RU2376604C2 - Method for detection of antigen or antibody in immune-enzyme analysis - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention is related to immunology. Substance of method consists in the fact that sensitin is immobilised on carrier, which represents submicron (0.1 mcm) monodisperse stable emulsion of perfluoroorganic compounds (PFOC) 10 wt % 0.5 ml. of PFOC prior to immobilisation is preliminarily released from salts and glucose by washing with tenfold volume of 0.15 M physiological solution pH 6.8. Sensitin is immobilised onto surface of purified emulsion drops (PFOC).

EFFECT: simplified performance of immune-enzyme analysis (IEA), with provision of high sensitivity of method at the same time.

2 ex

Description

The invention relates to medicine, in particular to immunology, and, in particular, can be used to detect various antigens and antibodies in an enzyme-linked immunosorbent assay in biological body fluids, environmental objects, etc.

From the practice of medicine, a method is known for detecting antigens and antibodies in radioimmunoassay (RIA) using plastics, cellulose, Sephadex as solid carriers of homologous antibodies or antigens (Fremel G. Immunological methods. - M .: Medicine, 1987. - 472 p.) .

However, the known method has the following disadvantages: the need to use radioactive isotopes, which pose a certain danger to personnel and the environment, a limited lifetime of the reagents. In addition, costly γ-ray recording equipment is required to take into account the results of the reaction.

Thus, the above disadvantages do not provide the opportunity to obtain a technical result - a simplification of the method.

There is also a known method for detecting antigens and antibodies in a homogeneous enzyme-linked immunosorbent assay - EMIT analysis, which consists in the fact that the immunological reaction of the interaction of antigens with antibodies and the detection of its course is carried out in a homogeneous solution. Under these conditions, there is no need for methods of physical separation of the antigen bound and not bound to antibodies. The main principles of this analysis are the mechanisms of steric exclusion of the substrate, antibody modulation of the action of substrates or inhibitors, induced by antibodies to the conformation of the active center of the enzyme (Gavrilova E.M. Journal of the D.I.Mendeleev All-Union Chemical Society, vol. XXVII, 4, 1982. - p. .90-97).

However, the known method has the following disadvantages: the need to use highly sensitive enzymatic systems, the significant influence of the physicochemical characteristics of antibodies on the possibility of the method (equilibrium and kinetic reaction constants of antigen-antibody), the content of a small admixture of free hapten or enzyme can significantly increase the background signal, a significant decrease in sensitivity method, if there are impurities in the preparation of the enzyme and the sample, such as an endogenous enzyme or substrate, cross natural reacting antigens, in the case of using macromolecular substrates (red blood cells, microbial cells), the sensitivity of the method is only 0.5-2 μg / ml.

Thus, the above disadvantages do not provide the opportunity to obtain a technical result - a simplification of the method.

The closest way to the proposed is a method for the detection of antigens and antibodies in an enzyme-linked immunosorbent assay using polystyrene tablets, tubes, beads, etc. as a solid carrier of sensitins (Ngo T., Lenhoff G. Enzyme-linked immunosorbent assay. - M.: Mir, 1988. - 446 p.).

However, the disadvantages of this method are:

- the complexity of the process of preparing a sensitized surface of a solid carrier;

- the need for the manufacture of solid polymer carriers of sensitins (tablets, beads, test tubes, etc.);

- the use of special metering devices in the process of mass sensitization of solid carriers;

- for specific sensitins, due to the different binding ability, a mandatory selection of a solid carrier is necessary;

- limited contact surface area of the solid carrier;

- low capacity of solid carriers, as a result of which, to determine the antigen content, it is necessary to take a large amount of antibody immunosorbent or immobilize only the high-affinity fraction of antibodies on it;

- inhomogeneity of the material of solid carriers (uncontrolled factor).

These shortcomings do not allow to obtain a technical result - a simplification of the method.

The similarity of the proposed method with the known one lies in the fact that they both relate to immunology and are based on the use of immobilized sensitins on a carrier.

The present invention solves the main problem - the simplification of the method. The invention is expressed by a combination of essential features sufficient for the technical result provided by the invention.

The solution to this problem lies in the fact that a submicron monodisperse stable emulsion of organofluorine compounds (PFOS) 10 vol.% 0.5 ml is freed from salts and glucose by washing 5 times by centrifugation at 6000 rpm for 5 min in a tenfold volume of 0.15 M physiological a solution of pH 6.8, then the corresponding sensitin is immobilized on the surface of the emulsion droplets.

