JPH01112161A - Immune agglutination determining device - Google Patents

Abstract

PURPOSE:To measure the degree of agglutination of the agglutinated lumps generated by agglutination by measurement of magnetic intensity with high accuracy by using a magnetic material as fine particles to conjugate antibodies. CONSTITUTION:100mul liquid suspension of the magnetic beads is added to 20mul serum contg. AFP of various concns. and is brought into reaction for 15min at room temp. This reaction liquid is transferred into the liquid to be measured part 2 of a sheath flow cell 1 and while a sheath liquid 3 is passed therein, the agglutinated lumps in the reaction liquid are dispersed and are introduced to a detecting part 4. The fine particles are magnetized by a magnet 5 in the detecting part 4 and the (x), (y), (z) components of magnetic moment are detected by a SQUID fluxmeter 6. The detected signal values are subjected to AD conversion in an A/D converter 11 and the converted signals are guided to a personal computer 12, by which the square root of the sum of the square values of the respective signal values is calculated and the magnitude of the magnetic moment is determined.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to an immunoagglutination quantitative device using a particle size measurement method for fine particle aggregates, and is particularly suitable for a quantitative method applying an immunoagglutination reaction. Regarding quantitative devices.

[Conventional technology]

Conventionally, there is a method of binding an antibody to the surface of fine particles such as latex beads, reacting the antibody with a sample, and measuring the amount of antigen in the sample from the difference in turbidity that changes due to the reaction. This principle is based on the fact that antigens on the surface of microparticles cause an antigen-antibody reaction and aggregate the microparticles, and that this aggregation changes quantitatively with respect to the amount of antigen.This method is used to quantify various proteins, etc. ing. However, since the entire reaction solution, which has a distribution of degree of aggregation, is measured at once, only the average particle size can be determined, which poses an accuracy problem in calculating the antigen concentration.

As a method to solve this problem, for example, there is a method of dispersing the reaction solution into aggregates using a sheath-flow method and measuring the scattering intensity of each aggregate (Journal of the Japanese Society of Clinical Laboratory Automation, Ni 1, 226 (1986)).

According to this method, the degree of aggregation of individual aggregates can be measured, and the degree of agglutination distribution can also be measured, which improves the accuracy of calculating antigen concentration.
can be made to

However, even in this method, the degree of aggregation is measured by optical measurement, and the influence of absorbers such as scatterers and dyes that coexist in the sample is inevitable, and the measurement accuracy is limited.

The above-mentioned conventional techniques do not take into account the effects of scatterers, absorbers such as dyes, and fluorescent substances coexisting in the sample, and have the problem of causing measurement errors.

[Problem to be solved by the invention]

The present invention solves the problems of the prior art described above, and is capable of accurately measuring the size and distribution of aggregates generated by antigen-antibody reactions, and is capable of determining antigen concentration with high accuracy. The purpose is to provide.

[Means to solve the problem]

The above object is achieved by measuring the degree of aggregation not by optical measurement but by magnetic measurement.

That is, this is achieved by using a magnetic material as the fine particles and using a magnetic measurement means such as a SQUID (superconducting quantum interference device) to measure the aggregation state in an immunoagglutination quantitative apparatus.

The present invention quantitatively measures antigen concentration by the following method.

Antibodies are bound to the surface of magnetizable microparticles, and the microparticles are reacted with antigens in the sample. Accompanying this antigen-antibody reaction, aggregates of fine particles are formed.

The measurement reaction solution prepared in this way is transferred to a sheath flow cell, and while the sheath liquid is flowing, aggregates of fine particles are dispersed and moved along with the flow of the sheath liquid. The antigen concentration can be quantitatively measured by magnetizing the aggregates in this liquid flow and measuring the magnetic moment.

Magnetizable fine particles include ferromagnetic materials such as Fe and Ni.
, Co, and other metal particles are typical examples. γ-ferrite particles and magnetite particles are easy materials to use. Alternatively, particles coated with a film of such a magnetizable material may be used.

The particle size of the fine particles is usually about 2 μm or less.

Furthermore, it is more preferably 1 μm or less. If the particle size is too small, the magnetic flux that generates the agglomerates will be small, so the selection of the particle size may be made in consideration of the sensitivity of the magnetic measurement system, the measurement ability such as the SN ratio, etc.

In order to bind antibodies to the surface of microparticles, the following known means can be used.

(1) Bind the antibody to the surface of the microparticles by adsorption.

(2) After adsorbing the antibody onto the surface of the fine particles, chemically cross-linking the antibody molecules. The crosslinking reaction makes the bond between the fine particles and the antibody stronger than in the case of mere adsorption.

Examples of useful crosslinking agents for chemical crosslinking include aldehyde groups and carbodiimide groups.

