JPS6035268A - Microcapsule reagent for immunological reaction and examination using the same - Google Patents

Abstract

PURPOSE:To make clear an agglutination transit image by mixing a microcapsule reagent with a relatively high specific gravity and a microcapsule reagent with a relatively low specific gravity to be used for the antigen-antibody reaction. CONSTITUTION:A microcapsule reagent comprises microcapsules with a high specific gravity d2 and microcapsules with a low specific gravity d1. Here, the gravities d1 and d2 are set at a value to meet the formulas of d1>=1.03 and d2<1.3, 0.08<=d2-d1<0.3 and 1.08<=(d1+d2)/2<0.3. In the antigen-antibody reaction using the microcapsule reagent, an agglutination image is formed by a microcapsule with the specific gravity d1 to fill the gap of a coarse agglutination image formed by the microcapsule reagent with the specific gravity d2. Thus, a clear and sharp agglutination image is obtained without roughness.

Description

Detailed Description of the Invention The present invention provides a group of microcapsule reagents in which a sensitized microcuff 0 cell exhibiting a high specific gravity is mixed with a sensitized microcapsule exhibiting a low specific gravity, and a method for producing antigens or antibodies using this reagent. Concerning testing methods for detection.

As a simple method for carrying out antigen-antibody reactions, immunological agglutination methods are widely used in which antigens or antibodies are supported on water-insoluble carriers and the state of agglutination based on the antigen-antibody reactions is observed with the naked eye. However, in the conventional method, animal-derived carriers are used, so there are disadvantages such as non-specific aggregation, variation in performance based on individual differences between animals, easy deterioration over time, and high cost. there were.

-Although the method using polystyrene latex as a carrier (latex agglutination reaction) eliminates the disadvantages mentioned above due to animal origin, it not only has lower sensitivity than the hemagglutination reaction, but also has poor long-term storage stability. , '-1 has drawbacks such as being prone to spontaneous agglutination reactions that are not based on antigen-antibody reactions.

For substitutes such as red blood cells and latex.

A method using microcapsules as a carrier was proposed. (Unexamined Japanese Patent Publication No. 55-94636, No. 57-196 (i)
1, 57-19662, etc.).

In this method, the above-mentioned drawbacks of solid carriers of animal origin and the disadvantages of synthetic carriers such as latex are solved, and if this microcapsule carrier method is fully applied, it will improve sensitivity, simplify operation, and enable on/off operation. New tests that are more difficult to improve than conventional methods are being developed, such as reliable discrimination of

JP-A-56-72346 describes a microcapsule reagent having a specific gravity within the range of 107 to 1.16.

Now, when a microcapsule reagent with a relatively high specific gravity outside this range and a microreagent with a relatively low specific gravity are mixed and used in an antigen-antibody reaction, the agglutination image obtained will be improved and the agglutination transition image will be clear. I found out that I can observe it.

The present invention is a pan-original or whole-sensitized antibody. High specific gravity t3zk
Showing microcapsules and low specific gravity d! The present invention relates to a high/low specific gravity microcapsule reagent comprising a microcapsule having the following characteristics, and having a specific gravity d1 and a specific gravity d2 satisfying the following formula.

d1≧103 ・(1) dl<1.3 ・・(2) 0.08≦d2−di(0,:3 ・(3) dl →−
d2 108≦□≦], , 18 (4) The present invention also relates to an antibody or antigen testing method characterized by using such a microcapsule reagent for an antigen-antibody reaction.

In the present invention, the microcapsules having a high specific gravity dz't may be smaller than 13 at the upper limit.

Microcapsules having a low ratio &d+ of 103 or more may be used. However, as shown in equation (3), d
ld, must be in the range of 0.08 or more and less than 03, and furthermore, the apparent specific gravity of the mixed microcassette as a whole must be 1.08 or more and 1 as shown in equation (4).
If you attack with 18 or less, there is no k.

