JPS5873866A - Immunological method for detection - Google Patents

Abstract

PURPOSE:To enable quick and highly sensitive detection and also preliminary preparation and preservation of a capillary tube with reagent therein by a method wherein a reagent for immune agglutination reaction is injected into the capillary tube, frozen and dried and are mixed with a solution to be analyzed, and then the capillary tube is laid horizontally in a stationary state to observe the state of agglutination. CONSTITUTION:A reagent (an antibody for an antigen, an antigen for an antibody, etc.) agglutinating in peculiar reaction to a substance to be measured (an antigen, an antibody, a complement component, etc.) is injected, as it is, into a capillary tube (a glass or plastic tube) having the inside diameter of 0.5-5mm. and the length of 50-100mm., or a reagent for immune agglutination reaction fixed on a carrier such as a glass or plastic grain or a red corpuscle is injected therein. Then it is frozen and dried in liquid nitrogen. The capillary tube, sealed up at both ends, can be preserved in the frozen state until an analysis is conducted. At the time of the analysis, the reagent dissolved or dispersed in water and then mixed with a solution to be analyzed or the four-times dilution thereof. Thereafter, the capillary tube is laid horizontally in a stationary state, and the state of agglutination thus generated is observed. When the agglutination (a) is present, the substance is positive, and when there is a state (b) that the reagent adheres to the wall of the tube, the substance is negative. Thus, the detection can be performed quickly and simply.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for detecting or measuring components in human or animal body fluids using immunological agglutination reactions. When using the reaction between an antigen and an antibody to immunologically detect or quantify either one of them, the substance that binds to the substance to be measured is immobilized on carrier particles of an appropriate size,
A highly sensitive measurement method that utilizes the phenomenon of particles aggregating in the presence of a substance to be measured has become an important means for immunological investigation. Conversely, the substance to be measured is immobilized on particles, and the presence of the substance to be measured prevents aggregation of the particles immobilized with the substance to be measured due to the presence of an antigen or antibody that specifically opposes the substance to be measured. Methods for detecting or positioning a substance to be measured are also widely used in immunological clinical tests.

Conventionally, it has been determined whether immunoactive substance-immobilized microparticles aggregate due to a reaction with a component to be measured in a sample fluid, or whether the immunoactive substance-immobilized microparticles aggregate due to a reaction between a pre-prepared reagent and immunoactive substance-immobilized microparticles in a sample fluid. Determination as to whether a component to be measured inhibits or not is mainly made by mixing the specimen and agglutination reaction reagent on a flat plate or microplate.

However, on flat plates, the agglutination reaction is very rapid or has low sensitivity. On the other hand, agglutination reactions on microplates have high sensitivity, but the time required for determination is long, 2 hours or more.

In order to solve these conventional drawbacks, the present inventors have studied a highly sensitive, short determination time, and simple inspection method, and as a result, they have arrived at the present invention.

That is, the present invention is a method for detecting or quantifying components in a body fluid by immunological agglutination reaction, which is characterized by injecting an agglutination reaction reagent into a capillary tube and freeze-drying it, and mixing the specimen and the agglutination reaction reagent in the capillary tube. This is an immunological testing method that

The agglutination reaction reagent used in the present invention is a substance that specifically binds to a substance to be measured, or a substance in which the substance to be measured is immobilized on a carrier. Substances to be immobilized on these carriers include, for example, Treponema pallidum antigen, rheumatoid factor, hepatitis B surface antigen (HBs antigen), antibody against HBs antigen (anti-HBs antibody), Toxoplasma antigen,
Streptolysin-○, anti-streptolysin 0 antibody, mycoplasma antigen, human ciliated gonadotropin (HCG)
), anti-HCG antibody, heat-agglutinated human immunoglobulin G1 nuclear protein, deoxynucleic acid, anti-C-reactive protein antibody, estrogen, anti-estrogen antibody, complement components (C1q, C1r
, C1s , C2゜C5, C4, C5, C6, C7, C
8, C9) and antibodies against them. Examples of carriers include blood cells (fixed with formalin, etc.), bacterial cells, polystyrene, latex, and other polymer microbeads.

Immobilization of the immunologically active substance onto the carrier is performed by physically adsorbed ionic bonding or covalent bonding.

