PL222575B1 - IgY polyclonal antibodies specific for the Map protein, process for the preparation and use thereof - Google Patents

Abstract

The subject of the invention are polyclonal IgY antibodies specific for the Map protein from Stephylococcus aureus isolated from the egg yolk of poultry immunized with a protein antigen in the form of a recombinant Map19 protein derived from Staphylococcus aureus with the specific sequence presented. The method of producing polyclonal IgY antibodies specific for the Map protein from Stephylococcus aureus consists in immunizing poultry with a protein antigen in the form of a recombinant Map19 protein derived from Staphylococcus aureus, and Freund's complete adjuvant is used as an adjuvant, and then antibodies are isolated using a known method with a yield of 100-160 mg/egg and a purity of 85-95%. The subject of the invention is also the use of polyclonal IgY antibodies specific for the Map protein for detecting Staphylococcus aureus bacteria.

Description

(12) PATENT DESCRIPTION (19) PL (11) 222575 (13) B1 (21) Application number: 398828 (22) Application date: 16/04/2012 (51) Int.Cl.

C07K 16/02 (2006.01) C07K 16/12 (2006.01)

The patent description was reprinted due to errors noticed

Polyclonal IgY antibodies specific for Map protein, method of their production and their use

(73) Patent holder: WROCŁAW UNIVERSITY OF TECHNOLOGY, Wrocław, PL UNIVERSITY OF LIFE SCIENCES IN WROCŁAW, Wrocław, PL (72) Inventor(s): (43) The notification was announced: MARCIN SIEŃCZYK, Wroclaw, PL 19.11.2012 BUP 24/12 AGNIESZKA ŁUPICKA, Milicz, PL KAMILA BOBREK, Łaziska Górne, PL RENATA GRZYWA, Wrocław, PL MACIEJ WALCZAK, Wrocław, PL (45) The patent was granted on: ERIC L. BROWN, Houston, US 31.08.2016 WUP 08/16 DARIA NOWAK, Oława, PL JOANNA ONIŚKIEWICZ, Nysa, PL JÓZEF OLEKSYSZYN, Siechnice, PL ANDRZEJ GAWEŁ, Wrocław, PL TADEUSZ STEFANIAK, Wrocław, PL (74) Proxy: attorney-at-law Katarzyna Paprzycka

PL 222 575 B1

Description of the invention

The subject of the invention are polyclonal IgY antibodies specific for Map protein, a method for their production and the use of polyclonal IgY antibodies specific for Map protein in immunotherapy of humans and animals against Staphylococcus aureus infections, both passive immunization and applications in external infections.

Map protein, also known as Eap or p70, produced by Staphylococcus aureus strains, is an extracellular adhesion protein that allows bacteria to adhere to the substrate and colonize body tissues. Map protein has both structural and functional analogy to the proteins of the human major histocompatibility complex (MHC II). Map protein also has the ability to modulate the immune system.

Staphylococcus aureus diagnostics is largely based on microbiological techniques - microscopic and culture tests - but they have a number of disadvantages, such as low differentiating power (within the species), difficulty in obtaining material for testing (internal organ abscesses) or abnormal results in people after antibiotic therapy [Colque-Navarro P. Soderquist B, Holmberg H. Blomqvist L. Olcen P. Mollby R. J. Med. Microbiol, 47. 1998. 217-25]. Serological diagnostic methods are based on the principle of antigen-antibody reaction, e.g. using ELISA test. One of the methods is measuring the amount of antibodies specific to target antigens in biological material (blood, serum). The use of whole Staphylococcus aureus cells or their selected antigens for coating a microtiter plate (ELISA test) and then performing an analysis to confirm the presence of specific antibodies (IgG or IgM classes) in the patient's blood has been described in the scientific literature [Joost I. Jacob S, Utermohlen O. Schubert U., Patti JM, Ong MF, Gross J. Justinger C. Renno H. Preissner KT. Bischoff M, Herrmann M., FEMS Immunol Med Microbiol 2011,62(1): 23-31; Kumar A, Ray P. Kanwar M. Sharma M., Varma S., Int J Med Sci. 2005, 2(4): 129-36]. The use of Staphylococcus aureus antigens for detecting staphylococcal enterotoxins in food has also been described [Fey II. Pfister O. Ruegg O., J Clin Microbiol, 1984, 19(1): 34-8]. High species specificity of Map protein is also used in the diagnosis of S. aureus infections, in which the presence of the map gene is confirmed [Hussain M, von Eiff C, Sinha B, Joost I, Herrmann M. Peters G. Becker K., J. Clin Microbiol. 2008, 46(2): 470-6).