Microcapsules with a high specific gravity dz't have a fast reaction rate and therefore tend to reach the bottom of the reaction vessel more quickly, and have a strong tendency to not cause a sufficient antigen-antibody reaction. Normally, the bottom of a flocculation reaction vessel is sloped so that it converges toward the center, but the flocculate formed by the microcapsule reagent with a high specific gravity d2 flows toward the center where the slope converges. It may roll off, causing disturbances in the aggregated image.

However, when one microcapsule with a low specific gravity d is mixed with this, an agglomerated image by the microcapsule reagent with a specific gravity dl is formed to fill the voids of the rough agglomerated image formed mainly by the microcassicle reagent with a high specific gravity d2. I can't stand it. Therefore, compared to an agglutinated image formed using only one type of microcassicle reagent having an intermediate specific gravity, a clear and sharp agglutinated image without any notches on one peripheral edge can be obtained.

When using the mixed microcapsule reagent of the present invention, such a clear agglutination image is clearly obtained, so that it is possible to easily distinguish between an agglutination image and a sedimentation without requiring any skill.

The preferred two roasting styles of the present invention will be explained in detail.

All microcapsules with a specific gravity of 12 are prepared as high specific gravity microcapsules. Since the wall thickness is at most 2% of the particle diameter, the specific gravity is almost determined by the specific gravity of the oily substance forming the core, so an oily substance that gives a specific gravity of 12 is appropriately selected.

Similarly, microcapsules with a specific gravity of 1.04 are prepared and mixed with high specific gravity microcapsules. The mixture of high and low specific gravity microcapsules is sensitized with an antigen or antibody according to the method described in "Immobilized Enzyme" by Kazue Chibata, Kodansha (1975), Japanese Patent Application Laid-Open No. 1966-1981. Note that high specific gravity and low specific gravity microcapsules may be sensitized and then mixed.

The microcapsules used as carriers in the present invention consist of a core of an oily substance (core material) and a wall material (insoluble in water and the core material) surrounding the core, and are generally prepared by the following procedure. The oily substance forming the core is mechanically emulsified and dispersed in a liquid with which it is not mixed, forming an oil-in-water emulsion. Liquids that do not mix with oily substances have the effect of aiding emulsification and improving the dispersion of oil droplets, and generally an aqueous polymer solution is used.

Microencapsulation is achieved by forming a polymer film wall insoluble in water and oily substance liquid on the surface of the oil droplets thus obtained.

Various emulsifying machines and dispersing machines can be used to emulsify and disperse the oily substance.

Typical methods for forming polymer films include: ■ chemical methods;
There are physicochemical methods and ■physical methods.

The wall material of the microcapsule is not particularly limited as long as it can chemically bond to the antigen or antibody without deactivating it and can be microencapsulated. For example, proteins (eg collagen) can be used as wall materials having amino groups or imino groups.

gelatin, casein, etc.), polyamino acids, polyacrylamide, polyamide, polyurethane.

Resin such as polyurea: As a wall material with hydroxyl groups.

Examples include cellulose and its derivatives (eg, methylcellulose, ethylcellulose, carboxymethylcellulose), Arabic comb, Denzon, and the like.

Oily substances that serve as core substances for microcapsules include natural mineral oils, animal oils, vegetable oils, and synthetic oils.

Since the surface of these core substances is completely covered with a wall material, they have no direct effect on antigens or antibodies, but it is preferable to avoid biochemically active substances. A specific example of the core material is disclosed in JP-A-56-72347.

57-19662, etc. For details on the production of microcapsules, see 1. For example, "Microcapsules" by Asashi Kondo, published by Nikkan Kogyo Shimbun (1971).
It is described in.

Also, the average size (diameter) of microcapsules is 0.5μ
It is desirable to select from the range of m to 20 μm, preferably 1 μm to 10 μm.

It is desirable that the solid content of the microcapsule carrier is usually within a range of about 1 to 3 by weight.

