JPS6217710B2 - - Google Patents

Description

[Detailed description of the invention]

JP-A No. 55-94636 proposes microcapsules having antigens or antibodies bound to their wall surfaces, and a method of detecting antibodies or antigens in a specimen by antigen-antibody agglutination using the microcapsules. . In the antigen-antibody agglutination reaction, the antigen is particulate,
For example, in the case of red blood cells or bacteria, or when antigens are bound or adsorbed to carriers such as red blood cell polystyrene latex or kaolin particles, these particulate antigens are crosslinked by antibodies and antigen particles aggregate together. , the reaction is about 100% lower than the antigen-antibody-precipitation reaction when the antigen is a soluble substance.
Because it is ~1000 times more sensitive, it is frequently used in clinical tests such as pregnancy tests and rheumatology tests. The subject of Japanese Patent Application No. 54-1555 is to use microcapsules as a carrier instead of the above-mentioned antigen-carrying carriers such as red blood cells and latex that have been conventionally used for antigen-antibody agglutination reactions. Proteins (e.g. collagen,
The use of gelatin, casein, etc.), polyamino acids, polyacrylamide, polyamide, polyurethane, polyurea, etc. is described. In this way, when microcapsules are used as carriers, the core material of the capsules is appropriately selected to achieve a desired specific gravity of 0.8 to 1.20.
Capsules of desired size 0.1-30μ preferably
Since it can be manufactured with a size of 0.5 to 10μ, it has generally been found that extremely significant improvements in sensitivity and accuracy have been observed in the detection of various antibodies or antigens in specimens using antigen or antibody agglutination reactions. Currently, when detecting antibodies or antigens in a specimen by antigen-antibody agglutination reaction using polyurethane wall, polyurea wall, or polyurethane urea wall microcapsules for detecting antigens or antibodies with antigens or antibodies bound to the wall surface, It was found that the detection sensitivity was extremely high. In other words, antibody detection sensitivity is determined by the minimum antibody concentration at which the antigen-bound capsules cause aggregation. This can be shown by measuring the minimum antibody concentration at which agglutination occurs (antibody detection sensitivity). That is, when polyurethane, polyurea or polyurethane urea resin is used as a wall material for microcapsules according to the present invention, a known diagnostic solution containing sheep red blood cells as carrier particles and an antigen chemically bound with glutaraldehyde, polystyrene latex, etc. Of course, when comparing diagnostic solutions made by using kaolin as carrier particles and adsorbing antigens to them, proteins (collagen, gelatin, casein), polyamino acid polyacrylamide resins, cellulose and its derivatives (methyl cellulose, carboxymethyl cellulose, etc.) It was found that the antibody detection sensitivity is extremely high even when compared to microcapsules whose walls are made of polymer compounds such as gum arabic, starch, and their derivatives. Furthermore, unlike when bacteria, sheep red blood cells, etc. are used as carriers, the microcapsules of the present invention exhibit no non-specific reactions or variations in quality, which are observed in such cases, and are highly accurate. It has advantages such as being able to be freely adjusted, being inexpensive, and being able to be stored for a long time. Oily substances that serve as capsule core materials include natural mineral oils, animal oils, vegetable oils, and synthetic oils. Since the surface of these core substances is completely covered by the capsule wall, they do not seem to have a 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 include 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-2153.635), 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. However, the capsule 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 preferably about 0.8 to 1.20, but is not particularly limited. A microcapsule carrier with an appropriate specific gravity is selected depending on the method for observing aggregation of capsule particles based on an antigen-antibody reaction. Furthermore, the average size of the microcapsule particles according to the present invention is between 0.1μ and 30μ, preferably between 0.5μ and 10μ.
Good μ range. EXAMPLES In order to further clarify the effects of the present invention, examples will be described below. However, the method for producing microcapsules according to the present invention is not limited to Example 1, Example 2, and Example 3. Example 1 0.1 g of ethylenediamine/propylene oxide adduct is dissolved in 25 g (specific gravity: approximately 1.10) of a mixed oil of 11.8 g of diisopropylnaphthalene and 13.2 g of chlorinated paraffin (degree of chlorination: 50%), and the mixture is cooled in advance with ice water. To this, an adduct of tolylene diisocyanate and trimethylolpropane, Desmodyur-L.
(trade name; manufactured by Bayer AG) 4 g of 50% methyl ethyl ketone solution was dissolved. Add this oily liquid to polyvinyl alcohol (saponification degree 88%, polymerization degree 500).
The mixture was poured into 65 g of % aqueous solution and stirred to emulsify. When the average size of the droplets became about 7 μm, the mixture was diluted with 100 g of water and reacted at 75° C. for 1 hour to encapsulate. The thus obtained polyurethane-walled microcapsules were soaked in phosphate buffer (8 g of NaCl, 0.2 g of KCl,