International patent application No. WO 2009023043 describes IgG antibodies isolated from mice or rabbits specific for selected Staphylococcus aureus proteins or whole cells - Abcam, product number ab20920, however their use in the diagnosis of Staphylococcus aureus infections has not been presented.

IgY antibodies belong to the main class of immunoglobulins found in birds, reptiles, amphibians and protoplatycodons. They perform functions analogous to IgG antibodies in mammals. They are not only present in the blood, but are also transported in large quantities to the egg yolk, which provides the offspring with protection against pathogens. Due to the presence of IgY antibodies in egg yolk, it is possible to use them as a source of specific antibodies. A number of methods for isolating IgY antibodies from bird egg yolk and using them as a source of specific immunoglobulins have been described in the literature.

So far, the production of IgY antibodies specific for the Map protein from Staphylococcus aureus has not been described in the literature.

Polyclonal IgY antibodies specific for Map protein isolated from egg yolk of poultry immunized with a protein antigen in the form of recombinant Map 19 protein derived from Staphylococcus aureus with the sequence presented in formula 1.

Preferably, the antibodies have a molecular weight of from 175,000 to 200,000 Daltons.

A method of producing polyclonal IgY antibodies specific for Map protein comprises immunizing poultry, preferably chickens of farm breeds, with a protein antigen in the form of recombinant Map19 protein derived from Staphylococcus aureus with the sequence presented in formula 1, wherein the immunization is carried out in three separate doses, and Freund's complete adjuvant is used as an adjuvant, and then antibodies are isolated in a known manner with a yield of 100-160 mg/egg and a purity of 85-95%.

Preferably, in order to enrich isolated antibodies with specific anti-Map antibodies, an affinity chromatography method is used, which consists in covalently binding the Map protein to a carrier, preferably γ-aminoagarose, and then applying a mixture of polyPL 222 575 B1 clonal IgY antibodies with anti-Map specificity to the substrate, and then, after removing unbound non-specific antibodies, antibodies with the desired specificity are obtained.

Preferably, the solution of IgY antibodies specific for the Map protein is dialyzed against a carbonate buffer, and in parallel, horseradish peroxidase in a carbonate buffer is mixed with cyanuric chloride, after which the reaction mixture is dialyzed against a carbonate buffer, and in the next step, the horseradish peroxidase solution obtained after dialysis is mixed with the IgY antibody solution obtained in the first step for 4 hours at 37°C, the pH of the reaction mixture is adjusted to 7.2-7.4 using a 1M citric acid solution, an equal volume of glycerol is added, and IgY-HRP conjugates are obtained.

Use of polyclonal IgY antibodies specific for Map protein for in vitro detection of Staphylococcus aureus bacteria.

The isolated antibodies are subjected to biochemical analysis to determine their purity, antibody titer, antigen specificity and concentration. During the immunization process, antibody analysis was performed for each collected egg over a period of 22 weeks. The body's response in the form of production of specific IgY antibodies appeared after 7 weeks from the moment of immunization.

The method according to the invention is characterized by low invasiveness because it does not require bleeding of animals at specific time intervals in order to isolate antibodies.

The antibodies of the invention find use in immunotherapy of humans and animals against Staphylococcus aureus infections, both in passive immunization and in applications in external, skin infections.

The diagnostic use of the obtained anti-Map IgY antibodies is based on their ability to specifically bind to the target antigen and then detect the resulting Map-IgY complexes using commercially available secondary anti-IgY antibodies (e.g. rabbit IgG anti-IgY antibodies) containing any label (peroxidase, alkaline phosphatase, biotin, fluorescein) enabling detection of antigen-antibody complexes. Conjugates of specific anti-Map IgY antibodies and horseradish peroxidase (anti-Map IgY-HRP) were also obtained for use in direct detection of Map protein in ELISA and western blotting tests.

