Erythrocyte diagnosticum from tick-borne encephalitis virus. Immunoglobulin against tick-borne encephalitis from horse serum, liquid (Russia)
MINISTRY OF HEALTH AND MEDICAL
INDUSTRY OF THE RUSSIAN FEDERATION
On permission for the medical use of medicines
immunobiological preparations
In accordance with Article 43 of the “Fundamentals of Civil Legislation of the Russian Federation on the Protection of Citizens’ Health”,
----------------
*Probably an error in the original. It should be read "Article 43 of the Fundamentals of the Legislation of the Russian Federation on the Protection of Citizens' Health." Note "CODE".
I order:
1. Department of State Control of Medicines and Medical Equipment:
1.1. Register medical immunobiological preparations and enter them into the State Register of Medicines approved for use in medical practice (Appendices 1,).
1.2. Transfer the relevant documentation (registration certificates, instructions for medical use, temporary pharmacopoeial monographs) for medical immunobiological drugs specified in the appendices to the following organizations:
1.2.1. LLC "Gritvak", Moscow (clause 1 of Appendices 1 and );
1.2.2. LLP MGP "Progress", Moscow (clause 2 of Appendices 1 and 2);
1.2.3. MNIIEM named after G.N. Gabrichevsky, Moscow (item 2 of Appendices 1 and );
1.2.4. State Unitary Enterprise for the Production of Bacterial Preparations named after G.N. Gabrichevsky (clause 2 of Appendices 1 and );
1.2.5. SE NPO "Virion", Tomsk (clause 3 of Appendices 1 and ).
2. To the developers and manufacturers specified in paragraphs 1.2.1. 1.2.5. transfer industrial regulations for medical immunobiological preparations to the National Authority for the Control of Medical Immunobiological Preparations.
3. Entrust control over the implementation of this order to the First Deputy Minister A.M. Moskvichev.
Minister
T.B.Dmitrieva
List
medical immunobiological preparations,
approved for medical use
1. Typhoid vaccine Vi-polysaccharide liquid (VIANVAK).
2. Acylact in suppositories.
3. Diagnosticum erythrocyte for tick-borne encephalitis virus, immunoglobulin rat dry for RNGA (erythrocyte diagnosticum for tick-borne encephalitis virus).
Head of Department
state control
medicines and
medical equipment
R.U. Khabriev
Brief annotations on medical
immunobiological drugs approved
for medical use by order
Ministry of Health
Russian Federation
Typhoid vaccine V-polysaccharide
liquid (VIANVAK)
Temporary pharmacopoeial article VFS 42-2944-97 was approved by order of the Ministry of Health of Russia dated September 16, 1997 N 276.
Registration No. 97/276/1.
Typhoid vaccine V-polysaccharide liquid (VIANVAK) is a solution of capsular polysaccharide extracted from the supernatant of the Salmonella typhi culture, purified by enzymatic and physicochemical methods, a colorless, transparent, slightly opalescent liquid with a phenol odor.
The vaccine is produced in ampoules of one vaccination dose or 5 vaccination doses. One dose (0.5 ml) contains 25 mcg of Vi-antigen.
The vaccine is administered once subcutaneously into the outer surface of the upper third of the shoulder. The vaccination dose is 0.5 ml.
The vaccine contains a preservative - phenol in a final concentration of no more than 0.25%.
Purpose: prevention of typhoid fever in adults.
The vaccine indicates the development of a pronounced immune response, seroconversion after 1-2 weeks of antibodies to the V antigen, which plays a key role in the development of typhoid infection; immunity to infection lasts for 2 years. Revaccination is carried out every 2 years.
The drug is well tolerated and non-pyrogenic. Reactions to the vaccine are quite rare and are considered mild. The vaccine is stored at a temperature from 2 degrees C to 8 degrees C. The shelf life of the drug is 2 years.
The development organization and manufacturer is the Limited Liability Company "GRITVAK".
Acylact in candles
Temporary pharmacopoeial article VFS 42-2941-97 was approved by order of the Ministry of Health of Russia dated September 16, 1997 N 276.
Registration No. 97/276/2.
Instructions for use approved 09/08/97.
Acylact in suppositories is a microbial mass of live acidophilic lactobacilli, dried in a cultivation medium with the addition of sucrose-gelatin-milk medium.
The suppository contains at least 10_7 live acidophilic lactobacilli (1 dose).