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

Example 1 4.5 g of noisozolobylnaphthalene and chlorination/'t' roughin (degree of chlorination 50%, Toyoparanox 150) 2
Oil-soluble red dye Oleanol Red BB (manufactured by Sumitomo Chemical) 0,25,!
7k dissolved. The obtained oily substance liquid was treated with maleic anhydride-methyl vinyl ether copolymer (GANTREZ).
AN-1'4.9, manufactured by General Aniline and Film Co.)2.5.9'! i = added to a solution dissolved in 75 ml of water. Stir, emulsify, and use Coulter Counter TA-
The size of oil droplets was measured using an It type, and the oil droplets were adjusted to have an average size of about 5 μm. Add to this 25 g of urea and 0.25 g of resorcinol! : A solution of 0.3 g of ammonium chloride dissolved in 25 ml of water was added. Additionally, 5 ml of water
+Add and dilute 7 ml of 37% formaldehyde aqueous solution
After adding lf, the mixture was reacted at 60° C. for 2 hours to perform microencapsulation. Thereafter, an IN sodium hydroxide aqueous solution was added to adjust the pH to 9.0, and the entire microcapsule was prepared.

The microcapsules thus prepared were centrifuged and washed with physiological saline to remove unreacted residues.

The microcapsule particles were dispersed in physiological saline to a concentration of 10%, and this was designated as microcapsule A.

Noisozorobylnaphthalene], 4.79 and chlorinated graphine (degree of chlorination 4.0%, Toyono Z Lux A-4
Microcapsules B'(z) were prepared under the same conditions as in the case of preparing microcapsules Af, except that a mixed oil (specific gravity: 104) with 103 g of 0) was used.

Preparation of comparative microcapsule C - 10.1 g of diisozorobylnaphthalene and 149 g of salt-X paraffin (degree of chlorination: 50%, Yoparax 150)
Microcapsules c1 were prepared under the same conditions as in the case of preparing microcapsules A, except that a mixed oil (specific gravity of about 1.12) was used.

When the specific gravity of the microcapsules was measured using a multilayer method in which specific gravity liquid was stacked in multiple stages on a column, microcapsules A and B were found.
, C had a specific gravity of 1.21°1.06 and 1.13, respectively.

Preparation of sensitized microcassicle reagent A: Propagate Rhesotosvira autumnalis desiccant strain A in Kortov's medium (containing 10% normal rabbit serum), and culture 6 to 6.
The culture solution on the 10th day was heated at 9.00 rpm for 20 minutes (5℃
) After centrifugation, the sediment was washed twice with physiological saline, redispersed in physiological saline, and crushed for 10 minutes using a 20 kHz sonicator (manufactured by Inugake Seisakusho).
The antigen solution was prepared so that the optical density at a wavelength of m was 0.2.

1.5 g of each of the microcapsules A and Bi prepared in Example 1 were mixed and dispersed in 20 ml of physiological saline. Next, 20 mNr of a 25% glutaraldehyde aqueous solution diluted 100 times with physiological saline was added, and after reacting for 45 minutes at 37°C, it was centrifuged and washed and redispersed in 3 ml of physiological saline.Aldehyde-treated microcapsules After adding 2 ml of antigen solution to 2 ml and incubating at 37°C for 90 minutes, it was left standing in a refrigerator at 4°C for 18 hours.Next, after washing three times with physiological saline containing 0.2% glycine, 2 ml of 3% Bovine serum albumin (BSA) and sodium nitride 00
1% 015M phosphate buffered saline (PBS, pi
-(==7.2) to obtain sensitized microcapsule reagent A.

Comparative sensitizing microcapsule reagent color: Comparative microcapsules C=i 1.5 g were taken and dispersed in 10 ml of physiological saline. To this dispersion, 10 ml of a 25% aqueous glutaraldehyde solution diluted 100 times with physiological saline was added, and the mixture was reacted at 37° C. for 45 minutes. After the reaction is complete, centrifuge and wash at 10m/! redispersed in physiological saline. The subsequent operations were carried out according to the conditions of Example 1 to obtain a sensitized microcassicle reagent color.