The subject of the invention is presented in more detail in the examples of embodiments and in the drawing in which:

Fig. 1 - Electrophoretic analysis (SDS-PAGE) under non-reducing conditions of the obtained IgY antibodies. Staining was performed using Coomassie R 250 dye.

Fig. 2 - Graph presenting the response in the form of production of IgY antibodies specific to Map protein. Arrows indicate the time of immunization.

Fig. 3 - Detection of Map protein by the obtained specific IgY antibodies (image obtained in the western blotting technique).

Fig. 4 - Graph showing the ability of IgY antibodies to detect Map protein: Abs* presented absorbance values after subtraction of the absorbance values obtained for IgY antibody control solutions.

Fig. 5 - Graph showing the ability of IgY antibodies (different dilutions) to detect Map protein; Abs* - absorbance values presented after subtraction of absorbance values obtained for IgY antibody control solutions.

Fig. 6 - Detection ability of Map protein by the obtained specific IgY antibodies: western blotting image (left) and obtained signal intensity values (right).

Fig. 7 - Detection of Map protein by the obtained specific IgY antibodies (western blotting).

Fig. 8 - Determination of the level of non-specific binding of the obtained IgY antibodies to selected proteins (western blotting).

Fig. 9 - Results of western blotting analysis of Staphylococcus aureus bacteria cultures to determine the presence of Map protein. Using the obtained IgY antibodies; rMap19 - recombinant Map protein used for immunization; fMap - native Map protein; h - culture fluid; o - sediment from culture fluid (description in the text); A - cultures A009 and E006; B - cultures E025 and E58; C - cultures E039 and E52.

Fig. 10 - Graph showing the detection of IgY conjugates by rabbit IgG anti-IgY antibodies; Abs* - absorbance values presented after subtraction of the absorbance values obtained for control solutions.

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Fig. 11 - Direct detection of Map19 protein by IgY antibodies conjugated with horseradish peroxidase (IgY-HRP) (western blotting).

Fig. 12 - Detection of Map19 protein by IgY antibodies before and after their purification by affinity chromatography (western blotting).

Example 1

1.1 Preparation of antigen for immunization

Map protein (rMapl9, recombinant. 22.5 kDa with the sequence shown in formula 1) was dissolved in phosphate buffer at pH 7.4 and then lyophilized. The protein was dissolved just before injection in a mixture of sterile physiological saline and Freund's complete adjuvant in a 1:1 (v/v) ratio.

1.2. Animal immunization

Breeding hens of the White Leghorn breed are immunized by intramuscular administration of antigen, 40 pg/animal (300 µl). Immunization is then repeated after 5 and 8 weeks using antigen, 10 µg per animal. The control group receives an injection of physiological saline solution containing Freund's complete adjuvant (1:1, v/V, 300 µl/animal).

1.3. Collection and storage of biological material

Eggs were collected daily from the day following immunization for 4 months and stored at 4°C until antibody isolation.

1.4. Isolation of IgY antibodies

The collected eggs were first washed with 50% isopropanol solution, and then the yolk was separated from the albumen. After removing the albumen membrane surrounding the yolk sac, the liquid content was diluted five times with phosphate buffer (100 mM, pH 7.4), and then polyethylene glycol 6000 was added to a final concentration of 3.5%. After thorough mixing, the whole was centrifuged at 11,000 rpm for 15 min at 4°C and the obtained supernatant was filtered through a filter paper. PEG 6000 was then added to the supernatant to a final concentration of 12%. After thorough mixing, the precipitate was centrifuged (11,000 rpm for 15 min at 4°C). The supernatant was discarded, and the precipitate containing IgY antibodies was thoroughly dissolved in phosphate buffer (pH 7.4) in a volume of 5 ml. After biochemical analyses, antibody solutions were stored at -20°C. The concentration of the obtained proteins was determined spectrophotometrically (A ₂₈₀ ).

Example 2. Analysis of the purity of isolated IgY antibodies

The purity of isolated antibodies was determined by performing electrophoretic separation of the obtained proteins in non-reducing conditions. For this purpose, 10 µl of the obtained solution of the analyzed protein diluted 100-fold was applied to the wells of a polyacrylamide gel (SDS PAGE, 4%-12%). Proteins were visualized using Coomassie R 250 dye. Based on the analysis of electrophoretic images, the purity of the obtained antibodies was determined, which ranged from 85% to 95%.