The drug is intended for the treatment of clinically pronounced dysbiosis and inflammatory processes of the female genitalia: nonspecific and hormonally dependent colpitis, vaginal dysbacteriosis, subacute and chronic stages of inflammatory processes in the genital area. Acylact in suppositories is also prescribed in preparation for planned gynecological operations in order to prevent postoperative infectious complications, during the period of prenatal preparation for pregnant women at risk, for the correction of normal microflora after specific antimicrobial therapy for sexually transmitted diseases.
The drug is prescribed as an independent remedy or after completing a course of antibacterial therapy.
For dysbacteriosis and senile vaginitis of a hormonal nature, acylact in suppositories is prescribed 1 suppository 2 times a day for 5-10 days. If the purity of the vaginal secretion in pregnant women is impaired to the III-IV degree, acylact in suppositories is used 1-2 suppositories per day for 5-10 or more days under the control of restoring the purity of the vaginal secretion to the I-II degree. In order to prevent purulent-septic complications, the drug is used 1 suppository 1-2 times a day for 5-10 days. before the proposed operation or delivery. Rehabilitation therapy after the use of antibiotics - 1-2 suppositories 1-2 times a day for 10 days. In the latter case, the course is repeated 2-4 times with an interval of 10-20 days.
Release form: 10 candles each, wrapped in wax paper and packed in boxes, or 5 candles in a blister pack.
Acylact in suppositories is stored and transported at a temperature of (5+/-3) degrees C. Shelf life - 1 year.
The developer institution is MNIIEM named after Gabrichevsky. The manufacturer is MGP-Progress LLP.
Diagnosticum erythrocyte for tick-borne virus
encephalitis immunoglobulin rat dry
for RNGA
Temporary pharmacopoeial monograph VFS 42-2874-97 was approved by order of the Russian Ministry of Health dated September 16, 1997.
Registration No. 97/276/3.
Instructions for use were approved on July 25, 1995.
Diagnosticum erythrocyte for TBE virus immunoglobulin rat dry for indirect hemagglutination reaction (IRHA) is a set of components, the main of which is erythrocyte diagnosticum (ED). ED is a freeze-dried 3% suspension of sheep erythrocytes, fixed with formalin or acrolein, treated with tannin and sensitized with a specific immunoglobulin.
Purpose - rapid detection of tick-borne encephalitis virus antigen in the blood of those suspected of being infected with the TBE virus or sick people, in suspensions of tick vectors and other virus-containing or potentially dangerous materials.
Release form:
1. Diagnosticum CE for RNGA - 5 amp. 1 ml.
2. TBE antigen (KA+) - 1 amp. 1 ml.
3. Normal antigen (KA-) - 1 amp. 1 ml.
4. Immunoglobulin against TBE (IG) 0.01 - 0.1% - 3 amp. 1 ml each
5. Normal rabbit serum (NRS), 10% - 2 amp. 1 ml each
Storage and transportation conditions: store in a dry, dark place at a temperature of (6+/-2) degrees C. Transportation is carried out by all types of covered transport under the same conditions.
Shelf life - 2 years. Developer and manufacturer: NPO Virion.
Head of the National
MIBP control body,
Director of GISC named after L.A. Tarasevich
N.V. Medunitsyn
The text of the document is verified according to:
"Collection of orders of the Ministry of Health
Russian Federation",
September, 1997
Owners of patent RU 2247991:
The invention relates to medicine and concerns a means, a method for its preparation and a method for express diagnostics of tick-borne encephalitis virus. The product is a 2% coagglutinating reagent obtained by adding an equal amount of 0.1% highly purified and highly avid immunoglobulin against tick-borne encephalitis to a 10% staphylococcal reagent dissolved in distilled deionized water, carrying out sensitization for 1-1.5 hours at a temperature of 25-30°C, resuspending the sediment in a 0.15 M sodium chloride solution pH 5.4-5.5 and bringing the resulting coagglutinating reagent to 2% content. A method for express diagnostics of the antigen of the tick-borne encephalitis virus consists of applying 15-20 μl of the test material to a glass slide, adding an equal amount of means for express diagnostics to it, thoroughly mixing by shaking the glass slide and visually determining after 2-7 minutes by the phenomenon of coagglutination presence of tick-borne encephalitis virus antigen. 3 n. and 3 salary files, 3 tables.
The invention relates to biotechnology and medicine, concerns the production of medical immunobiological preparations and laboratory diagnosis of the causative agent of a human infectious disease, in particular a means and method for express diagnostics (indication) of the tick-borne encephalitis virus.
A known means and method for rapid diagnosis of tick-borne encephalitis virus antigen by enzyme-linked immunosorbent assay (ELISA) using prepared immunoglobulin against tick-borne encephalitis and its conjugate with horseradish peroxidase.
