JPH0361906B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a carrier for adsorbing immunoactive substances, and more specifically to a carrier for adsorbing immunoactive substances made of benzoguanamine particles (hereinafter simply referred to as benzoguanamine particles), which are spherical fine particles of cured resin, or benzoguanamine particles whose surface has been modified with silica sol. Regarding carriers for use. Currently, carriers for adsorbing immunoactive substances, especially carriers for reagents used in immunological clinical tests, are made of polystyrene, polyacrylamide, poly- 2
- Fine particles made of materials such as hydroxy methacrylate are frequently used. Among them, polystyrene latex was selected empirically from a variety of polymer materials,
Although it can be said to be the mainstream carrier for current immunological diagnostic reagents, because the particle surface is originally hydrophobic, it tends to cause self-agglutination reactions (non-specific agglutination reactions), and therefore, after sensitization with antigens and antibodies, There are problems such as detachment of . On the other hand, known methods for modifying the hydrophobic surface of fine particles include a topochemical method using the concept of in situ polymerization, a coating method using a precipitation reaction, a microencapsulation method, and a mechanochemical method. Also, JP-A-56-121322
``Surface treatment method for plastic fine powder'' discloses a colloid method in which the surface of 12 nylon fine particles is directly coated with titania sol or syria sol. The present inventors considered utilizing the colloid method as described below. By the way, in addition to synthetic polymer compounds, sheep red blood cells have been used as carriers for adsorbing immunoactive substances, but these have problems such as having heterophilic antigens and poor storage stability. In view of the above-mentioned current situation, the inventors of the present invention have conducted extensive research on various powder materials, and have found that they themselves have no immunological activity, do not cause agglutination reactions in liquid, and have uniform particle size. As a carrier material that is chemically stable and can withstand storage,
The present invention was developed after finding that benzoguanamine particles were suitable. The carrier for adsorbing immunoactive substances according to the present invention is a benzoguanamine formaldehyde condensate, and the main component is the following structure: It is a white fine powder represented by benzoguanamine particles with a specific gravity of 1.41 measured with a pycnometer. These particles are a carrier for adsorbing immunoactive substances that do not have the major drawback of polystyrene latex, which is a carrier for adsorbing microscopic immunoactive substances, that is, non-specific aggregation in liquid. In particular, benzoguanamine particles having an average particle size of 1.22 μm can be used as a carrier for immunological agglutination reactions. Furthermore, when the contact angle of benzoguanamine particles coated with silica sol in the present invention is measured on tablets obtained by vacuum compression molding of both particles, the contact angle is smaller than that of untreated benzoguanamine particles, and an increase in hydrophilicity is observed. This results in improved dispersibility of benzoguanamine particles in an aqueous medium and increased protein adsorption ability by coating with silica sol. Furthermore, benzoguanamine particles are cheaper than polystyrene latex, which is currently commonly used as carrier particles for immunological clinical test reagents, and can be said to be an excellent carrier for adsorbing immunoactive substances from an economical point of view. The present invention will be explained in more detail with reference to Examples below, but the present invention is not limited thereto. Example 1 Surface modification of benzoguanamine particles by silica sol treatment First, 1.0 g of benzoguanamine particles was placed in a beaker, and a silica sol aqueous solution diluted with dilute hydrochloric acid of PH 2.4 to concentrations of 0.125, 0.25, 0.5 and 1.0% (v/v) was added to it. After adding an appropriate amount and kneading well to form a paste, an aqueous silica sol solution was further added to bring the total volume to 50 ml. The benzoguanamine particles used here were cured resin spherical fine particles (average particle size 1.22 μm,
It has a specific gravity of 1.41) and is manufactured by Nippon Shokubai Chemical Co., Ltd. The silica sol was manufactured by Nissan Chemical Industries, Ltd., with an average particle size of 64 nm and a SiO ₂ content of 20.
%, (SiO ₂ /Na ₂ O) = 165, PH2.4. this
After stirring for 15 minutes to ensure good dispersion, the dispersion was allowed to stand at 25°C for 30 minutes and filtered. Furthermore, the benzoguanamine particles were washed several times with 30 ml of diluted hydrochloric acid having a pH of 2.4, and finally washed with water and air-dried to obtain benzoguanamine particles treated with silica sol. by nitrogen adsorption
From S _N2 specific surface area measurements, benzoguanamine particles
Values of 15.7 m ² /g and 20.2 m ² /g were obtained for benzoguanamine particles treated with 1% (v/v) silica sol. Example 2 Measurement of the amount of silica sol adsorbed on benzoguanamine particles and the contact angle of untreated and treated benzoguanamine particles with water The amount of silica sol adsorbed on benzoguanamine particles calculated from the amount of unadsorbed silica sol is as shown in Table 1. . The amount of silica sol in water, which is a dispersion medium, is determined by measuring the absorbance at 600 nm, and the adsorption amount is calculated by calculating the amount of silica sol in the dispersion liquid and cleaning solution after treatment from the amount of silica sol in the silica sol aqueous solution for treatment. I deducted it.