Example 3. ELISA - general description of the test

To confirm the presence of antibodies specific for the Map protein, an ELISA test was performed for each of the obtained preparations. 96-well microtiter plates were coated with antigen dissolved in carbonate buffer (50 mM, pH 9.6) and incubated at 4°C for 12 hours. After washing away the unbound antigen, free sites were blocked using a 5% solution of skimmed milk in phosphate buffer containing 0.05% (v/v) Tween-20. Blocking was performed at 37°C for 60-120 minutes. After washing off the blocking agent, specific and control antibodies diluted in 0.5% skimmed milk in phosphate buffer containing 0.05% (v/v) Tween-20 (v/v) (100 mM, pH 7.4) were applied to the antigen-coated microtiter plate (100 µl) and incubated for 1-2 hours at 37°C. Detection of Map protein-IgY antibody complexes was performed using a rabbit anti-IgY antibody conjugated to horseradish peroxidase using O-phenyldiamine as a chromogenic substrate. Absorbance was measured at 490 nm using a microplate reader. All measurements were performed in duplicate.

Example 4. Analysis of the response to the administered antigen.

To analyze the immune response of the immunized animals and determine the titer of the produced antibodies, the ELISA test was used according to the method described in Example 3 for each isolated antibody at a dilution of 1:100, 1:1000 and 1:10000. The antibody titer was the dilution at which the absorbance value was at least 2.1 times higher than the absorbance of the control antibody solution. All measurements were performed

PL 222 575 B1 in duplicate for each antibody and each dilution. The amount of antigen used in the assay was 6 ng/microtiter plate well.

Example 5. Specificity analysis - western blotting.

In order to confirm the specificity of the obtained antibodies specific for the Map protein, western blotting analysis was performed. For this purpose, electrophoretic separation was performed under denaturing conditions (SDS PAGE, 4%-12%) of the Map protein dissolved in a sample buffer containing a reducing agent (39.4 ng of protein per well). Then, the proteins were electrotransferred onto a nitrocellulose membrane in a semi-dry transfer apparatus. Blocking of free binding sites on the nitrocellulose membrane was performed using a 5% solution of skimmed milk in a phosphate buffer containing 0.05% Tween-20 (v/v) for 12 h at 4°C. Then, the antibodies specific for the Map protein were diluted (1:100) in a 0.5% solution of skimmed milk in a phosphate buffer containing 0.05% Tween-20 (v/v) and incubated with the membrane for 1-2 h at 37°C. After washing the membrane with PBST buffer, detection of antigen-bound specific antibodies was performed using a rabbit anti-IgY antibody conjugated to horseradish peroxidase using chemiluminescence. Image analysis was performed using a molecular imaging system equipped with a CCD camera.

Example 6. Detection limit of Map - ELISA

In order to determine the detection limit of Map protein by the obtained specific IgY antibodies, a solid-phase enzyme-linked immunosorbent assay (ELISA) was used as described above. For this purpose, a 96-well microtiter plate was coated with Map protein at various concentrations ranging from 2000 ng/ml to 0.15 pg/ml (100 µl/well). After washing the wells, a solution of the Map-specific antibody diluted 2500-fold with a solution of skim milk in phosphate buffer containing 0.05% (v/v) Tween-20 was added. Detection of Map-IgY complexes was performed using a rabbit anti-IgY antibody conjugated to horseradish peroxidase using O-phenyldiamine as a chromogenic substrate. Absorbance was measured at 490 nm using a microplate reader. All measurements were performed in duplicate.

Example 7. Detection limit - antibody titer (dilution) - ELISA

The titer of the antibodies obtained specific for the Map antigen used was determined using ELISA. A microtiter plate was coated with 6 ng/well of antigen (100 µl of a 60 ng/ml solution per well). Then, a series of dilutions of IgY antibodies (specific for the Map protein and control) ranging from 1:10 to 1:100,000 were prepared and incubated with the antigen-coated plate. Detection of Map-IgY complexes was performed using a rabbit anti-IgY antibody conjugated with horseradish peroxidase using O-phenyldiamine as a chromogenic substrate. Absorbance was measured at a wavelength of 490 nm using a microplate reader. The titer of Map protein-specific antibodies was defined as the highest dilution for which the absorbance value was at least 2.1 times higher than the absorbance value of the antibody solution isolated from egg yolk of non-immunized hens. All measurements were performed in duplicate.

