JPH06148189A - Magnetic particulate for laser magnetic immunological measurement and its manufacture - Google Patents

Abstract

PURPOSE:To provide a magnetic particulate for laser magnetic immunological measurement having strong magnetic characteristic and excellent dispersing property by bonding a plurality of aminated dextran-coated magnetic particulates. CONSTITUTION:A surfactant is added to aminated dextran-coated magnetic particulates and a bifunctional cross-linking agent, mixed and stirred in oil to generate droplets. The cross-linking reaction is caused between the amino groups of aminated dextran in the droplets to mutually bond a plurality of aminated dextran-coated magnetic particulates. The plurally bonded particulates of large particle size remarkably increase the sensitivity in laser magnetic immunology. This reason is considered that the magnetic characteristic per magnetic particulate is proportional to the contained magnetic body quantity, and it can be avoided by the larger particle size that the sucking force of magnetic field is arrested arrested by Brownian movement. Thus, the magnetic particulate of large particle size having strong magnetic characteristic and good dispersing property is applied to laser magnetic immunological measurement, whereby its sensitivity is enhanced, and the reliability can be improved.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a magnetic fine particle for laser magnetic immunoassay used in a laser magnetic immunoassay, which is one of immunoassays utilizing an antigen-antibody reaction, and a method for producing the same.

[0002]

2. Description of the Related Art A conventionally known detection method utilizing an antigen-antibody reaction is radioimmunoassay (RI
A), enzyme immunoassay (EIA), fluorescent immunoassay (FIA), laser immunoassay (LIA) and the like have already been put to practical use. These methods are methods for detecting the presence or absence of an antigen to which isotopes, enzymes, and fluorescent substances are added as labels, respectively.

However, the above EIA method, FIA method,
The sensitivity of the LIA method is 10 ^-6 g to 10 ^-10 g at most, and the sensitivity is insufficient in the immunoassay for detecting the antigen itself such as the initial diagnosis of virus infection and a small amount of hormone test, which is a practical problem. was there. In addition, the RIA method has a sensitivity of 10 ⁻¹² g, which is sufficient for ultratrace analysis and antigen testing, but requires special equipment because it uses radioactive substances, and therefore safety, versatility, and There was a problem in terms of price, etc.

Therefore, the inventors of the present application can perform antigen test 1
A laser magnetic immunoassay method has been proposed as a highly versatile assay method having a sensitivity of 0 ^-12 g or more. (JP-A-63-79070, JP-A-63-106559, JP-A-63-108265, JP-A-63-188766, JP-A-63-315951, JP-A-1-29768, etc.)
In these new measurement methods, after antigen-antibody reaction, the antigen bound to polymer beads is labeled with magnetic fine particles, the polymer beads labeled with the magnetic fine particles are aggregated on the water surface by a magnetic field, and the amount of protrusion of the water surface due to aggregation is laser interference. This is a method of measuring the intensity of light. Therefore, since the above-mentioned laser magnetic immunoassay method is a highly sensitive displacement measurement due to the agglutination effect due to the magnetic field and the interference of the laser light, it is possible to detect an ultratrace amount whose sensitivity is equal to or higher than that of the RIA method.

Here, in the laser magnetic immunoassay method,
One of the important factors that influence the sensitivity is magnetic fine particles used as a labeling material, as is clear from the measurement principle. Regarding magnetic microparticles applied to laser magnetic immunoassay methods, Molday et al., US Pat.
There are magnetic particles coated with dextran disclosed in No. 73. The present invention shows that the magnetic fine particles are coated with dextran, and the dextran can be oxidized to bind an antibody or other protein to be used in a laser magnetic immunoassay method.

[0006]

Therefore, the inventors of the present application have improved this patent and have made earnest studies on magnetic fine particles applied to a laser magnetic immunoassay method. As a result, magnetic fine particles having a high magnetic susceptibility can be obtained.
41119, "Method for producing magnetic fine particles", Japanese Patent Laid-Open No. 3-2.
No. 42327 has filed a patent application as a "method for producing magnetic fine particles".

