UA144483U - METHOD OF STRENGTHENING REACTION OF PRECIPITATION BY IMMUNOELECTROPHORESIS - Google Patents

Abstract

A method of enhancing the precipitation reaction by immunoelectrophoresis involves the use of standard equipment for immunoelectrophoretic study of the interaction of specific antibodies with the antigen. To identify precipitation zones, a 1.0% solution of fluorescein copolymer (finished product) is added at a dose of 5 μl to 10 ml of antigen (or inactivated pathogen in the vaccine).

Description

The useful model belongs to the field of biology, in particular to immunology and biotechnology and clinical diagnostics, namely, it concerns methods of detecting the precipitation zone by counter electrophoresis by adding a fluorescein copolymer solution to the causative agent (antigen or inactivated causative agent in the vaccine). A useful model can be used in immunology and clinical practice to establish the presence of antibodies in the blood and persistent immunity to the disease.

Immunoelectrophoresis is widely used in laboratory practice. It is known that under certain conditions antigen and antibody move in opposite directions during electrophoresis. For this, the reacting components are introduced into separate wells in the agar gel, which is prepared in a buffer. Since the majority of precipitating antibodies belong to the class of immunoglobulins (Id), which have a weak negative charge at pH 8-9, they move to the cathode. Antibodies have a positive charge and move towards the anode. Accordingly, the antibodies in the wells of the gel are placed closer to the anode, and the antigen - to the cathode. Under the action of an electric field, antigen-antibody migration occurs towards each other. With immunoelectrophoresis (counter electrophoresis, electrosynthesis), visible precipitates can appear after 30-60 minutes. electrophoresis (sometimes after 2 hours; Gaal 9.

Electrophoresis in the separation of biological macromolecules/ Z. Gaal, G. Medyesha, L. Veretsky. -

M.: Mir, 1982. - P. 446).

The reaction of immunoelectrophoresis (RIEF) is used for intravital diagnosis of Aleutian disease in mink (Spo N. u., Ipdgat 0. 9. Apiidep apa apiibodu ip AIeshiap aibzease ip tipkK. I.

Rgesirianop Veasiyop Bu Adag!-Siye! EiIesigtorpogeziv// TNe y. And so on. - 1972. - M. 108, Mo. 2. - R. 555-557; Spo N. ., Ipagat 0. U. Assytsgaie apa garia iezi og ayeyesiiipu AiIeshiiap dizease ipiesivy tipKk// Yeig.

That's where it arrived. - 1972. - M. 50. - R. 8.; Spo N. u., Ipdgat 0. 9. Apiidep apa apiirodu ip AIeshiap dibvaze ip tipK. II. Tpe geasiip oi apiibodu m/p AIeshiap aizease adepi ivipd ittipodiyviop apa ittipoeYIestooztorpogeziv// Sap. ). Satr. Mea. - 1973. - M. 37, Mo. 3. - R. 217-223). Freon extracts of the liver, spleen, and lymph nodes of experimentally infected minks are used as an antigen. RIEF is characterized by high specificity and allows detection of sick minks 6-15 days after infection. In suckling puppies, it is positive within the first hour after sucking milk from sick females. On average, for all forms of the disease, with help

98-100,956 sick animals are found in the RIEF. For experimental infection of animals RIEF

Zo detects the disease after 6-7 days. Under the conditions of the farm, with direct contact between experimental and diseased animals, infected animals are detected after 30-60 days. (Parvovirus infections of dogs and fur animals/ L.E. Kornienko, V.I. Holovakha, B.M. Yarchuk, etc. - Bila Tserkva, 2001. - p. 55).

The disadvantage of using RIEF for disease diagnosis: in the absence of visible antigen-antibody precipitates, it is recommended to immerse the gel in 12.5 95 mass for 7-10 minutes. and wash in distilled water for at least 12 hours. Additional manipulations with the agar gel lead to an increase in the time of establishing the diagnosis, and in some cases, due to the destruction of the fragile gel, to the loss of results and the conduct of repeated studies.