The capillary tube used in the testing method of the present invention is 0.5 to 5 TIrI
A transparent tubular body made of glass or plastic such as polymethyl methacrylate, polystyrene, polycarbonate, polyvinyl chloride, polysulfone, etc., preferably having a uniform inner diameter of about 1 mm, and more preferably a length of 50 to 100 mm. be.

This capillary is connected to a piston or cap-shaped pipettor for the purpose of inhaling a fixed amount of a sample or agglutination reaction reagent.
Furthermore, it is preferable to have a scale.

In the present invention, an agglutination reaction reagent is injected into a capillary tube.

Afterwards, the reagent is lyophilized. The freeze-drying method is usually 'etc.

However, it is preferable to freeze in liquid nitrogen. The agglutination reaction is performed by injecting a sample such as serum or plasma into a capillary tube and mixing it with an agglutination reaction reagent. It is preferable to dissolve the freeze-dried agglutination reaction reagent in water before mixing it with the specimen. It is of course possible to add conventionally known additives for aggregation reactions to the aggregation reaction. After thoroughly mixing the sample and reagent, if the capillary tube is left standing in an almost horizontal direction, a (+) image and a (
→A sedimentation image that can be distinguished is obtained.0 The present invention is an excellent inspection method that exhibits high sensitivity comparable to that of a U-shaped microplate and can shorten the time required for determination. Reaction reagents have a good shelf life, so if they are prepared in advance, they can be tested by simply injecting the sample into a capillary tube.

The present invention will be described in more detail below with reference to Examples.

Example 1 A 10μ sample of a suspension of syphilis HA antigen (Fuji Organ Pharmaceutical Co., Ltd.) sensitized sheep blood cells was taken into a glass hemadocrit tube with an internal diameter of 1.2cm and a height of 75mm, frozen in liquid nitrogen, and dried under reduced pressure. did. After injecting 10 μt of Vζ distilled water into the tube to redisperse the sensitized blood cells, 10 μt of serum diluted with serum diluent was injected, the tube was moved up and down in the axial direction to mix thoroughly, and the tube was left standing on a horizontal surface. . The determination results after 30 minutes are shown in Table 1 and FIG.

In Figure 1, (a) is positive (-F), (b) is negative (=)
In FIG. 2, as in FIG. 1, (a) indicates positive and (b) indicates negative. In the case of a microplate, it took 18 hours for the determination.

Table 1 *Final dilution ratio in the tube Example 2
10 μt of the BSA-immobilized polymer microbead dispersion described in No. 8677 was taken and freeze-dried. Distilled water in the tube 10
After redispersing the BS/immobilized polymer microbeads by injecting μt 200μ Ab/m7? 10 μt of a phosphate-buffered saline solution of anti-BSA antiserum (rabbit) was taken, mixed thoroughly by moving it up and down in the axial direction of the tube, and the tube was placed on a horizontal surface. In addition, as a negative control, anti-BSA antiserum (rabbit) was used as a negative control.
Phosphate buffered saline solution (BSA111+9/m/
) and diluted to the same ratio and used.

BS in phosphate buffered saline solution as determined after 20 minutes.
The negative control added with A shows the (-) image in Figure 1, and the BS
The sample to which A was not added showed a (-11 image).

Example 5 In a hematocrit tube, 10μ of the syphilis antigen-immobilized polymer microbead dispersion described in Japanese Patent Application No. 58677/1986 was placed.
The tube was collected with caps on both ends and stored in the refrigerator.

The caps at both ends of the tube were removed, and 1 μm of serum with a TPHA titer of 1280 diluted 40 times with a serum diluent was taken, mixed thoroughly by moving up and down in the axial direction of the tube, and the tube was allowed to stand still on a horizontal surface.

L The same procedure was repeated with negative serum as a control 02
When judged after 0 minutes, a (-+) image was obtained for positive serum, and a (-) image was obtained for negative serum.

[Brief explanation of drawings]

FIG. 1 is an enlarged longitudinal sectional view showing the test results obtained by the method of the present invention, and FIG. 2 is an enlarged plan view showing the test results obtained by the conventional method using a microplate. (a) shows ten images, and (b) shows - images, respectively. Patent applicant Toshi Co., Ltd. (phantom)
(!71 Figure (0L) 2nd wa

Claims (1)

[Claims] (1) A method for detecting or quantifying components in body fluids by immunological agglutination reaction, which is characterized by injecting an agglutination reagent into a capillary tube, freeze-drying it, and mixing the sample and agglutination reaction reagent in the capillary tube. Immunological testing method.