As a result of electron microscopic observation, it was found that a plurality of magnetites having a diameter of 10 nm or less were contained in the dextran having a diameter of about 100 nm or less as a result of the electron microscopic observation. However, since the amount of antigen bound to one polymer bead is small in the region where the antigen concentration is low, the amount of labeled magnetic fine particles also decreases accordingly, and the attraction force by the magnetic field to one polymer bead decreases. Therefore, in order to further increase the sensitivity, it has been desired to improve the “cohesiveness against magnetic field” (hereinafter referred to as “magnetic property”) of the magnetic fine particles.

As a method for improving magnetic characteristics,
(1) To improve the magnetic susceptibility of magnetite itself.
(2) There was a method such as increasing the size of dextran burying magnetite. However, with the method described in (1) above, the current magnetic susceptibility has been obtained at a value close to the theoretical limit, and improvement cannot be expected unless the material is changed. Although the method described in the above (2) has been studied up to now, it has become clear that it is difficult to increase the particle size of the magnetic fine particles themselves with good dispersibility by the present manufacturing method. It was Therefore, the present invention has been made in view of the above circumstances, and an object thereof is to provide magnetic fine particles for laser magnetic immunoassay having large magnetic properties and excellent dispersibility, and a method for producing the same.

[0009]

In order to solve the above-mentioned problems, the magnetic magnetic fine particles for laser magnetic immunoassay according to claim 1 capture an antigen with an antibody labeled with the magnetic fine particles, and then, this is subjected to a magnetic field on the water surface. The magnetic microparticles in the laser magnetic immunoassay method of agglomerating on top and measuring the amount of protrusion of the water surface by laser interference light intensity are characterized in that a plurality of aminated dextran-coated magnetic microparticles are bound together. It is a thing.

In order to solve the above-mentioned problems, the method for producing magnetic fine particles for laser magnetic immunoassay according to claim 2 is characterized in that the magnetic fine particles for laser magnetic immunoassay described in claim 1 are aminated dextran-coated magnetic fine particles. Prepared by mixing and stirring a bifunctional cross-linking agent in oil to form droplets, and then causing a cross-linking reaction between the amino groups of aminated dextran in the droplets to bond a plurality of aminated dextran-coated magnetic fine particles. It is characterized by doing.

In order to solve the above-mentioned problems, the method for producing magnetic fine particles for laser magnetic immunoassay according to claim 3 produces droplets in the method for producing magnetic fine particles for laser magnetic immunoassay according to claim 2. In this case, a surfactant is further added to the aminated dextran-coated magnetic fine particles and the bifunctional crosslinking agent, and the mixture is stirred in oil to generate droplets.

[0012]

The magnetic fine particles for laser magnetic immunoassay of the present invention are composed of a plurality of aminated dextran-coated magnetic fine particles bonded together. It is intended to improve the magnetic properties by increasing the body mass. Further, as described above, the magnetic fine particles of the present invention in which a plurality of magnetic fine particles are bonded together are prevented from being obstructed by the Brownian motion of the attractive force due to the magnetic field due to their large size, and the magnetic properties are remarkably improved. Is made possible.

Further, in the method for producing the magnetic fine particles for laser magnetic immunoassay of the present invention, the aminated dextran-coated magnetic fine particles and the bifunctional crosslinking agent are mixed and stirred in oil to form droplets, and then the droplets are formed. By causing a cross-linking reaction between the amino groups of the aminated dextran within, by binding a plurality of aminated dextran-coated magnetic fine particles, it is possible to produce a large particle size of the magnetic fine particles of the present invention, When the above-mentioned aminated dextran-coated magnetic fine particles and the bifunctional cross-linking agent are mixed and stirred in oil to form liquid droplets, a surfactant is further added to the magnetic fine particles having a large particle size to improve dispersibility. Can be efficiently manufactured.

Therefore, by applying the magnetic fine particles having a large particle size and good dispersibility to the laser magnetic immunoassay method as described above, the sensitivity of the laser magnetic immunoassay method can be increased.
The reliability of the data can be increased.

