CS223295B1 - Immunoreangent - Google Patents

Description

The invention relates to the use of microparticles based on hydrophilic polymers in immunology, in particular for the determination of phagocytic activity.

Phagocytosis is one of the immunological amplification systems an important means of defense of the organism. Clinical testing of the level of phagocytosis is becoming an important criterion for assessing the patient's immune status. There are a number of diseases that are accompanied or directly caused by defects in the phagocytic ability of macrophages and microphages. These are primary defects, ie directly in the functionality of these cells, usually their cytoplasmic membranes, and second defects consisting of dysfunction of opsonization or chemotactic factors such as agammaglobulinaemia, defects of the complement system, septic conditions, cancer, etc.

Currently, a number of methods based on phagocytosis of various materials are used to determine phagocytic activity. Unfortunately, there are shortcomings in each of the prior art techniques that make it difficult to interpret the results correctly and obscure this significant clinical immunology test.

Phagocytosis of cadmium carbonate microcrystals (CdCCU), Candida albicans or Saccharomyces cerevisiae is the most widely used in clinical practice. When using CdCO 3 microcrystals, the basic drawback is the difficulty in preparing a standard suspension of cadmium microcrystals, since the possible release of free cadmium leads to a sharp inhibition of phagocytosis. Another drawback of this procedure is the dependence on autologous serum. In addition, some cell damage occurs during the test, so the interpretation of the results is highly unreliable. The disadvantages of the two remaining techniques are common: difficult preparation of phagocytosed particles, necessity of their constant cultivation, some non-standardity of the method in different environments and also dependence on opsonization.

The aforementioned disadvantages of the conventional methods are eliminated by the method according to the invention.

The object of the invention is to use microspheres based on hydrophilic polymers 0.05 to 3.0 µm in size as an immunoreactive agent for determining the properties and functions of the plasma membrane of cells involved in immune processes and for cell separation.

Microsphere particles of hydrophilic polymers 0.05 to 3.0 μπι suitable for

22329S used in immunology are prepared by radiation polymerization, under the influence of χ radiation from a cobalt source, a composition composed of 90 to 99% by weight. water or a mixture thereof with from 0.01 to 20% by volume of an alcohol having a C number of 1 to 4, 1 to 10% by weight of water; % monomer mixtures, composed of 25 to 99.5 wt. % of hydrophilic monomers, 0.01 to 40 wt. % ionogenic comonomers and 0.1 to 35 wt. % crosslinker and 0.01 to 2 wt. a polymer additive having a molecular weight of 10,000 to 200,000 g / mol.

Hydrophilic monomers which may be used are glycol monomethacrylate, glycol monoacrylate, wherein glycol means not only ethylene glycol but also propylene glycol, butylene glycol, as well as diethylene glycol, triethylene glycol and other homologous polyglycols; glycerin monomethacrylate, N-monosubstituted methacrylamide, N-monosubstituted acrylamide, Ν, Ν-disubstituted acrylamide, wherein the substituent may be C 1 -C 5 alkyl or C 1 -C 6 hydroxyalkyl containing 1 to 2 OH groups; 4-vinylpyridine, 2-vinylpyridine, 5-vinyl-2-methylpyridine; acrylamide, methacrylamide.

Ionogenic comonomers are methacrylic acid, acrylic acid, itaconic acid, maleic anhydride, diethylaminoethyl methacrylate, dimethylaminoethyl methacrylate, p-diethylaminostyrene; methacryloyloxyethyltrimethylammonium chloride.

It is preferred to use glycoldimethacrylate as the crosslinker, wherein the glycol is as defined above; glycol diacrylate, methylene-bis-acrylamide, ethylene-bis-acrylamide, trimethylolpropane triacrylate, triacryloylhexahydrotriazine.

The additive is preferably poly [N- (2-hydroxypropyl) methacrylamide], furthermore N-monosubstituted methacrylamide may be used, polyols N-monosubstituted acrylamide, polymers Ν, ub-disubstituted acrylamide, where the substituent may be C 1 -C 3 alkyl or hydroxyalkyl of 1 to 5 carbon atoms containing 1 to 2 OH groups, polyvinyl alcohol, polyacrylamide, polyethylene glycols, polypropylene glycols. The molecular weight of the additives is preferably in the range of 10,000 to 200,000 g / mol.