Examples include bifunctional substances having a maleimide group and the like.

(3) Chemically bond the antibody to the surface of the microparticles.

(4) The surface of the fine particles is coated with an organic polymer material having a functional group, and the antibody is covalently bonded using this functional group.

As the organic polymer material having a functional group, for example, methacrylic acid polymer or the like is usually used.

Crosslinking agents used for chemical crosslinking treatment include:
Bifunctional substances having an aldehyde group, a carbodiimide group, a maleimide group, etc. are effective examples.

Preferred examples of the functional group include an amino group, a carboxyl group, an epoxy group, and a hydroxyl group.

The sheath flow cell may be of a normal type, for example, a mechanism equivalent to that used in flow cytometers.

The detection part consists of a magnet that magnetizes particles and a magnetometer that measures magnetic strength.
It is preferable to use an ID flux meter.

A magnetometer can perform particularly precise measurements when the detection coils are arranged in two or three directions orthogonal to each other.

Examples of antigens to which the present invention can be applied include human α-fetoprotein (AFP), carcinoembryonic antigen (CEA), ferritin, β2 microglobulin (β2-m), human chorionic gonado 1-lopin (HCG), C-reactive protein (CR
P), rheumatoid factor, etc.

[Effect]

If a magnetic material is used as the fine particles to which antibodies are bound, the degree of aggregation of aggregates produced by aggregation can be determined by measuring the magnetic intensity. In this case, since the above-mentioned influencing factors in optical measurement do not matter, highly accurate measurement is possible.

〔Example〕

An embodiment of the present invention will be described below with reference to FIG.

Anti-human α-fetoprotein antibody (anti-human AFP) was coated on the surface of spherical γ-ferrite particles (average particle size 0.75 μm).
An immobilized antibody magnetic piece was obtained by adsorbing antibodies) and cross-linking the antibody molecules using glutaraldehyde. The immobilization method may be a conventionally known method, for example, [Protein Hybrid] edited by Yuji Inada, p1
99-122, Kyoritsu Shuppan (Tokyo), 1987, etc.

100 μQ of the above magnetic bead suspension was added to 20 μQ of serum containing various concentrations of AFP and allowed to react at room temperature for 15 minutes.

Transfer this reaction liquid to the test liquid part 2 of the sheath flow cell 1,
While flowing the sheath liquid 3, aggregates in the reaction liquid are dispersed and guided to the detection section 4. Although physiological saline was used as the sheath fluid, an appropriate buffer solution or water may be used. The sheath flow cell used is equivalent to the mechanism applied to, for example, a flow cytometer. The flow path portion of the sheath flow cell had an inner diameter of 100 μm, and the sheath liquid flow rate and the pressure difference between the sample and sheath liquid were set so that the flow rate was approximately 10 m/s.

The dispersed aggregates are guided to the detection section 4 according to the flow.

The detection unit 4 includes a magnet 5 for magnetization and a SQUID magnetometer 6.

The magnet 5 magnetizes the particles, and by using a ring-shaped electromagnet, the magnetization can be oriented in one direction along the flow direction. An example of the magnetization direction is shown by arrow 7.

The magnetometer 6 is a SQUID magnetometer, and is configured by arranging three pairs of detection coils in mutually orthogonal directions. That is, this state is shown in FIG. FIG. 6 shows a cross section taken along line AA' in FIG. Two pairs (81,
82) A pair (83) were provided in the direction perpendicular to the flow direction so that the X+V+Z components of the magnetic moment could be detected.

The detected signal values were AD converted by an A/D converter and led to the personal computer 12, and the square root of the sum of the square values of each signal value was calculated to determine the magnitude of the magnetic moment.

This process avoids the occurrence of errors in measured values due to the rotation of the particle mass to be measured, which would occur if only the unidirectional component of the magnetic moment was detected.

In addition, in order to simplify the apparatus, it is also possible to arrange the detection coils in only one direction or only in two directions for measurement.

FIG. 7 shows an example of the arrangement of coils having a substantially i configuration. FIG. 8 is a sectional view taken along the line AA' in FIG. 7. This figure shows a case where one set of detection coils is used.

At this time, it is necessary to take care not to rotate the fine particle mass, and it is necessary to take into account errors caused by rotation.

Incidentally, it is also possible to use, for example, an oxide superconductor as the superconductor material used in the SQUID. For example (R
Tl:1. A high temperature superconducting material having an oxygen-deficient perovskite structure represented by a general formula such as -X 2 Mx 2 xuo3-X or a 2NiF type structure may be used.

In the above general formula, RE is La, Y, Sr.

Yb, Lu, Tm, Dy, Sc, Ce, Pr.