Example 8. Detection limit of Map - western blotting

In order to determine the detection limit of Map protein in the western blotting technique, in the first step, electrophoretic separation (SDS PAGE, 4%-12%) of Map protein was performed under reducing conditions in the amount ranging from 2.1 ng to 39.4 ng/well. Then, semi-dry transfer of proteins to a nitrocellulose membrane was performed. After blocking free binding sites using a 5% solution of skimmed milk in PBST buffer, the membrane was incubated with a solution of the obtained IgY antibody specific for Map protein diluted 1:1000 with 0.5% solution of skimmed milk in PBST buffer. After washing the membrane with PBST buffer, detection of specific antibodies bound to the antigen was performed using a rabbit anti-IgY antibody conjugated with horseradish peroxidase using a chemiluminescent substrate. Image analysis was performed using a molecular imaging system equipped with a CCD camera.

Example 9. Detection limit - antibody titer (dilution) - western blotting

In order to determine the concentration range of IgY antibodies obtained in the western blotting technique, necessary for the detection of Map protein, Map protein was applied to the wells of a polyacrylamide gel in an amount of 4.8 ng/well and then electrophoretic separation of proteins was performed under reduced conditions.

PL 222 575 B1 cations (SDS PAGE, 4%-12%). After semi-dry transfer of proteins to a nitrocellulose membrane and blocking of free sites with a 5% solution of skimmed milk in PBST buffer, the membrane strips were incubated for 1 h with solutions of an IgY antibody specific for Map protein diluted in the range of 1:100 to 1:50,000 with PBST buffer containing 0.5% skimmed milk. After washing the membrane strips with PBST buffer, detection of specific antibodies bound to the antigen was performed using a rabbit anti-IgY antibody conjugated with horseradish peroxidase using a chemiluminescent substrate. The images were analyzed using a molecular imaging system equipped with a CCD camera.

Example 10. Nonspecific binding of IgY antibodies - western blotting

In order to determine the possibility of non-specific binding of the obtained IgY antibodies to proteins other than Map, the Western blotting test was used. For this purpose, protein solutions were applied under reducing conditions to a polyacrylamide gel (4%-12%. SDS PAGE) in the amount of 1 pg/well and a semi-dry transfer of proteins to a nitrocellulose membrane was performed. After blocking the free binding sites, the membrane was incubated with a solution of the IgY antibody specific to the Map protein, diluted 1:10,000 in PBST buffer containing 0.5% skimmed milk. After washing the membrane with PBST buffer, detection of specific antibodies bound to the proteins was performed using a rabbit anti-IgY antibody conjugated with horseradish peroxidase using a chemiluminescent substrate. The images were analyzed using a molecular imaging system equipped with a CCD camera.

Example 11. Detection of Map protein in S. ureus culture

In order to determine the ability of the obtained IgY class antibodies to detect the Map protein in the culture of Staphylococcus aureus bacteria - environmental isolates from the collection of the Department of Epizootiology with the Clinic of Birds and Exotic Animals of the University of Environmental and Life Sciences in Wrocław: A009 and E006 (pigeon cloaca), E025 (cat eyes), E039 (pigeon crop). E052 (dog tonsils). E058 (horse skin) the western blotting technique was used. The following culture medium was used: soy peptone (3 g/l), casein peptone (17 g/l), sodium chloride (3 g/l), potassium hydrogen phosphate (2.5 g/l). sodium pyruvate (10 g/l), pH 7.3. The bacteria were cultured at 37°C. After 24 h and 48 h / culture, the culture medium was collected and electrophoretic separation was performed under reducing conditions (4%-12%. SDS PAGE). Analysis of the presence of the Map protein in the culture sediment was also performed. For this purpose, 25 ml of the culture fluid was centrifuged (4500 rpm, 4°C, 15 min). The sediment was then dissolved in 2% SDS solution in water and boiled for 5 min, then centrifuged (4500 rpm, 4°C, 15 min) and the collected supernatant was centrifuged again (4500 rpm, 4°C, 15 min) and transferred to a clean tube. In order to perform the western blotting analysis, its separation was performed under reducing conditions. After electrophoretic separation, semi-dry transfer of proteins to a nitrocellulose membrane was performed. After blocking the binding sites with 5% skim milk in PBST buffer, the membrane was incubated with a solution of IgY antibody (1:1000) specific for the Map antigen. After washing the membrane with PBST buffer, detection of specific antibodies bound to the Map protein was performed using a rabbit anti-IgY antibody conjugated to horseradish peroxidase using a chemiluminescent substrate. Image analysis was performed using a molecular imaging system equipped with a CCD camera.