[0015]

The present invention will be described in detail below with reference to the drawings, but the embodiments disclosed below are merely examples of the present invention and do not limit the scope of the present invention. (Example 1) Hereinafter, a method for producing magnetic fine particles for laser magnetic immunoassay of Example 1 of the present invention will be described in detail.
First, aminated dextran was synthesized by the following method. Pharmacia Dextran 2 with an average molecular weight of 40,000
0 g of 0.05 M acetate buffer (pH 6.5) 1
It was dissolved in 00 ml and this was oxidized with 2.14 g of sodium periodate at room temperature for 1.5 hours. Oxidized dextran was diluted to 180 ml with pure water and adjusted to pH 9 or higher with glacial acetic acid.
Was reacted at 23 ° C. for 1 hour. After the reaction, the product was reduced with NaBH ₄ and quenched, and the solution was dialyzed against 4 liters of pure water for one day using a nitrocellulose dialysis membrane to remove unreacted substances.

Then, 5 ml of the aminated dextran obtained as described above was weighed and 0.75 g of ferric chloride hexahydrate and 0.32 g of ferrous chloride tetrahydrate were added to the aminated dextran. Was further mixed with 7.5% aqueous ammonia solution at room temperature with vigorous stirring and carefully 1 m.
l is added. When the pH is increased after the addition, magnetic fine particles are generated to form a black film. After the black film was formed, this temperature was gradually raised to 70 ° C. for 1 hour, and a total of 20 ml of 7.5% aqueous ammonia solution was added with vigorous stirring to prepare aminated dextran-coated magnetic fine particles.

Therefore, the method for producing large-diameter magnetic fine particles in droplets by the crosslinking agent of the present invention using the aminated magnetic fine particles obtained by the above method will be described in detail below. First, aminated dextran magnetic fine particles (iron concentration 10 ⁻⁶ g / ml) prepared as described above were diluted 10 times with a phosphate buffer solution (PBS), and 1 cc of this dilution
0.5 mg of Bis (sulfosuccinimidyl) suberate (hereinafter abbreviated as BS ₃ ) manufactured by Pierce is dissolved in the solution. In order to make the above-mentioned BS ₃ more soluble, it is advisable to mix BS ₃ previously dissolved in 30 μl of dimethylformamide.

Then, to the above mixture, commercially available salad oil 5
0 cc and 500 μl of a commercially available surfactant TWEEN 20 (0.1%) are added, and the mixture is vigorously stirred with a homogenizer to prepare a suspension of reverse micellar droplets containing magnetic fine particles. Further, this suspension is left to stand in a constant temperature bath at 37 ° C. for 30 minutes for crosslinking reaction. Then add 50 cc of ethanol,
Shake well, remove the lower layer (oil layer) with a separating funnel, add 50 cc of ethanol, and add 1 at 5000 rpm.
Centrifuge for 0 minutes and discard the supernatant. This operation is repeated twice, and finally the ethanol is replaced with a buffer solution containing 0.05% of bovine serum albumin (BSA-HEPES).

The average particle size of the droplets when the two liquids are suspended by a stirrer is R. Arshady's "Microspheres and Microcaps".
ules: A Survey of Manufacturing Techniques. '' (POLYM
ER ENGINEERING AND SCIENCE, VOL. 29. NO. 24, pp174
6-1758), it is known that it is expressed by the following equation. <D> ∝ k (Dv ・ R ・ vd ・ S) / (Ds ・ P ・
vm · C) where <d>: average particle size, k: device constant, Dv: diameter of container, Ds: diameter of stirrer, R: volume ratio of droplet to suspension, P: rotation of stirring Number (or power), vd:
Droplet clay, vm: suspension clay, S: surface tension between suspension and droplets, C: stabilizer concentration.