It represents an element such as Nd, Sm, Eu, Gd, Tb, Ho, Er, etc., and M represents an element such as Ba, Sr, Ca, etc.

In this example, in order to obtain a superconducting state, SQUI
D (detection coil and SQUID element) is cooled with liquid nitrogen, and in order to prevent the flow cell from freezing, a heat insulating material 13 with good magnetic permeability is installed between the detection coil and the flow cell.
was placed.

Using the high temperature superconducting materials described above, such cooling and insulation can be simplified or eliminated.

By measuring the magnetic intensity of the agglomerates using the magnetometer 6, the number of particles (agglomeration degree) contained in each agglomerate can be easily determined. At the same time, the distribution of agglomeration degree is also determined. The measurement results are shown in FIGS. 2 and 3. As seen in FIG. 2, the output signal varies in size depending on the number of particles collected in the aggregate.

From this distribution of the degree of aggregation, the proportion of agglomerates consisting of two or more fine particles is calculated to determine the aggregation ratio.

When the determined aggregation ratio was plotted against the degree of AFPa, a correlation as shown in FIG. 4 was obtained.

Similarly, A in heparin-treated blood (including red blood cells, etc.)
FP measurement was performed, and the size of the aggregate could be accurately measured without any problems, and a correlation curve similar to that for serum was obtained.

Therefore, according to this example, the state of aggregation of microparticles due to antigen-antibody reaction can be measured without being affected by light scatterers and absorbers and two light emitters such as dyes that coexist in the sample.

It is also effective for measuring hemolyzed samples and chyle serum samples.

〔Effect of the invention〕

According to the present invention, it is possible to accurately measure the size and distribution of aggregates generated by antigen-antibody reactions without being affected by light scatterers such as proteins and blood cells, absorbers such as dyes, and fluorophores contained in the sample. It is effective in measuring antigen concentration with high accuracy.

[Brief explanation of the drawing]

FIG. 1 is a conceptual diagram of the configuration of an apparatus according to an embodiment of the present invention, FIG. 2 is a diagram showing an example of a detection signal, FIG. 3 is an example of measurement of particle size distribution, and FIG. 4 is A F P ? 1III Diagram showing the regular rules, No. 5
The figure shows the configuration of the magnetic measuring section, FIG. 6 is a partial cross-sectional view of the magnetic measuring section of FIG. 5, FIG. 8 is a partial cross-sectional view of the magnetic measurement section of FIG. 7. FIG. DESCRIPTION OF SYMBOLS 1... Sheath flow cell, 2... Test liquid part, 3... Arrow showing the flow of sheath liquid, 4... Magnetic detection part, 5... Magnet, 6... SQUID magnetometer, 7...・Arrow indicating magnetization direction, 8 (81, 82, 83)...Detection coil, 9...
SQUID element, 10... Signal processing circuit, 11... A/D converter, 12... Personal computer, 13... Heat insulating material. 3rd country/The total cost was $25 [7th month]

Claims (1)

[Claims] 1. In an immunoagglutination quantitative device that measures the antigen concentration in a sample by binding an antibody to the surface of fine particles and reacting the antibody with a sample to measure the aggregation of the fine particles, Immunoaggregation quantification characterized by having means for dispersing the generated fine particle aggregates into aggregates and moving them according to the liquid flow, and a detection means for measuring the size of the aggregates in the liquid flow by magnetic measurement. Device. 2. The immunoagglutination quantitative device according to claim 1, wherein the antibody is bound to the surface of the fine particles by adsorption. 3. The immunoagglutination quantitative device according to claim 1, wherein the antibody is adsorbed onto the surface of the fine particles, and then the antibody molecules are bonded by chemical crosslinking treatment. 4. The immunoagglutination quantitative device according to claim 1, characterized in that an antibody is chemically bonded to the surface of the fine particles. 5. The immunoagglutination quantitative device according to claim 1, characterized in that the surface of the fine particles is coated with an organic polymer having a functional group, and the antibody is covalently bonded using this functional group. 6. The immunoagglutination quantitative device according to claim 1, wherein the fine particles are made of a magnetizable substance. 7. The immunoagglutination quantitative device according to claim 6, wherein the magnetizable substance is a ferromagnetic substance. 8. The immunoagglutination quantitative device according to claim 6, wherein the magnetizable substance is a metal. 9. The immunoagglutination quantification device according to claim 1, wherein the detection portion comprises a magnet that magnetizes the fine particles and a magnetometer that measures magnetic intensity. 10. The immunoagglutination quantitative device according to claim 9, wherein the magnetometer is a SQUID magnetometer. 11. The magnetometer has two or three detection coils orthogonal to each other.
11. The immunoagglutination quantitative device according to claim 10, characterized in that the immunoagglutination quantitative device is configured to be arranged in a direction.