However, this method is time-consuming (6-7 hours), labor-intensive, since it is necessary to perform five stages of research, and also expensive equipment is required when performing the method.
The closest in technological essence (prototype) to our proposed method for express diagnostics of tick-borne encephalitis virus antigen is the method by performing an indirect hemagglutination reaction (IRHA). The closest means and method for express diagnostics of tick-borne encephalitis virus antigen (prototype) is an erythrocyte diagnosticum and a method for its preparation . Erythrocyte diagnosticum is a 3% suspension of sheep erythrocytes, fixed with formalin or acrolein, treated with tannin and sensitized with immunoglobulin to tick-borne encephalitis virus.
Erythrocyte diagnosticum is obtained as follows:
A 3% suspension of acrolein- or formalin-fixed and toned sheep erythrocytes is sensitized with 0.05-0.1% immunoglobulin against tick-borne encephalitis at 56°C for 30 minutes. Then centrifuge for 10 minutes at 2000 rpm, wash twice in 1% NCS followed by centrifugation, the sediment is resuspended with filler: PBS pH 7.2, peptone - 3%, sucrose - 1%.
Sensitized erythrocytes specifically bind to the pathogen antigen in the test material, which is visually expressed in the manifestation of the phenomenon of hemagglutination in the RNGA.
The RNGA method is performed using the micromethod in a total volume of 75 μl, using U plates of the Takachi system from Matrimpex or any other plates intended for serological analyses.
The RNGA method is set up as follows:
25 μl of a 1% NCS solution is added to all wells of the plate. 25 μl of antigen (K+, K-, test material) is added to the first well of each row. Mix and transfer sequentially to all wells of the corresponding rows. Dilutions of antigens with a factor of 2 are obtained, then 25 μl of 1% NCS and a 1% suspension of erythrocyte diagnosticum are added to all wells. Erythrocytes are monitored for spontaneous agglutination: 50 μl of 1% NCS and 25 μl of a 1% suspension of erythrocyte diagnosticum are added to 2-3 wells. The tablet is shaken in a horizontal plane to mix the ingredients and placed at a temperature of (20±2)°C for 2-2.5 hours or for 1-1.5 hours at a temperature of (37±2)°C.
In the absence of agglutination in the erythrocyte controls, the reaction is counted visually using the 4-cross system. A positive result in the RNGA is considered to be agglutination of erythrocytes at +3 +4. The antigen titer is taken to be its maximum dilution, which gave hemagglutination at +3 +4. RNGA with K+ should be positive, with K- - negative.
However, this agent, the method for its preparation and the method for express diagnostics of tick-borne encephalitis virus antigen have the following disadvantages:
The duration of preparation of the erythrocyte diagnosticum, the sensitivity of which depends on many factors: the physiological state of sheep erythrocytes, the purity of acrolein (or formalin), tannin, temperature conditions of each stage of the technological process and, finally, on the purity, specific activity and avidity of the immunoglobulin;
The implementation of the RNGA method takes several hours, while time is lost and the effectiveness of emergency seroprophylaxis for victims, which is highest in the first day after an infected tick bite, decreases;
The RNGA method is not sensitive enough and thus does not give the desired result, which is one of the important conditions for the rapid diagnosis of tick-borne encephalitis virus antigen.
The problem solved by the proposed invention is to reduce the time, increase the sensitivity, accessibility and cost-effectiveness of the method while maintaining its high specificity.
The problem is solved by a means for express diagnostics containing a 2% coagglutinating reagent obtained by adding an equal amount of 0.1% highly purified and highly avid immunoglobulin against tick-borne encephalitis to a 10% staphylococcal reagent dissolved in distilled deionized water, performing sensitization for 1-1.5 hours at a temperature of 25-30°C, resuspending the sediment in a 0.15 M sodium chloride solution pH 5.4-5.5 and bringing the resulting coagglutinating reagent to 2% content and using the express diagnostic method antigen of the tick-borne encephalitis virus by applying 15-20 μl of the test material to a glass slide and adding to it an equal amount of an agent for express diagnostics containing a 2% coagglutinating reagent obtained by sensitizing a 10% staphylococcal reagent with highly avid and highly purified immunoglobulin against tick-borne encephalitis, thorough mixing by shaking the glass slide and visual determination after 2-7 minutes by the coagglutination phenomenon of the presence of tick-borne encephalitis virus antigen and its quantity according to a 3-point system.
In the patent and scientific-medical literature analyzed by the authors, this set of distinctive features leading to a new technical result was not found, and they do not clearly follow for a specialist from the prior art. The method, a means for express diagnostics of the antigen of the tick-borne encephalitis virus and the method for its preparation were tested in laboratory conditions. Thus, these technical solutions meet the criteria of the invention: “novelty”, “inventive step”, “industrially applicable”.