[Table] To measure the contact angle, take approximately 0.3 g of each of untreated benzoguanamine particles and silica sol-treated benzoguanamine particles, and press them under a pressure of 200 kg/cm ² using a disk press (manufactured by Hitachi, Ltd.).
The test was carried out using tablets obtained by compressing tablets under reduced pressure for 20 minutes. The tablet was first placed horizontally on a sample stage of a precision contact angle measuring device (manufactured by Kyowa Kagaku Co., Ltd.), and a certain amount of purified water was gently dropped onto the tablet from a syringe equipped with a micrometer. Immediately after dropping, photographs are taken at appropriate time intervals to show the change in the advancing contact angle of the droplet over time. Next, the film is projected using a slide projector to determine the width and height of the droplet. The shape of the droplet was regarded as a part of a sphere, and the contact angle was calculated from the following formula. tanθ/2=2h/r θ: Contact angle h: Droplet height (mm) r: Droplet width (mm) The obtained contact angle values are plotted over time on a graph, and the droplet width is The extrapolated contact angle of the tablet forming raw material powder was determined by extrapolating from the time when the contact angle became apparently constant to 0 seconds. Untreated benzoguanamine particles, and 1.0%
The extrapolated contact angle values of benzoguanamine particles treated with (v/v) silica sol aqueous solution are 43° and 43°, respectively.
It was 30 degrees. From this result, it was considered that the surface of the benzoguanamine particles was modified to be more hydrophilic by treatment with the silica sol aqueous solution, contributing to improved dispersibility in an aqueous medium. In addition, the droplets dropped on the tablets of both raw material powders are
12-14 with untreated benzoguanamine particles after deposition
The benzoguanamine particles treated with a 1.0% (v/v) silica sol aqueous solution penetrated into the tablet in 6 to 7 seconds, and their wetting properties were compared to carrier particles using conventional organic polymer materials. This shows a significant improvement, suggesting that it is also useful as a carrier for column packing. Example 3 Adsorption of immunoactive substances by benzoguanamine particles and benzoguanamine particles treated with silica sol aqueous solution 0.2 g of benzoguanamine particles was placed in a test tube.
Add phosphate buffer (PH7.0, ionic strength I=
0.7: hereinafter abbreviated as PBS), 4.0×10 ^-2 %
(w/v) diluted antigen (human IgG) solution.
Add 20ml. After shaking the test tube for 2 hours, the dispersion is centrifuged at 3000 rpm for 15 minutes. The precipitate is 10
After washing twice with twice the volume of PBS, 0.5-1.0% (w/
It was redispersed at a concentration of v) to obtain an antigen-adsorbed benzoguanamine particle dispersion. The same operation was performed on the benzoguanamine particles treated with the silica sol aqueous solution to prepare antigen-adsorbing silica sol-treated benzoguanamine particles. The amount of adsorbed antigen calculated per 1 g of each benzoguanamine particle was determined by measuring the absorbance at 280 nm of the amount of antigen in the dispersion medium after the reaction.
When calculated by subtracting this from the amount of antigen before reaction, it was calculated to be 4.4 mg for benzoguanamine particles and 11.0 mg for benzoguanamine particles treated with silica sol aqueous solution.
The benzoguanamine particles treated with the silica sol aqueous solution adsorbed about three times as much human Ig-G as the untreated benzoguanamine particles. From this, it is clear that surface modification of benzoguanamine particles with silica sol increases their protein adsorption ability. Example 4 Aggregation reaction test using human Ig-G sensitized benzoguanamine particles Human Ig-G sensitized benzoguanamine particles obtained in Example 2 and Ig-G The presence or absence of specific aggregation ability and non-specific agglutination reactivity of unsensitized benzoguanamine particles was assayed using a glass agglutination reaction plate. The results are shown in Table 2. In addition, in the judgment, 3 minutes after mixing at a volume ratio of benzoguanamine particle dispersion liquid: test liquid: PBS = 1:1:1, a marked aggregation reaction is confirmed as +, and a case where the agglutination reaction is not clear is ± , The case where no agglutination reaction was observed was evaluated as -.
Contains 0.1% (w/v) fetal bovine serum albumin (hereinafter abbreviated as BSA) for non-specific agglutination reaction
Confirmed using PBS.

【table】

[Table] *2 Untreated benzoguanamine As is clear from Table 2, the human Ig-G-sensitized benzoguanamine particles did not cause a nonspecific aggregation reaction against BSA, regardless of whether they were treated with an aqueous silica sol solution. In addition, for anti-human IgG rabbit serum, the agglutination image was clear even in a 320-fold diluted serum, and these results indicate that benzoguanamine particles and benzoguanamine particles treated with a silica sol aqueous solution adsorb immunoactive substances. The usefulness as a carrier was confirmed.

Claims (1)

[Claims] 1. A carrier for adsorbing an immunoactive substance, wherein the carrier particles are benzoguanamine particles in the form of hardened resin particles. 2. The carrier for adsorbing an immunoactive substance according to claim 1, characterized in that benzoguanamine particles, which are spherical fine particles of a cured resin, are coated with silica sol.