The closest analogue of a useful model is the method of detecting antigen-antibody precipitates by

RIEF for diagnosis of Aleutian disease in mink (Instructions for the use of antigen sets and control serum for serological diagnosis of Aleutian disease in mink in the reaction of immunoslectroosmophoresis. - M., 1994. - p. 7). The peculiarity of this method is the introduction of reacting components (serum of the blood of a sick or healthy animal and specific antibodies) into separate wells in the agar gel and carrying out immunoelectrophoresis for 30-60 minutes.

However, after conducting immunoelectrophoresis and with questionable results, the agar gel is immersed in 12.5 95 Mass for 7-10 minutes, and then washed in distilled water for at least 12 hours. or conduct repeated studies. The specified features of research slow down the diagnosis of the disease and the culling of sick animals, and also slow down the application of preventive measures to eliminate the disease.

The useful model eliminates the shortcomings of the closest analog and by adding a solution of fluorescein copolymer (finished product; patent of Ukraine Mo 98749, МПК СОов8о 77/46, СО8Оо 63/66, СОвО 63/12, Сова 63/685, Сов 63/668. Method of obtaining copolyesters of natural dibasic c-amino acids and polyether glycols/ I.A. Dron, R.S. Taras, S.A. Voronov, S.M. Varvarenko,

Nagornyak M.I., Figurka N.V., Nosova N.G., Samaryk V.Ya., Voronov A.S., Ferens M.V., Tarnavchik

I.T. (Ukraine); Lviv Polytechnic National University; statement 13.10.2014, publ. 12.05.2015. - Bull. Mo 9) to the antigen or inactivated pathogen as part of the vaccine by immunoelectrophoresis provides visualization of precipitation zones and eliminates the need to immerse the gel in 12.5 95 mass for 7-10 min. and washing in distilled water for at least 12 hours.

The useful model is based on the task of ensuring the detection of precipitation zones by immunoelectrophoresis and increasing the efficiency and probability of disease diagnosis by additional introduction of a fluorescein copolymer solution (finished product) to the antigen or inactivated pathogen in the vaccine.

The technical result is achieved by adding to the antigen or inactivated pathogen in the vaccine 1.0 95 solution of fluorescein copolymer (finished product) in a volume of 5 μl to 10 ml of antigen.

According to the patent and information search, an analogue was found, which has essential features common to the useful model: the use of an antigen or an inactivated pathogen as part of a vaccine for immunoelectrophoresis, which ensures antigen-antibody precipitation in an agar gel; similarity of the agar gel composition in the closest analogue and useful model.

However, the presence of the specified features in common with the closest analogue are not sufficient to obtain a technical result of a useful model. No analogues were found that would completely match the features of the useful model in terms of the set of features.

In the sources of patent and scientific and technical information, there are no analogs with features that distinguish the claimed method from the closest analog and provide a technical result: detection of precipitation zones by adding a 1.09 solution of fluorescein copolymer (finished product) in a dose of 5 μl to 10 ml of antigen or inactivated pathogen in the vaccine.

The task is solved by the fact that in the method of enhancing the precipitation reaction by immunoelectrophoresis, which includes the use of standard equipment for the immunoelectrophoretic study of the interaction of specific antibodies with an antigen, according to a useful model, to detect precipitation zones, a 1.095 solution of fluorescein copolymer (finished product) is added in a dose of 5 μl up to 10 ml of antigen (or inactivated pathogen in the vaccine).

