RU2169924C2 - Method for applying agglutination and immunological analysis - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: method involves applying hydrosol surface modification with soluble salts of metals having d-electron layer according to the periodical law when the surface has organic cations. Biologically specific ligand sorption on the hydrosol surface is applied with following modification of hydrosol compounds with biological liquid under test. Analysis results are interpreted. EFFECT: high sensitivity of analysis. 5 tbl

Description

 The invention relates to the field of biotechnology and medicine, can be used in the diagnosis of various infectious and somatic pathologies. With these diseases, antibodies arise in the patient’s body that are complementary to the antigens underlying the pathogenesis of the disease.

 These antibodies and antigens can be found in peripheral blood, however, to date, diagnostic preparations have not been created that are distinguished by the stability of immunochemical properties and allow this reaction to be carried out non-instrumentally. There are no reliable and simple methods for screening studies of patients and the population belonging to risk groups, which is important for the correction of therapy and determining the prognosis of the disease.

 The purpose of the invention is the development of a non-instrument express method for determining the presence and type of antibodies and antigens - markers of various nosologies, increasing the sensitivity of the analysis, reducing the time of analysis.

 An analog of this method is a method for determining various antigens or antibodies by labeling one of the components: antigens or antibodies complementary to them, hereinafter referred to as bioligands, with various labels - radiological, enzymatic, fluorescent, erythrocytic [1], latex [2].

 An analog of this method is a method for determining antibodies against tick-borne encephalitis virus in blood serum using the indirect hemagglutination reaction [3].

 An analogue of this method is also a method for determining anti-Rhesus antibodies in autoimmune hemolytic anemia [4] using a direct hemagglutination reaction.

An analogue of this method is a method for performing agglutination immunological analysis using hydrosols [5], ultrafine particles of an inorganic nature, for example, iron (III) oxide, Fe ₂ O ₃ , on the surface of which antigens or antibodies are adsorbed.

 The prototype of this technical solution is the method of setting agglutination immunochemical analysis described in [6].

This method involves the preparation of a suspension of iron oxide (III) Fe ₂ O ₃ in a buffer solution composed of a pair: an organic acid, for example, acetic acid, and its salt with some strong base, for example sodium acetate, according to this method, appropriate buffer solutions are prepared, for example, acetic acid is sodium acetate with a pH of from 4.0 to 5.0. Next, dispersion is carried out in this buffer: in 2 ml of 50 mg of iron oxide. Iron (III) oxide is incubated in this buffer for at least 12 hours for pre-activation. In this case, the surface of iron oxide is modified in an acidic medium and becomes a cation exchanger. After that, the bioligand to be sorbed is introduced; antigens or antibodies and adsorb by the mechanism of ion adsorption [5] for 1 to 24 hours, depending on the nature of the desired bioligand. At the end of sorption, iron oxide preparation with adsorbed biospecific preparations is washed from unbound bioligands - antigens (antibodies) - by washing repeatedly with the same buffer, followed by centrifugation, removing the supernatant and resuspending in the same buffer based on carboxylic acid and its salt. The resulting preparation is called a hydrosol, for example, an iron (III) oxide hydrosol, with a tuberculosis antigen adsorbed on it to detect complementary antibodies against M. tyberculosis antigens in human biological fluids.

 Then, the hydrosol is incubated with the studied sample of biofluid, for example, blood serum, and the content is determined by the agglutination of particles in the last separation using phosphate-saline [6] buffer solution with a pH from 6.8 to 7.6.

The disadvantages of the prototype can be considered:
1. The multi-stage and duration of the preparation of reagents;
2. Insufficient sensitivity of agglutination immunological analysis;
3. Long time analysis of the results of the analysis.

 These shortcomings are overcome if dispersed solid phases are used as dispersed solid phases for the subsequent sorption of biospecific preparations (bioligands): particles of metal oxides, hydroxides, phosphates, metal carboxylic acids, the surface of which is modified with soluble metal salts containing the d-electron layer of the Periodic system of elements D .AND. Mendeleev [7].

 These modifiers are used in the official pharmacopoeia as additives to multivitamins [8] (zinc, copper salts), specific antidotes for red phosphorus poisoning [9], lotions (bismuth and lead salts) [10], and specific disinfectants for surgical instruments [10] ( mercury salts). The use of these water soluble salts as modifiers of hydrosol particles has not been previously described.

 The invention is illustrated by the following examples of specific performance.

 Example 1

Carry out the sorption of bioligands on a dispersed preparation of iron oxide (III) Fe ₂ O ₃ . As the bioligand use measles γ-globulin (its F _{with a} fragment) according to the prototype and the proposed method. In the case of the prototype, a buffer solution of acetic acid and sodium acetate is used to ionically modify the surface of iron oxide, followed by the formulation of the agglutination reaction in phosphate-buffered saline pH 6.8-7.6.