Dissolve 0.9 g of Na ₂ HPO ₄・12H ₂ O2 and 0.2 g of KH ₂ PO ₄ in water to make 1), then 0.5 g of the solution.
was dispersed in 5 ml of phosphate buffer. To this dispersion, 1 ml of 25 mg/ml of ovalbumin as an antigen was added, followed by 100 μl of glutaraldehyde (25%).
After addition, each sample was allowed to react for 1 hour at room temperature, centrifuged, washed with phosphate buffer, and then added to each sample again.
Disperse 0.5g in 5ml of phosphate buffer to give a concentration of approximately 10%.
Antigen-binding microcapsules were obtained. On the other hand, add one drop (25μ) of the phosphate buffer to each hole of the microplate using a dropper for 12 hours.
After dropping it over the column, take 25μ of rabbit antiserum against ovalbumin using a direuter.
Add to the diluted solution in the first column and stir thoroughly.
A 25μ sample was added to the diluted solution in the second column and thoroughly stirred, and then a 25μ sample was taken again and added to the diluted solution in the third column and thoroughly stirred. Continue this operation, 2 ¹²
The rabbit antiserum was diluted up to 1:1 to prepare a multiple dilution solution of the rabbit antiserum. Next, antigen-binding microcapsules diluted to 1%
25μ was added dropwise to the diluted antiserum solution in each row using a dropper, and the microplate was shaken well to thoroughly mix the antigen and antiserum, and then allowed to stand at room temperature. Thereafter, the bottom image of the tube was observed, and the minimum antibody concentration at which particle aggregation occurred (antibody detection sensitivity) was determined. Example 2 Carboxy-modified polyvinyl alcohol (molecular weight
100,000, degree of saponification 90%, carboxy group content 5-6%)
Add 2.5 g of urea, 0.25 g of resorcinol, and 0.3 g of ammonium chloride to 25 g of a 10% aqueous solution consisting of
After stirring and dissolving, the pH of the system was adjusted to 4.0 with a 0.1N aqueous hydrochloric acid solution. 25 g of the same mixed oil having a specific gravity of 1.10 as used in Example 1 was emulsified in the above aqueous solution with vigorous stirring to prepare an O/W type emulsion, and the drop size was adjusted to about 7 μm in diameter. To this emulsion, 6.4 g of a 37% formaldehyde aqueous solution was added, and the temperature of the system was adjusted to 60° C., followed by encapsulation for 2 hours. The prepared polyurea wall microcapsule solution was washed three times with phosphate buffer to remove residual formalin, urea, protective colloid, etc. Next, the antigen was bound in the same manner as in Example 1, and the detection sensitivity of the antibody was measured. Example 3 0.1 g of ethylenediamine/propylene oxide adduct is dissolved in 25 g (specific gravity: approximately 1.10) of a mixed oil of 11.8 g of diisopropylnaphthalene and 13.2 g of chlorinated paraffin (degree of chlorination: 50%), and the mixture is cooled in advance with ice water. To this, an adduct of tolylene diisocyanate and trimethylolpropane, Desmodyur-L.
(trade name, manufactured by Bayer AG) 4 g of 50% methyl ethyl ketone solution was dissolved. Add this oily liquid to polyvinyl alcohol (saponification degree 88%, polymerization degree 500).
The mixture was poured into 65 g of a 1% aqueous solution and stirred to emulsify. When the average drop size reached about 7 μm, 100 g of a 1% aqueous solution of hexamethylene diamine was added and reacted at 75° C. for 1 hour to perform encapsulation. The thus obtained polyurethane/urea wall microcapsules were washed three times with phosphate buffer, and then antigen was bound in the same manner as in Example 1, and the antibody detection sensitivity was measured. Comparative Example 1 5g of acid-treated gelatin and 5g of gum arabic at 40℃
Dissolved in 40 g of warm water and emulsified therein 50 g of a mixed oil having the same specific gravity of 1.10 as used in Example 1,
Make an emulsion with an average drop size of 7.0μ. 213 g of water at 40° C. are added to this emulsion and the pH is then adjusted to 4.60 with acetic acid. After cooling the system to 10° C., 2 g of 3.7% formalin was added for dura mating. Further, 40 g of a 10% solution of carboxymethyl cellulose (degree of polymerization 220) was added, and the mixture was adjusted to Ω=10 with a 10% aqueous casein soda solution, heated to 50° C., and left to stir for 1 hour. The gelatin-walled microcapsule solution thus prepared was washed three times with phosphate buffer to remove residual formalin and protective colloid. An antigen was bound to the capsule thus obtained in the same manner as in Example 1, and then the detection sensitivity of the antibody was measured. The results of measuring the antibody detection sensitivity of Example 1, Example 2, Example 3, and Comparative Example are shown in the table below.

[Table] As is clear from the above table, polyurethane wall, polyurea wall, and polyurethane/urea wall antigen-binding microcapsules have an extremely high antibody detection rate of 8 times or more than gelatin wall microcapsules, and in the case of polyurethane, 32 times as much. showed sensitivity.

Claims (1)

[Scope of Claims] 1. A polyurethane wall, a polyurea wall, for antigen or antibody detection, which has an antigen or antibody bound to the wall surface.
or polyurethaneurea-walled microcapsules. 2. A method for detecting antibodies or antigens in a specimen by an agglutination reaction, which is characterized by using polyurethane-walled, polyurea-walled, or polyurethane-urea-walled microcapsules with antigens or antibodies bound to the wall surface.