Examples of specific execution of the method

Example 1

Since the antigen or inactivated pathogen in the composition of the vaccine are proteins (or parts of protein molecules), in order to confirm the effectiveness of the claimed method in the conditions of the laboratory of molecular biology and clinical biochemistry of the Institute of Animal Biology of the National Academy of Sciences, studies were conducted on the interaction of fluorescein copolymer (finished product) with serum proteins (bovine serum albumin; Fig. 1). In fig. 1 shows electrophoresis

From a solution of fluorescein copolymer with bovine serum albumin, where 1 is bovine serum albumin stained with bromophenol blue; 2 - 1.0 95 fluorescein copolymer solution (finished product); C - fluorescein copolymer complex with bovine serum albumin; -; - the zone of interaction of the components introduced into the well is indicated (the zone of binding of the fluorescein copolymer with bovine serum albumin).

It was found that in the first well, during electrophoresis in agar gel, bovine serum albumin stained with bromophenol blue moved to the anode. A similar result was obtained by electrophoresis of complexes: in the second well - 1.0 96 solution of fluorescein copolymer, and in the third - fluorescein copolymer with bovine serum albumin. At the same time, in the third well, after electrophoresis of the copolymer of fluorescein with bovine serum albumin, a zone with more intense staining was established, which characterizes the formation of a complex between the components. Free (not bound to the protein) fluorescein moves under the action of the electric field in the same way as in the second well.

To confirm the result, vertical electrophoresis of samples of bovine serum albumin and a complex of fluorescein copolymer with bovine serum albumin was performed. For this, two samples were prepared: Mo 1 - bovine serum albumin (5 mg/ml), and Mo 2 - a complex of fluorescein copolymer with bovine serum albumin (5 μl of 1.0 95 copolymer per 10 ml of bovine serum albumin solution containing 5 mg/ ml). After electrophoresis, the gel was photographed (Fig. 2): polyacrylamide 12.5 95 gel with unstained (A) and stained (B) samples: Mo 1 - bovine serum albumin; Mo 2 is a complex of fluorescein copolymer with bovine serum albumin. Stained bovine serum albumin is marked with arrows.

It was established that, by electrophoresis, in the well of the unstained gel (A), the sample Mo 1 (bovine serum albumin) has no content, and in the sample Mo 2, the polymer remains at the start (Fig. 2, A and

B; sample Mo 2). After staining (Fig. 2, B), it was found that the bovine serum albumin under the action of the electric field in the hole Mo 1 traveled a greater distance from the start than in the hole Mo 2.

Therefore, the fluorescein copolymer is able to interact with bovine serum albumin, which leads to an increase in the size of the complex and slowing down of its mobility during electrophoresis. The revealed ability of the fluorescein copolymer to bind protein served as a basis for its use as a marker of the precipitation zone and a method of enhancing the precipitation reaction for immunoelectrophoresis.

Example 2

To test a useful model, the established ability of the fluorescein copolymer complex to bind proteins was used, and as an example of the use of the antigen application method: the vaccine against hemorrhagic disease of rabbits (Lapimun HEM-2). The vaccine includes inactivated viruses of two strains: BG-04»640 GAO and GBK-22640 GAO and auxiliary components.

Young rabbits of the 1-month-old Termonskaya Bila breed were selected for research.

Rabbits were injected with the vaccine subcutaneously behind the scapula in a dose of 1.0 ml according to the manufacturer's instructions.

Before vaccination (control), after 14, 21 and 28 days, blood was taken from the marginal ear vein of rabbits in tubes with heparin. Up to 10 ml of antigen (vaccines against hemorrhagic disease of rabbits - Lapimun

HEM-2) added 5 μl of 1.095 fluorescein copolymer solution (finished product) in two modifications: MF-37 and MF-47. Modifications of the fluorescein copolymer solution differ in fluorescein content.

In fig. C shows the results of counter-immunophoresis to determine the intensity of immunity of young rabbits within 21 days after vaccination using a useful model: samples MoMo 1, 2, Z, respectively, Mo 1 - without use and with the use of fluorescein copolymer in two modifications: Mo 2 - MF- 37 and Mo Z - MF-47.