The synthesis of the immunochemical preparation according to the claimed method is carried out using copper sulfate, mercury acetate, lead acetate, bismuth nitrate, chromium chloride, manganese nitrate, cobalt chloride. All the claimed modifiers — salts — contain a d-electron layer in the cations of their valence shells. Hydrosol agglutination reactions are also carried out here in phosphate-buffered saline. These reactions are carried out in the wells of a sloping bottom plate in the presence of "positive" and "negative" sera. In the wells of the tablet, serum called biological fluid is diluted. Serum is obtained by taking a small (not more than 0.1 ml) blood volume. The resulting blood is centrifuged to precipitate shaped elements. Acceptance of blood by venipuncture or capillary taking is allowed. Subsequent dilutions of "positive" and "negative" sera in titers from 1: 2 to 1: 1280 by dilution of both "positive" and "negative" sera with a buffer solution are performed so that the volume of the titrated biological product in the well is 50 μl. Then, 50 μl of a hydrosol preparation of iron oxide with sorbed γ-globulin is added to each separation. After incubation in a thermostat at 37 ^o C for 20 minutes, a visual assessment of the reaction or microscopy of the precipitate. A positive reaction of immunological agglutination analysis is the presence of agglutination at a dilution of 1: 2 and higher. The results of this experiment are presented in table 1.

 The presented example shows the advantages of the claimed technical solution in comparison with the prototype for greater sensitivity of the reaction and less time for its formulation.

 Example 2

The bioligands are sorbed on a dispersed preparation of iron oxide Fe ₂ O ₃ , as in Example 1. The same salts are used as modifiers, diphtheria toxoid and affinity-purified soapy antibodies against cancer-embryonic antigen are used as bioligands. Otherwise, the loading and subsequent analysis methods do not differ from those as in example 1. The results of this experiment are presented in table 2.

 Example 3

 Carry out the sorption of bioligands on a dispersed preparation of iron hydroxide (III) similarly to examples 1, 2, according to the prototype and the proposed method. As modifiers use compounds similar to examples 1, 2; as a bioligand, the antigen tuberculosis PPD in standard dilution is used. The results of this experiment are presented in table 3.

 Example 4

 Synthesize hydrosols based on calcium oxalate according to the prototype and the proposed method, similarly to the above examples 1-3. As biospecific ligands, mouse anti-rabbit immunoglobulin, cardiolipin antigen and diphtheria toxoid are used. The very formulation of the agglutination reaction according to the prototype and the proposed method does not differ from examples 1-3. The results of the experiment are presented in table 4.

 Example 5

Adsorption on dispersed particles of calcium phosphate by human γ-globulin (its F _with fragment) is carried out. As modifiers use salts of d-elements of copper and mercury, as well as salts of metals that do not contain d-electrons: sodium chloride and lithium, potassium and calcium nitrates, the experimental results are presented in table 5; An analysis of these data shows that the absence of d-valence electrons does not lead to satisfactory results in sensitivity and specificity.

 Thus, from the presented examples, the advantages of the technical solution are clearly visible compared with the prototype in terms of providing greater sensitivity of immunological agglutination analysis and less time for its formulation. Along with this, the expansion of the raw material base is ensured: it is possible to produce dispersed preparations not only on iron oxide, but also on hydroxides, phosphates and salts of carboxylic acids.

 The presented results indicate the possibility of using the proposed method in the diagnosis of titer and dynamic changes in the level of specific antigens and antibodies associated with a particular nosological unit. The data obtained can be important in screening diagnostics, determining the prognosis of the disease and correcting treatment.

Literature
1. Roy A. Fundamentals of immunology. - M.: Mir, 1991 .-- S. 91-93.

 2. Ed. Pokrovsky V.I. Immunology of the infectious process. - M .: Medicine, 1994 .-- S. 230.

 3. Nikitin V.M. Handbook of immunology methods. - Chisinau, 1982. - 304 p.

 4. Nikitin V.M. Handbook of immunology methods. - Chisinau, 1982. - 304 p.

 5. Meshandin A.G. Physico-chemical aspects of sorption processes in the synthesis of solid-phase bioligands. - M., 1994 .-- S. 31-34.

 6. R.U. 2044320 C1. September 20, 1995

 7. Slesarev V.I. Fundamentals of the chemistry of the living. - S. Petersburg, 2000 .-- p. 344-355.

 8. Mashkovsky M.D. Medicines (t. 2). - M.: 1993 - p. 8-18.

 9. Ed. Zakusov V.V. Clinical Pharmacology - M .: Medicine. 1978 - p. 334.

 10. Belikov V.G. Pharmaceutical Chemistry. - M .: Higher school. 1985 - p. 250-257.

Claims (1)

 A method of agglutination immunological analysis using water-insoluble hydrosol dispersed preparations containing inorganic cations, including surface modification of hydrosols, sorption of biospecific ligands on their surface, subsequent connection of the hydrosol with the biological fluid to be tested and evaluation of the analysis results, characterized in that soluble metal salts containing the d-electron layer of the Periodic system of elements are used as modifiers Mendeleev.

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