Example 12. Preparation of IgY-HRP conjugates

In the first stage, the solution of IgY antibodies specific to the Map protein is dialyzed against carbonate buffer (50 mM, pH 9.6) and diluted to a concentration of 5 mg/ml. In the second stage, 1.2 mg of horseradish peroxidase (concentration 6 mg/ml) is weighed into a test tube and dissolved in cold (4°C) carbonate buffer (50 mM, pH 9.6) and transferred to a test tube containing 670 pg of cyanuric chloride. The whole is gently mixed at room temperature for 6 h, after which the reaction mixture is dialyzed against carbonate buffer (50 mM, pH 9.6). In the next stage, the entire solution of horseradish peroxidase obtained after dialysis is mixed with 90 µl of the IgY antibody solution obtained in the first stage and gently mixed for 4 h at 37°C. After this time, the pH of the reaction mixture is adjusted to 7.2-7.4 using 1M citric acid solution and then an equal volume of glycerol is added.

To confirm the presence of IgY-HRP conjugates, an ELISA test was performed. For this purpose, the wells of a 96-well microtiter plate were coated with rabbit IgG anti-IgY antibody dissolved in carbonate buffer (50 mM, pH 9.6) at a dilution of 1:5000 and incubated at 4°C for 12 hours. After washing away the unbound antibody, the free sites were blocked with a 5% solution of skimmed milk in phosphate buffer containing 0.05% (v/v)

PL 222 575 B1

Tween-20. Blocking is performed at 37°C for 60-120 minutes. After washing off the blocking agent, the obtained IgY-HRP conjugate diluted in 0.5% solution in phosphate buffer containing 0.05% (v/v) Tween-20 (v/v) (100 mM, pH 7.4) was applied to a microtiter plate (100 µl) coated with antigen and incubated for 1-2 hours at 37°C. Detection of IgG-IgY-HRP complexes was performed using O-phenyldiamine as a chromogenic substrate. Absorbance was measured at 490 nm using a microplate agent. All measurements were performed in duplicate.

Example 13. Direct detection of Map protein using IgY-HRP conjugate western blotting.

In order to confirm the specificity and the ability to recognize the Map protein by the obtained IgY-HRP conjugates, the western blotting technique was used - the Map protein was applied to the wells of a polyacrylamide gel in the amount of 4.8 ng/well and then the electrophoretic separation of proteins was performed under reducing conditions (SDS PAGE, 4%-12%). After semi-dry transfer of proteins to a nitrocellulose membrane and blocking of free sites using a 5% solution of skimmed milk in PBST buffer, the membrane strips were incubated for 1 h with a solution of the IgY-HRP conjugate in the amount of 33 pg in PBST buffer (5 ml) containing 0.5% skimmed milk. After washing the membrane strips with PBST buffer, the detection of Map-IgY-HRP complexes was performed using a chemiluminescent substrate. The images were analyzed using a molecular imaging system equipped with a CCD camera.