The industrial production in Russia of PFOS emulsion as a blood substitute with a gas transport function is carried out by OAO NPO Perftoran. Its constituent components are perfluorodecalin and perfluoromethylcyclohexylpiperidine with the addition of proxanol, glucose and a set of salts as an emulsifier. The diameter of the emulsion droplets is about 0.1-0.15 microns. It is stable, chemically inert, the total surface of 1 ml is about 60 m ² . An undesirable effect of an emulsion as a blood substitute is the ability of drops to bind proteins, lipids, and other substances on its surface. This property of the emulsion was used by the authors to create droplets - sorbents of antigens or antibodies used in enzyme-linked immunosorbent assay.

The proposed method makes it possible to simplify the process of detecting antigen or antibody in an enzyme-linked immunosorbent assay due to the absence of a laborious procedure for preparing a sensitized surface of a solid carrier. PFOS emulsion is a universal carrier of sensitins and reduces the cost of research.

The proposed method is as follows.

The submicron monodisperse stable emulsion PFOS 10 vol.% 0.5 ml is freed from salts and glucose by washing 5 times by centrifugation at 6000 rpm for 5 min in a ten-fold volume of 0.15 M saline pH 6.8, then the corresponding sensitin is immobilized to the surface drops of emulsion.

The proposed method has been successfully tested at the laboratories of the Research Institute for Regional Infectious Pathology of the State Educational Institution of Higher Professional Education Astrakhan State Medical Academy of Roszdrav and the Federal State Institution of Research Institute for the Study of Leprosy of Roszdrav Astrakhan.

Example No. 1. In order to detect hepatitis B virus surface antigen (HBsAg), the prepared precipitate of the PFOS emulsion is resuspended in 1 ml of glycine buffer pH 8.8.

Preparation of buffer: 46.7 ml of 2M glycine (15 g per 100 ml of distilled water) is mixed with 10 ml of 1M NaOH (4 g per 100 ml of distilled water) and an equal volume of saline is added.

150 μg of goat anti-HBsAg antibodies produced by ImBiO, Nizhny Novgorod, with a protein content of 1 mg / ml, titer 1: 128 in double Ochterlooni immunodiffusion, are introduced into a PFOS emulsion balanced with glycine buffer. The resulting mixture was incubated for 2 hours at room temperature and 16 hours in a domestic refrigerator at 8 ° C, followed by five times washing with glycine buffer 5 ml of pH 8.8 by centrifugation at 3000 rpm for 10 min to E ₂₈₀ = 0 for remove unbound protein. The pellet was resuspended in 4 ml of glycine buffer pH 8.8. A sensitized emulsion of PFOS is used as the sorbent of HBsAg. The positive and negative controls, the immunoperoxidase conjugate for HBsAg are the components of the Vektohep B-HBs-antigen-strip commercial kit (kit 3), manufactured by Vector-Best CJSC, Novosibirsk. As the studied samples, the sera of 34 patients with acute hepatitis B who were treated at the Regional Infectious Clinical Hospital in Astrakhan are used. The diagnosis of acute hepatitis B was confirmed in an enzyme-linked immunosorbent assay for HBsAg according to the standard method. In addition, serum of blood donors was tested - 18 people, rejected by the results of an enzyme-linked immunosorbent assay for the presence of HBsAg. To test the specificity of the reaction, a study was conducted of the blood serum of healthy individuals - 11 people, and patients with chronic hepatitis C - 8 people.

Before performing an enzyme immunoassay, 0.1 ml of an antibody sensitized emulsion is taken for each test sample and after centrifugation at 6000 rpm for 5 min in Eppendorf tubes, positive and negative controls for HBsAg are added to the pellet, as well as blood serum samples in a volume of 100 μl with the addition of 50 μl of a phosphate-saline solution of 0.05 M pH 7.2 containing tween-20 0.05% (PSRT). The contents of the tubes are mixed with a single-channel dispenser with interchangeable tips, after which the rack with tubes is placed in a thermostat for 2 hours at a temperature of 37 ° C. During incubation, every 20-30 minutes, the tubes are shaken for 7-10 seconds. At the end of the incubation, the tubes containing the contents are centrifuged at 5000 rpm for 5 minutes. The supernatant is removed with a single-channel dispenser and the emulsion is washed three times with stirring with PSRT. Then, 150 μl of immunoperoxidase conjugate diluted in accordance with the kit instructions are pipetted into each tube. After 1-hour incubation of the tubes at a temperature of 37 ° C and periodic mixing of the emulsion, as described above, a four-time washing of the PSRT follows. The reaction results are evaluated after adding and mixing 100 μl of substrate-chromogenic mixture with the contents of the tubes. After 20 minutes of incubation at room temperature in the dark, the reaction is stopped by the addition of 50 μl of stop agent. The tubes are again centrifuged at 5000 rpm for 5 minutes and the resulting supernatant in a volume of 100 μl is transferred to a 96-well plate to record the optical density of the products of the enzymatic reaction at 450 nm.

