> Anti-A serum and anti-B serum were added at 25 ttf/well to the type B blood cell solid-phase plate prepared by the above method and stirred. Subsequently, type B photoreceptors encapsulated with magnetic particles similar to those used in the solid phase brake 1 were added at 25μj/
Added well by well. At this time, as in the production of the solid phase plate, the support plate is attached to the magnet (diameter 8III
111 (thickness: 3 mm, residual magnetic flux density: 2000 Gauss) was moved so as to approach the bottom of each well of the solid phase plate, so that a magnetic field was applied in the perpendicular direction to the bottom of each well. Due to the action of this magnetic field, a reaction pattern that spread uniformly across the bottom of the well containing anti-B serum was formed, whereas a pattern converging at the center of the bottom surface was formed in the well containing anti-A serum. Ta.

According to the above-mentioned embodiment, the red blood cells to be immobilized are encapsulated with a magnetic substance, and the red blood cells are collected by applying a magnetic field to the bottom of the well to which they are added, thereby reducing the amount of time required for conventional immobilization of red blood cells. It can significantly reduce time.

In addition, after reacting various serums with immobilized red blood cells,
The time required for pattern formation can be shortened by adding magnetically encapsulated red blood cells similar to those solidified and collecting them in front of the solid phase by the action of a magnetic field.

〔Effect of the invention〕

As described in detail above, since the blood cell particles of the present invention have a structure in which a magnetic material is encapsulated within the red blood cells, the dispersion state of the blood cell reagent can be magnetically controlled without employing centrifugation, and as a result, ABO type blood cell particles can be used. It has remarkable effects, such as being able to be effectively used for high-speed processing of immunological analyzes such as pattern background testing and irregular antibody screening.

[Brief explanation of the drawing]

FIGS. 1 to 3 are schematic diagrams showing application examples of the blood cells of the present invention, and FIG. 4 is a front view showing an immunoassay device used in an example of the present invention. 1.11.21...Reaction container, 2...A antigen, 3.
12.23... Blood cell particles, 4... Anti-A antibody, 14.
25.33... Magnet, 31... Microplate, 32... Well. Applicant's representative Patent attorney Atsushi Tsuboi

Claims (1)

[Claims] A blood cell particle characterized in that a magnetic substance is encapsulated inside at least the cell membrane of the red blood cell.