JPS6219703B2 - - Google Patents

Description

[Detailed description of the invention]

JP-A No. 55-94636 proposes a microcapsule with an antigen or antibody bound to its wall surface, and a method of detecting an antibody or antigen in a specimen by an antigen-antibody agglutination reaction using the microcapsule. . In the antigen-antibody agglutination reaction, when the antigen is particulate,
For example, in the case of red blood cells or bacteria, or when antigens are bound to or adsorbed to carriers such as red blood cell polystyrene latex or kaolin particles, these particulate antigens are crosslinked by antibodies, causing antigen particles to aggregate with each other. Because it is about 100 to 1000 times more sensitive than the antigen-antibody-precipitation reaction when the antigen is a soluble substance, it is frequently used in clinical tests such as pregnancy tests and rheumatoid arthritis tests. Microtiter systems and glass plate methods are known for detecting antigens or antibodies in serum using agglutination reactions, but the former is extremely useful for clinical testing because it can measure them semi-quantitatively. This is a useful method. According to this method, serum from a patient and a healthy individual are diluted and arranged on a microplate, and then a certain amount of antigen-binding particles is added. After shaking this, observe the precipitation state of the particles and observe whether it is negative or positive. If particle precipitation clearly appears in a dilution series of the patient's serum, the test is negative, and if the particles aggregate and spread over an area, the test is positive. However, when a patient tests negative, it takes a long time, about 4 to 5 hours, for particles to precipitate using conventionally used carriers. Therefore, it would be highly desirable to reduce the time required for particle precipitation by even one hour for the implementation of the microtartar system. The specification of JP-A No. 55-94636 describes the use of microcapsules as carriers in place of the above-mentioned carriers for carrying antigens or antibodies, such as red blood cells or latex, which have been conventionally used in antigen-antibody agglutination reactions. However, the use of proteins (eg, collagen, gelatin, casein, etc.), polyamino acids, polyacrylamide, polyamide, polyurethane, polyurea, etc. as capsule wall materials is described.
The particle size of these capsules is 0.1 to 30.
μ is preferably 0.5 to 10μ, and the specific gravity is
A value of 1.05 to 1.20, preferably 1.10 to 1.17 is proposed. Therefore, it is clear that the invention had a great advantage in that it could be freely selected and manufactured. Now, microcapsules for detecting antigens or antibodies with a specific gravity in the range of 1.07 to 1.16, more preferably 1.11 to 1.15, which have antigens or antibodies bound to the wall surface, can be used to detect antigens or antibodies using a microtiter system. It was found that it can be used advantageously for measurement. That is, the object of the present invention is a microcapsule for detecting an antigen or antibody having a specific gravity in the range of 1.07 to 1.16, more preferably in the range of 1.11 to 1.15, and having a wall material carrying an antigen or antibody. It is also a method for detecting antigens or antibodies using a microtiter system characterized by using a capsule. In the present invention, proteins (eg, collagen, gelatin, casein, etc.), polyamino acids, polyacrylamide, polyamide, polyurethane, polyurea, etc. can be used as wall materials for microcapsules. Moreover, examples of the oily substance serving as the core substance of the capsule include natural mineral oil, vegetable oil, and synthetic oil. Since the surface of these core substances is completely covered by the capsule wall, they have no direct effect on antigens or antibodies, but it is preferable to avoid biochemically active substances. Examples of mineral oils include petroleum, kerosene, gasoline, naphtha, and paraffin oil; examples of animal oils include fish oil and lard oil. Examples of vegetable oils are peanut oil, linseed oil, soybean oil, castor oil and corn oil. Examples of synthetic oils include biphenyl compounds (e.g. isopropyl biphenyl, isoamyl biphenyl) and terphenyl compounds (e.g.
OLS-2153635), naphthalene compounds (e.g. diisopropylnaphthalene, US-4003589), alkylated diphenylalkanes (e.g. 2,4-dimethyldiphenylmethane, US-3836383), phthalic acid compounds (e.g. diethyl phthalate, dibutyl phthalate, dioctyl phthalate), chlorinated paraffin, etc. The capsule inner core material used in the present invention is not limited to those described above. Capsules can be manufactured, for example, by the general method described in "Microcapsules" by Kondo et al., Sankyo Publishing Co., Ltd. (1978). In addition, the bond between microcapsules and antigens or antibodies is explained in "Immobilized Enzymes" by Ichiro Chibata, published by Kodansha (1975).
The method described in, etc. may be used. The specific gravity of the microcapsule particles used in the present invention is
It can be easily adjusted by changing the specific gravity of the core material, which is selected within the range of 0.07 to 1.16. Further, the average size of the microcapsule particles according to the present invention is 0.1μ to 30μ, preferably 0.5μ to 10μ.
A range of is good. EXAMPLES Hereinafter, in order to make the effects of the present invention even clearer, examples will be described. However, the method for producing microcapsules according to the present invention is not limited to Examples 1 to 5. Example 1 10% aqueous solution of maleic anhydride-methyl vinyl ether copolymer (GANTREZ-AN139. Molecular weight approximately 25,000 General Aniline/manufactured by And Film Co., Ltd.)25
2.5 g of urea, 0.25 g of resorcinol, and 0.3 g of ammonium chloride are added to 10 g, stirred, and dissolved to 20%.
The pH of the system was adjusted to 4.0 with aqueous caustic soda solution. To this, a mixed oil of 11.8 g of diisopropylnaphthalene and 13.2 g of chlorinated paraffin (chlorine content 50%) was emulsified and dispersed in the above aqueous solution to form an oil-in-water emulsion, and stirring was stopped when the drop size was around 7 μm on average. To this emulsion were added 25 g of water and 6.7 g of a 37% formaldehyde aqueous solution, and after adjusting the temperature of the system from room temperature to 65° C., it was allowed to react for 2 hours. The thus obtained microcapsules having a specific gravity of about 1.10 were washed three times with a phosphate buffer having the composition shown below to remove residual formalin protective colloid and the like. Composition of phosphate buffer NaCl 8g KCl 0.2g Na ₂ HPO ₄ .12H ₂ o 2.9g KH ₂ PO ₄ 0.2g Add H ₂ o to 1 Take 0.5g of washed microcapsules and add 5ml
redisperse in phosphate buffer. To this was added 1 ml of a 1% aqueous solution of egg albumin (Egg Albumin 5X Crystal Seikagaku Kogyo KK), which is an antigen, followed by 100 μl of glutaraldehyde (25%), followed by reaction binding for 1 hour at room temperature. After centrifugation, the microcapsules are washed with phosphate buffer, and 0.5 g of microcapsules are again dispersed in 5 ml of phosphate buffer. Examples 2 to 5 and Comparative Examples 1 to 2 were carried out in the same manner as in Example 1, except that the composition of the core oil (mixing ratio of diisopropylnaphthalene/chlorinated paraffin) was changed as follows.
Microcapsules and antigen conjugates of various specific gravities were produced. Then, sheep red blood cells are used as a carrier.
1.10 was used as a comparative sample as Comparative Example 3.