In the polymerization, the monomer mixture (polymerization mixture) is filled into a polymerization vessel, for example a glass vial, purged with nitrogen for about 15 to 20 minutes, and sealed (or otherwise sealed). At 0 to 40 ° C, the vial is placed in the center of the cobalt source and irradiated to a dose of 2 to 30 k Gy. After irradiation, the vial is opened, the contents of 10X are decanted with excess distilled water (decantation can also be carried out in two stages, first with a mixture of ethanol and distilled water, then with distilled water) and stored at 4 ° C.

The advantage of this polymerization process is the possibility of preparing pure microspheres. This is due to the fact that the process takes place in the absence of initiators or emulsifiers.

The principle of the newly proposed method is the phagocytosis of synthetically produced particles. This method removes the above-mentioned disadvantages of the processes currently used. The preparation of the particles is standard, the resulting suspension is completely homogeneous and stable (several years). Since it is a synthetic material, the method is not dependent on opsonins.

The particles are spherical, homogeneous in size, and can be prepared in various sizes by simply varying the concentration of the starting reagents. In the basic embodiment, they have a slight negative charge, which, due to the negative charge of the cells, ensures their low non-specific adherence to the cell surface. The particles are readily stainable by conventional optical and electron microscopy staining techniques. The particles can be fluorescently labeled and then labeled to detect cells using fluorescent techniques. Fluorescent labeling can be accomplished by conventional methods, for example using fluorescein isothiocyanate or dansylallylamine (in an amount of 0.01 to 5% by weight).

The particles contain free functional groups and can be additionally modified for specific binding to selected cell surface determinants.

The particulate stock suspension can be stored at 4 ° C and kept intact, ie non-agglutinated and uncontaminated, without sterile handling or use of antibiotics or preservatives for at least 2 years.

The durability of the suspension was verified at the Department of Immunology of the MBU CSAV from December 1978 to November 1981.

The phagocytosis test according to the invention is carried out by mixing 0.1 ml of fresh peripheral blood collected into heparin (5 µl / ml) with 0.05 ml of the particles of Example 1 (undiluted to a ratio of 4-5 X 10 ⁸ / ml in buffered saline). The mixture was allowed to incubate for 60 minutes at 37 ° C with constant shaking, after incubation, smeared on degreased slides and scored after May-Grunwald-Giemsy staining. The percentage of total phagocytic cells, 0 / o phagocytic cells of a particular type (FA) and phagocytic phagocytic index (FIF) are calculated.

The advantages of the novel phagocytosis test according to the invention are that the method is rapid and at the same time a blood count can be routinely evaluated. The method is not instrument-intensive and can be performed within the normal laboratory equipment. Only 0.1 ml of peripheral blood is needed per test, which prevents unnecessarily high blood donations, often leading to additional donor stress, which is especially important in children. Using May-Grunwald-Giemsa staining method, the particles stain strongly blue to blue-red, which makes it easy to differentiate from the nucleus and cytoplasm of cells. The method can be further standardized nationally, the techniques used so far have not allowed this step. The method was tested both at the immunological department of the MBU on healthy voluntary donors and at the Na Bulovce Hospital of Infectious Diseases on healthy donors aged 5 to 6 years and on donors (3 to 6 years) with otitis media chronica. The results show the difference in phagocytosis of white blood cell cells not only in young and adult donors, but also between healthy and sick individuals. According to the results so far, this method appears to be standard, reliable, inexpensive and fast, so it can be recommended for routine clinical practice.

In the following, the preparation of microparticles is illustrated by examples and the results of clinical tests are shown in the tables.

Example 1 g of a blend consisting of 77.8 wt. % Of 2-hydroxyethyl methacrylate, 0.1 wt. % ethylene dimethacrylate, 2 wt. % methylene-bis-acrylamide, 0.1 wt. poly [N- (2-hydroxypropyl) methacrylamide] having an average molecular weight of 70,000 g / mol and 20% by weight methacrylic acid was dissolved in 95 ml of water, filtered, the solution was placed in a glass vial, bubbled with purified nitrogen for 15 minutes. The mixture was irradiated at room temperature from a cobalt source with a total dose of 10 k Gy, after which the vial was opened, the contents poured into an Erlenmeyer flask and decanted repeatedly to obtain microspheres having a diameter of about 0.9 µm.