Example 14. Isolation of IgY antibodies specific to Map protein

ω-aminobutyl agarose (5.2 pmol/ml) was used as a solid protein carrier in the affinity chromatography method. In the first step, the amino groups of the carrier were substituted with S-acetylthioacetate groups using W-succinimido-S-acetylthioacetate, and then the S-acetyl group was removed using a 0.5 M hydroxylamine solution in PBS buffer containing 25 mM EDTA. Free thiol groups of the carrier were modified with a heterobifunctional linker GMBS (10-fold molar excess) and after washing off the excess linker, the carrier suspension in PBS buffer was incubated with Map protein. The reaction was carried out for 2-6 h at room temperature with gentle mixing. The reaction was then stopped with 1 M glycine solution and the whole was washed with PBS buffer. A mixture of IgY antibodies specific for Map protein (200 µl) is applied to the prepared substrate (75 µl) and washed with PBS buffer. After washing away all unbound antibodies, 500 µl of citrate buffer (20 mM, pH 2.5) is applied to the top of the column and each fraction is neutralized using 1M Tris-base (pH 8.5). The presence of antibodies in the collected fractions is confirmed using electrophoresis (SDS PAGE. non-reducing conditions) and silver staining. Fractions containing antibodies are combined and the protein concentration is determined using the microBCA test. In order to determine the immunoreactivity of the antibodies obtained against Map19 protein, a western blotting test is performed as described above. Map protein is applied to the wells of the polyacrylamide gel in an amount of 4.8 ng/well. After the electrophoretic separation, a semi-dry transfer of proteins to a nitrocellulose membrane is performed and free binding sites are blocked with a 5% solution of skimmed milk in PBST buffer (4°C, 12 h). After washing the membrane with PBST buffer, the detection of Map protein is performed using the obtained IgY antibodies with anti-Map specificity before and after affinity chromatography. For this purpose, the antibody (before purification by affinity chromatography) in the amount of 5 pg and the purified antibody in the amount of 5 pg and 1 pg are measured into 10 ml of a 0.5% solution of skimmed milk in PBST buffer and incubated with nitrocellulose membrane strips containing Map protein transferred from a polyacrylamide gel (37°C, 1 h). After washing the membrane with PBST buffer, detection of protein-bound specific antibodies was performed using a rabbit anti-IgY antibody conjugated to horseradish peroxidase using a chemiluminescent substrate. Image analysis was performed using a molecular imaging system equipped with a CCD camera.

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Claims (8)

Patent claims 1. Polyclonal IgY antibodies specific for Map protein isolated from poultry egg yolk immunized with the protein antigen in the form of recombinant Map19 protein derived from Staphylococcus aureus with the sequence shown in formula 1. 2. Antibodies according to claim 1 2. The process of claim 1 having a molecular weight of 175,000 to 200,000 Daltons. A method of producing polyclonal antibodies of the IgY class specific to the Map protein, characterized by immunizing poultry with the protein antigen in the form of a recombinant Map19 protein derived from Staphylococcus aureus with the sequence represented by the formula 1, where the immunization is carried out in three separate doses, and as an adjuvant Freund's complete adjuvant is used, then the antibodies are isolated by a known method with a yield of 100-160 mg / egg and a purity of 85-95%. 4. The method according to p. A method according to claim 3, characterized in that chickens of breeding breeds are immunized. 5. The method according to p. 3. The method according to claim 3, characterized in that in order to enrich the isolated antibodies with specific anti-Map antibodies, the affinity chromatography method is used, in which the Map protein is covalently bound to a carrier, and then a mixture of polyclonal IgY antibodies with anti-Map specificity is applied to the resin. -Map, followed by removal of unbound nonspecific antibodies to obtain antibodies with the desired specificity. 6. The method according to p. The process of claim 5, characterized in that γ-aminoagarose is used as the carrier. 7. The method according to p. 3. A method according to claim 3, characterized in that the solution of IgY antibodies specific to the Map protein is dialyzed against a carbonate buffer, while horseradish peroxidase in a carbonate buffer is simultaneously mixed with cyanuric chloride, and then the reaction mixture is dialyzed against a carbonate buffer, in the next step obtained after dialysis the horseradish peroxidase solution is mixed with the IgY antibody solution obtained in the first step for 4 hours at 37 ° C, the pH of the reaction mixture is adjusted to 7.2-7.4 with 1M citric acid solution, an equal volume of glycerol is added to obtain IgY-HRP conjugates become apparent. 8. Application of IgY class polyclonal antibodies specific for Map protein for in vitro detection of Staphylococcus aureus bacteria.