Example 2 The performance of the microcapsule reagent prepared in Example 1 was evaluated using the antibody titer obtained by the microtiter method using the antiserum described below.

(Preparation of antiserum) Rabbit' (f
Antiserum was prepared by immunization with 'i% degree. The Kortov medium culture solution of Desire A strain was centrifuged, and the precipitated bacterial bodies were suspended in physiological saline. This was subcutaneously injected into rabbits twice with anesthesia at intervals of 4 to 5 days, and intravenously injected 9 times over a further 4 to 5 days. 7 to 8 weeks after the first subcutaneous injection,
After confirming that the antibody had a predetermined antibody titer, whole blood was collected and antiserum was prepared.

(Test by microtiter method) The antibody titer of the reagent prepared in Example 1 was determined by the microtiter method. That is, tubes in which clear agglutination was observed were considered positive, and the highest dilution factor of the positive serum was calculated as the total antibody titer.

Add 10 ml of 25 μt of antiserum to each well of the microplate.
0x diluted solution containing 1% bovine serum albumin 0.15M
A series of multiple dilutions was prepared with further dilutions at 2-fold intervals with phosphate buffered saline.

Next, 25 of the microcapsule reagent A prepared in Example 1
The samples were collected using a μ7t drone and dripped into the tube holes of the antiserum dilution series of the micrograte. Microplate total 5
After shaking for a minute to advance the antigen-antibody reaction, the plate was left standing in a refrigerator at 4°C for 18 hours. Thereafter, it was taken out and placed on a light table with Micro Grate '6 to observe the agglomerated image at the bottom of the tube.

The same procedure was carried out for the comparative microcapsule reagent color, and the color was compared with the microcapsule reagent of the present invention.

Table 1 shows the antibody titer values obtained at the highest dilution of the serum that showed agglutination.

Microcapsules with high and low specific gravity are subjected to mixed wave sensitization, and the agglomerated image produced by the microcapsule reagent A of the present invention has less roughness compared to the comparative microcapsule reagent B.
A clear pattern is easy to see and there is no chance of misjudgment. Furthermore, the 1-microcapsule reagent A according to the present invention had high detection sensitivity.

Applicant for the above patents: Fuji Photo Film Co., Ltd. Agent: Patent attorney Gobe Sunakawa (and one other person) Procedures by Masako Sode (voluntary) Relationship with patent application (3) filed on August 5, 1980: Patent applicant fJE r'l! 210 Nakanuma, Minamiashigara City, Kanagawa Prefecture (520) Fuji Photo Film Co., Ltd. Name (
Name) Representative Minoru Ohnishi 4, Agent 5 Date of amendment order Self-issued 6-→Ejishi m-axis-11, 1 knowledge 7, subject of amendment, page 5, line 12, the following line is missing again Insert a verse.

[In the present invention, the mixing ratio of microcapsules having a high specific gravity dzi and microcapsules having a low specific gravity d+ may be 1 within the range where the specific gravity after mixing fully satisfies formula (4), and generally 1=3 to 1. Completion, preferably 1:2
The range is from ↓ to l:↓. ” and

Claims (1)

[Claims] 1. Sensitized with antigen or antibody. A microcapsule reagent for immune reactions comprising a microcapsule exhibiting a high specific gravity d2 and a microcapsule exhibiting a low specific gravity dl, wherein the specific gravity d and the specific gravity d2 satisfy the following formula. d1≧1.03 a2 (i3 0.08≦d 2 d 1 < 0.3 2 Sensitized with antigen or antibody. Microcapsule reagent consisting of microcapsules exhibiting high specific gravity d2 and microcapsules exhibiting low specific gravity d1) A method for testing antibodies or antigens, characterized in that microcapsules whose specific gravity d1 and specific gravity d2 satisfy the following formula are used for the antigen-antibody reaction: d1≧103 dz<1.3 0.08≦d2dt< 0.3