Example 2 g of a mixture consisting of 50 wt. % Of 2-hydroxyethyl methacrylate, 8.5 wt. % ethylene dimethacrylate, 40 wt. % acrylamide and 1.5 wt. poly [N- [2-hydroxypropyl] methacrylamide] having an average molecular weight of 30,000 g / mol was dissolved in 97 ml of water. The procedure was as in Example 1.

Example 3 g of a mixture consisting of 77.9 wt. % Of 2-hydroxyethyl methacrylate, 0.1 wt. % ethylene dimethacrylate, 2 wt. % methylene-bis-acrylamide and 20 wt. methacrylic acid was dissolved in 95 ml of water. The procedure was as in Example 1. Particles with a diameter of about 1 ₍ .mu.m) were obtained.

Example 4 g of a mixture consisting of 76.7 wt. % 2-hydroxyethyl methacrylate, 10 wt. % 2- (hydroxyethoxy) ethyl methacrylate, 10 wt. % diethylaminoethyl methacrylate, 0.3 wt. % ethylene dimethacrylate, 3 wt.

(ethoxyethyl) dimethacrylate was dissolved in 96 ml of water. The procedure was analogous to Example 1.

Example 5 g of a mixture consisting of 60 wt. 2-hydroxyethyl methacrylate, 15% by weight, N- (2-hydropropyl) methacrylamide, 0.5% by weight, ethylene dimethacrylate, 3.5% by weight methylene-bis-acrylamide, 20% by weight 4-vinylpyridine and 1% by weight polyethylene glycol Dissolve in 97 ml of a solvent consisting of 90% water and 10% methanol and proceed as in Example 1 except that the total dose of irradiation was 7 k Gy.

Table I

Effect of particle size on the degree of phagocytosis:

particle size μιη% phagocytic monocytes% phagocytic granulocytes

AND 0.35 63.7 58.4 (B) 0.45 57.9 66.7 C 0.8 55.2 68.7 D 0.9 66.7 65.8 E 1 60.2 69.2 F 1 60.0 64.7

Note: Particles A and B are too small for this test, the worse the particle count per cell, the less suitable for the phagocytosis test. A and B are specially designed for electron microscopy.

Table II

Comparison of some methods routinely used to determine phagocytic activity.

% phagocytic leukocytes

Note:

The values for Saccharomyces and CdCCb are for adult donors.

* particles according to Example 3;

** Otitis Chronic Media

Saccharomyces cerevisiae 44 to 62 CdCO3 13 to 15 Dec T “7 * 33 to 41 T-7 children 17 to 20 May Γ-7 patients ** (children) 7 to 8

Table III

Average values of phagocytic activity of peripheral blood cells in different donors.

Number of donor experiments *% phagocytic% phagocytic monocytes or granulocytes

13 R. P. J.H. X adults 57.6 44.2 46.0 62.1 59.9 60.0 health adults 12 A. L. 38.3 28.0 healthy P. H. 35.8 39.2 children X children (5 to 6 years) 38.8 32.6 12 J. L. 25.4 11.1 sick P. P. 17.3 17.7 children X patients (3 to 6 patients) ) 20.1 13.5 * was monitored 13 (12) donors in a group; two of them are listed as an example

Figure 1 shows a positive macrophage (cell left) and two negative lymphocytes from mouse peritoneal washings. Crystal violet staining. Incubate for 60 minutes, 37 ° C, particles prepared according to Example 3.

Fig. 2 shows the individual phases of phagocytosis of the microspheric particle by mouse peritoneal macrophage.

P - Particles prepared according to Example 3 V - Vacuum Incubation 60 minutes, 37 ° C Electromicroscopic image

Claims (1)

SUBJECT Use of microspheres based on hydrophilic polymers 0.05 to 3.0 µm in size as an immunoreagent for the determination of OF THE INVENTION The properties and functions of the plasma membrane of cells involved in immune processes and for cell separation.