The material factors that determine the droplet size from the above equation are the clay of the droplet, the clay of the suspension, and the surface tension between the droplet and the suspension. Therefore, in the present invention, the concentration of aminated dextran-coated magnetic fine particles, the type of oil to be used, the type of surfactant, the concentration, etc. were investigated in a wide range.
The representatives illustrated in the above examples are these representative types,
It is a numerical value and is not regulated by this. For example, the oil that becomes a suspension is sesame oil in addition to commercially available salad oil,
Cottonseed oil, toluene / chloroform, cyclohexane / chloroform, etc. can be used. Further, the cross-linking agent may be any one as long as it cross-links amino groups with each other, and in addition to BS ₃ , disuccinimidyl suberate, dithiobissuccinimidyl pionate, or glutaraldehyde can be used.

Therefore, the average particle size of the large-sized magnetic fine particles obtained as described above was determined by the static optical confusion method. Table 1
Is the average particle size of the magnetic fine particles, and it can be seen that the magnetic fine particles obtained by the method of the present invention have a particle size about 4 times that of the conventional method. From this, it is considered that the magnetic fine particles prepared by the method of the present invention are obtained by combining the magnetic fine particles before the polymerization with about 4 to the third power and increasing the size.

[0022]

[Table 1]

The magnetic characteristics were evaluated by the laser magnetic immunoassay method. The laser magnetic immunoassay method is a method in which magnetic particles are aggregated at one point on the water surface in a constant magnetic field, and the aggregation force is obtained from the laser interference intensity corresponding to the amount of swelling. Therefore, the magnetic characteristics of the magnetic fine particles with respect to a constant magnetic field can be evaluated by the relationship between the amount of magnetite in the magnetic fine particles and the interference light intensity.

The measurement results are shown in FIG. (A) shows the measurement results of large-diameter magnetic fine particles obtained by the present invention, and (b) shows the measurement results of magnetic fine particles obtained by a conventional polymerization method. The vertical axis of FIG. 1 represents the intensity of the interference light, that is, the amount proportional to the magnitude of the magnetic characteristics, and the horizontal axis represents the measured iron concentration of the magnetic substance.
As is clear from FIG. 1, the large-diameter magnetic substance obtained in Example 1 has a magnetic substance amount for obtaining the same interference light intensity as compared with the magnetic fine particles synthesized by the conventional polymerization method. Is almost 1/100.

From the above results, it was found that increasing the size of the magnetic fine particles has the effect of dramatically increasing the sensitivity in the laser magnetic immunoassay method. this is,
This is because the magnetic property per magnetic fine particle is proportional to the amount of magnetic substance contained, and in the conventional polymerization methods,
It is considered that the magnetic fine particles obtained had a particle size of 100 nm or less, and this was influenced by the fact that the attractive force of the magnetic field was largely hindered by Brownian motion.

Example 2 In Example 2, the detection sensitivity of the magnetic fine particles for laser magnetic immunoassay obtained in the present invention in the immunoassay was measured using α-fetoprotein, which is one of the tumor markers. did. The sample preparation was performed by the following method. First, 20 μl of an antibody-bound polymer beads (bead concentration: 0.2 wt%) suspension, and a diluted solution of α-fetoprotein as an antigen (protein concentration: 10 ⁻¹⁵⁾
20 μl of each step of 10-fold dilution in the range of -10 ⁻⁸ g / ml, 8 steps) was reacted at 37 ° C. for 2 hours. Next, 20 μl of anti-α-fetoprotein antibody (antibody concentration 0.3 mg / ml) bound with biotin was added to each reaction solution, and 37
After reacting at 0 ° C. for 1 hour, the unreacted biotinylated antibody was separated by B / F.

Further, in each of the B / F separated reaction liquids, adipic magnetic particles (magnetite concentration: 0.1 mg / ml) obtained by previously binding adipine to the magnetic particles obtained in the present invention using BS ₃ manufactured by Pierce Co. ) Is added to 20 μl and 3
The reaction was carried out at 7 ° C for 1 hour. Then, after the reaction, the unreacted magnetic fine particles were separated by B / F, and the reaction product was recovered with 30 μl of the buffer solution and used as a measurement sample.