[Table] Lopilnaphthalene
(g)

[Table] Evaluation of particle aggregation by antigen-antibody reaction 25μ of 1% antigen-binding microcapsules prepared by the above method were collected with a dropper, and dilution series of rabbit antibodies (positive sample) was collected on a microplate.
and a dilution series without antibody (negative samples), and shake the plate well to mix. The time until a positive image and/or negative image appeared was measured while the sample was left at room temperature. In addition, for discrimination ability, we observe whether positive and negative samples cause agglutination reaction or sedimentation, and determine whether it is (+) or (-) and whether or not the discrimination between positive and negative samples is correct. The discrimination ability of microcapsules was evaluated. The evaluation of particle aggregation based on the above is summarized in the table below.

【table】

[Table] As can be seen from the above results, the antigen-binding microcapsules of the present invention having a specific gravity in the range of 1.07 to 1.16 only have a small amount of discrimination time required for sheep red blood cells in the case of a specific gravity of 1.07; All of them are extremely good, lasting about 1 to 2 hours, especially from 1.11 to 2 hours.
Those having a specific gravity in the 1.15 range have a discrimination ability comparable to that of sheep red blood cells, but the time required for discrimination is only about 1 hour, which is extremely excellent. The method using microcapsules as a carrier did not cause non-specific reactions and had extremely high reproducibility compared to the method using sheep red blood cells. In other words, considering the inspection accuracy as well, the specific gravity is 1.07.
Antigen- or antibody-bound microcapsules with a specific gravity in the range of ~1.16 are superior to sheep red blood cells as a method for detecting antibodies or antigens, and further, antigen- or antibody-bound microcapsules with a specific gravity in the range of 1.11 to 1.15 are extremely superior.

Claims (1)

[Claims] 1. Specific gravity of antigen or antibody bound to wall surface
Microcapsules for antigen or antibody detection within the range of 1.07 to 1.16. 2 Specific gravity of antigen or antibody bound to the wall surface
Microcapsules for antigen or antibody detection according to claim 1 within the range of 1.11 to 1.15. 3 Specific gravity of antigen or antibody bound to the wall surface
1. A method for detecting an antibody or antigen using a microtiter system, characterized by using a microcapsule for detecting an antigen or antibody bound to an antigen or antibody within the range of 1.07 to 1.16. 4 Specific gravity of antigen or antibody bound to the wall surface
4. A method for detecting an antibody or antigen using a microtiter system according to claim 3, characterized in that a microcapsule for detecting an antigen or antibody bound with an antigen or antibody within the range of 1.11 to 1.15 is used.