The testing of blood serum samples of patients with acute hepatitis B and potential blood donors in whom HBsAg was detected shows a complete coincidence of the ELISA results using polystyrene plates as an antibody carrier and the PFOS emulsion proposed by the authors. The value of OD _{450 of the} negative control of the sera of healthy individuals and patients with chronic hepatitis C (CHC) does not exceed 0.15 optical units (p.u.). The OD value of ₄₅₀ blood serum samples containing HBsAg is 0.315 p.u. and higher.

Thus, the proposed method allows to detect HBsAg in serum in an enzyme-linked immunosorbent assay using an antibody-sensitized emulsion of perfluororganic compounds.

Example No. 2. To wash 0.5 ml of PFOS emulsion washed by centrifugation at 6000 rpm for 5 min, ten times the volume of 0.15 M physiological solution of pH 6.8 from glucose and salts that make up Perftoran, 3000 TE are added to 1 ml of physiological buffered a solution of 0.1 M pH 7.2 (PBS) of dry tuberculin, manufactured by the St. Petersburg Institute of Vaccines and Serums. Sensitization of PFOS is carried out at room temperature for 3 hours and at a temperature of 8 ° C for 48 hours. At the end of it should be washed by centrifugation ten times the volume of PBS at 3000 rpm for 10 min to E ₂₈₀ = 0. After the last centrifugation, the emulsion precipitate was dissolved in 4 ml of the same buffer and used for ELISA in Eppendorf tubes in a volume of 100 μl with preliminary centrifugation at 5000 rpm for 5 min per 1 sample. The research material is blood serum samples from 13 healthy individuals and from 17 patients with pulmonary tuberculosis, the diagnosis of which is confirmed bacteriologically. All blood serum samples are pre-tested in ELISA according to the instructions for the kit "IFA-Anti-Tub", produced by the St. Petersburg Research Institute of Vaccines and Serums named after L. Pasteur. When stating ELISA with sensitized emulsion Perftoran, an immunoperoxidase conjugate and substrate-chromogenic mixture from the IFA-Anti-Tub kit are used. The test sera are diluted 1:10 with PSRT and 100 μl are added to each tube. After one-hour incubation in a thermostat at a temperature of 37 ° C, the emulsion is washed three times by centrifugation at 5000 rpm for 5 minutes with 200 μl of PSRT. The procedure for incubation of the immunoperoxidase conjugate and subsequent washing of the PFOS emulsion is carried out under the same conditions and parameters as the serums. The reaction results are evaluated by the magnitude of the OD ₄₅₀ products of the enzymatic reaction. A comparison of the OD ₄₅₀ values obtained by ELISA using 96-well plates and Perftoran emulsion shows their coincidence. The magnitude of the negative results for the presence of antibodies in the blood serum against tuberculin was not more than 0.2 p.u. OD ₄₅₀ values exceeding 0.5 pu indicate the presence of antibodies to tuberculin.

The proposed method identifies antibodies to tuberculin in an enzyme-linked immunosorbent assay in the blood serum of patients with tuberculosis and healthy individuals using an emulsion of perfluororganic compounds sensitized with dry tuberculin.

Thus, the authors presented a method in which they use a submicron emulsion of PFOS, which was not previously proposed by anyone as a carrier of sensitins to detect antigen or antibody in ELISA.

Storage of diagnostic products for six months does not reduce their effectiveness (observation period).

The proposed method simplifies the process of preparing sensitized drops-sorbents, provides high sensitivity of the method, the standard and stability of the preparations, the relatively low cost of the emulsion PFOS. The PFOS emulsion has a large reactive surface area. The implementation of the proposed method is safe for personnel, does not require the use of expensive equipment.

The proposed method can be recommended for use by immunological laboratories of TB and infectious health care institutions.

Claims (1)

A method for detecting antigen or antibody in an enzyme-linked immunosorbent assay (ELISA), which involves the use of immobilized sensitins on a carrier and taking into account the results of ELISA, characterized in that a submicron monodisperse stable emulsion of perfluororganic compounds (PFOS) of 10 vol.% 0.5 ml liberated is used from salts and glucose by washing with a tenfold volume of 0.15 M physiological solution, pH 6.8, and the corresponding sensitin are immobilized on the surface of the drops of PFOS emulsion.