The analysis of the results shows that before vaccination, the protein content of the antigen-antibody precipitation zones (without and with the use of fluorescein copolymer as an antigen) in the animals was in the range of 6.9-39.5 95 (Fig. 4 - densitograms of the antigen-antibody precipitation zones for immunophoresis to determine the intensity of immunity of young rabbits within 21 days after vaccination. Precipitation zones are indicated by arrows. Table 1).

Table 1

Protein content in the precipitation zone for immunoelectrophoresis to determine the intensity of immunity of young rabbits within 21 days after vaccination

Conditions of vaccination studies)

M (unpolarized 17/77/7681 11111890 Y111117171112001

14 days after vaccination, the protein content of the precipitation zone with the use of fluorescein copolymer as part of the antigen was almost the same (32.9-37.8 95), and without the use of the polymer it was 12.6-28.8 96 lower. The established changes were preserved during the study of immune tension after 21 days. The difference between the results for the use of fluorescein copolymer in the composition of the antigen compared to the antigen - 5.8-6.2 95.

Repeated studies of the content of proteins in the fluorescein copolymer complex as part of the antigen-antibody confirmed that when antigen is added to the samples in the complex with the fluorescein copolymer, precipitation zones are more intensively manifested (Figs. 5, 6). In fig. 5 - results of counter-immunophoresis to determine the intensity of immunity of young rabbits within 28 days after vaccination using a useful model: samples MoMo 1, 2, Z, respectively, Mo 1 - without use and with the use of fluorescein copolymer in two modifications: Mo 2 - MF- 37 and Mo

Z - MF-47. In fig. 6 - densitorgami of antigen-antibody precipitation zones to determine the intensity of immunity of young rabbits within 28 days after vaccination. Arrows indicate precipitation zones.

The analysis of the results shows that before vaccination, the protein content of antigen-antibody precipitation zones (without use and with the use of fluorescein copolymer as part of the antigen) in animals was in the range of 14.0-30.2 95 (Table 2).

Table 2

The content of proteins in the precipitation zone during counter-immunoelectrophoresis to determine the intensity of immunity of young rabbits during 28 days after vaccination

Protein content in samples by immunoelectrophoresis, 90

Conditions Control of studies (until the day of studies after vaccination vaccination) 1 (without polymer)

C (polymer MF-47 2 (polymer MF-37 1 (without polymer)

14 days after vaccination, with the use of the MF-37 fluorescein copolymer complex as an antigen, the protein content of the precipitation zone was 49.5595, less (33.7 90) with the addition of the MF-47 polymer, and without the use of the polymer by 22.5-43, 9 95 lower than with the addition of polymers. The established changes were preserved for the study of the intensity of immunity after 21 and 28 days between the samples to which the fluorescein copolymer was added, compared to the samples without the polymer. The difference between the results for the use of the fluorescein copolymer complex in the composition of the antigen compared to the antigen was 19.6-60.5 90 and 8.5-22.9 9b, respectively.

Therefore, the use of the fluorescein copolymer complex as an antigen for diagnostic studies using immunoelectrophoresis enhances the precipitation reaction with antibodies of the blood serum (plasma) of immunized animals (rabbits), which is manifested by an 8.5-60.5 96 higher protein content in the specified zone. More effective manifestation of precipitation zones was established by using the MF-37 polymer sample as part of the antigen complex with fluorescein copolymer.

Claims (1)

USEFUL MODEL FORMULA The method of enhancing the precipitation reaction by immunoelectrophoresis, which includes the use of standard equipment for the immunoelectrophoretic study of the interaction of specific antibodies with an antigen, which differs in that for the detection of precipitation zones, a 1.0 95 solution of fluorescein copolymer (finished product) is added in a dose of 5 μl to 10 ml of antigen (or inactivated pathogen in the vaccine). 1 2 Z Fig.1 Me: 12 12 Y -- start of bovine serum sh- A B Fig. 2 blood plasma antigen 111: g g ; 3 t s: control 14 21 days (unvaccinated) (vaccinated) Fig. 3