Each of the measurement specimens obtained as described above was measured by a laser magnetic immunoassay device to obtain the interference light intensity. The result is shown in FIG. On the other hand, using the magnetic fine particles obtained by the conventional method, prepare a specimen exactly the same as above,
Figure 2 shows the interference light intensity measured by the laser magnetic immunoassay system.
It shows in (b). As is clear from FIG. 2, the specimen using the large-diameter magnetic fine particles obtained in the present invention has a sensitivity of 1 in the low concentration region of the antigen as compared with the specimen using the conventional magnetic fine particles.
It can be seen that it has improved by more than an order of magnitude.

As described above, since the magnetic material for laser magnetic immunoassay according to the present invention has a large particle size and good dispersibility, when it is applied to the laser magnetic immunoassay method, its sensitivity is remarkably improved. Can be made.

[0030]

INDUSTRIAL APPLICABILITY The magnetic fine particles for laser magnetic immunoassay according to the present invention are composed of a plurality of aminated dextran-coated magnetic fine particles bonded together. The amount of magnetic material is increased to improve the magnetic characteristics. Further, as described above, the magnetic fine particles of the present invention in which a plurality of magnetic fine particles are bonded together are prevented from being obstructed by the Brownian motion of the attractive force due to the magnetic field due to their large size, and the magnetic properties are remarkably improved. Is made possible.

Further, in the above-mentioned method for producing the magnetic fine particles for laser magnetic immunoassay of the present invention, the aminated dextran-coated magnetic fine particles and the bifunctional cross-linking agent are mixed and stirred in oil to form droplets, and then the droplets are formed. By causing a cross-linking reaction between the amino groups of the aminated dextran within, by binding a plurality of aminated dextran-coated magnetic fine particles, it is possible to produce a large particle size of the magnetic fine particles of the present invention, When the above-mentioned aminated dextran-coated magnetic fine particles and the bifunctional cross-linking agent are mixed and stirred in oil to form liquid droplets, a surfactant is further added to the magnetic fine particles having a large particle size to improve dispersibility. Can be efficiently manufactured.

Therefore, by applying the magnetic fine particles having a large particle size and good dispersibility as described above to the laser magnetic immunoassay method, the sensitivity of the laser magnetic immunoassay method can be increased.
The reliability of the data can be increased.

[Brief description of drawings]

FIG. 1 shows the relationship between the amount of magnetite in magnetic particles and the intensity of coherent light based on Example 1 of the present invention, in which curve (a) is obtained by the production method of the present invention. The large-diameter magnetic fine particles and (b) are the measurement results using the magnetic fine particles obtained by the conventional method.

FIG. 2 is an α-based on Example 2 of the present invention.
It is an experimental result of the detection of fetoprotein antigen, and (a) in the figure is a large magnetic fine particle obtained by the production method of the present invention,
(B) shows the results using the magnetic fine particles obtained by the conventional method.

Claims (3)

[Claims] 1. The magnetic fine particles in the laser magnetic immunoassay method, wherein an antigen is captured by an antibody labeled with magnetic fine particles, and the antigen is aggregated on the water surface by a magnetic field to measure the amount of protrusion of the water surface by laser interference light intensity. A magnetic fine particle for laser magnetic immunoassay, comprising a plurality of magnetic dextran-coated magnetic fine particles bonded together. 2. The aminated dextran-coated magnetic fine particles and the bifunctional cross-linking agent are mixed and stirred in oil to form droplets, and then a cross-linking reaction occurs between the amino groups of the aminated dextran in the droplets to aminate. The method for producing magnetic fine particles for laser magnetic immunoassay according to claim 1, wherein a plurality of magnetic fine particles coated with dextran are bonded. 3. A droplet is formed by adding a surfactant to the aminated dextran-coated magnetic fine particles and the bifunctional cross-linking agent and mixing and stirring in oil when the droplet is formed. The method for producing magnetic fine particles for laser magnetic immunoassay according to claim 2.