A tool for rapid diagnosis of tick-borne encephalitis virus antigen is prepared as follows:
Dry 10% staphylococcal reagent is dissolved in distilled deionized water, kept for 1.5-2.0 hours at room temperature for swelling and added to the 10% staphylococcal reagent dissolved in distilled deionized water, an equal amount of 0.1% highly purified and high-avidity immunoglobulin against tick-borne encephalitis and sensitization is carried out for 1-1.5 hours at a temperature of 25-30 ° C, then centrifuged for 20 minutes at 3000 rpm, the sediment is resuspended in a 0.15 M sodium chloride solution pH 5.4- 5.5 to 2% content of the coagglutinating reagent, add sodium merthiolate to 0.001% content and store at a temperature of (7±3)°C for 4-6 months.
The method for rapid diagnosis of tick-borne encephalitis virus antigen is carried out as follows:
15-20 μl of the test material is applied to a glass slide and an equal amount of rapid diagnostic agent is added to it, containing a 2% coagglutinating reagent obtained by sensitizing a 10% staphylococcal reagent with highly avid and highly purified immunoglobulin against tick-borne encephalitis, mix thoroughly using shaking the slide and visually determine after 2-7 minutes by the coagglutination phenomenon the presence of tick-borne encephalitis virus antigen using a 3-point system.
“3+” - the formation of an agglutinate and intense transillumination of the reaction mixture (the reaction is sharply positive);
“2+” - clear clearing of the mixture (positive reaction);
“+” - slight clearing of the mixture (slightly positive reaction);
“-” - lack of enlightenment (negative reaction).
The proposed method makes it possible to detect both the presence and quantitative content of the tick-borne encephalitis virus antigen in the test material. For quantitative determination, dilutions of the test material are made in a 0.15 M sodium chloride solution with a coefficient of 2 and 15-20 μl are applied to a glass slide, an equal amount of rapid diagnostic agent is added, containing a 2% coagglutinating reagent obtained by sensitization A 10% staphylococcal reagent with highly avid and highly purified immunoglobulin against tick-borne encephalitis is mixed by shaking the slide and after 2-7 minutes, the titer of the antigen of the tick-borne encephalitis virus in the test material is determined visually by the coagglutination phenomenon using a 3-point system.
Coagglutination at +3 +2 is considered a positive result of the proposed method. The antigen titer (1 coagglutinating unit - COAU) is taken to be its last dilution, which gave intense coagglutination (by +2).
Our proposed method and tool for rapid diagnosis of tick-borne encephalitis virus antigen is based on the phenomenon of coagglutination during the interaction of specific antibodies and antigen.
To confirm the specificity of the proposed method and means for express diagnostics of the tick-borne encephalitis virus antigen, the material (substrate) containing and not containing the tick-borne encephalitis virus antigen was titrated (see Table 1).
The proposed method and means for express diagnostics of the tick-borne encephalitis virus antigen were compared in terms of sensitivity, determining the amount (titer) of the tick-borne encephalitis virus antigen by diluting the test material with a factor of 2 in a 0.15 M sodium chloride solution with the rapid diagnostic methods ELISA and RNGA. In this case, the same material containing the antigen of the tick-borne encephalitis virus was titrated in three ways: by ELISA, RNGA and the proposed one (see Table 2). The material under study was a tick-borne encephalitis vaccine at different stages of the technological process.
From the above material it follows that the proposed method for rapid diagnosis of tick-borne encephalitis virus antigen, performed using a product containing a 2% coagglutinating reagent obtained by sensitizing a 10% staphylococcal reagent with a highly avid and highly purified immunoglobulin against tick-borne encephalitis, is specific in quantity identified tick-borne encephalitis virus antigen and a minimal amount of protein E is many times more sensitive than the RNGA method and on average 2-4 times more sensitive than the ELISA method.
Thus, the proposed method for rapid diagnosis of tick-borne encephalitis virus antigen, performed using a product containing a 2% coagglutinating reagent, obtained by adding an equal amount of 0.1% highly purified staphylococcal reagent to a 10% staphylococcal reagent dissolved in distilled deionized water and high-avidity immunoglobulin against tick-borne encephalitis, carrying out sensitization for 1-1.5 hours at a temperature of 25-30°C, resuspending the sediment in a 0.15 M sodium chloride solution pH 5.4-5.5 and bringing the resulting coagglutinating reagent to 2 % content, like the ELISA and RNGA methods, is specific, but surpasses them in sensitivity and many times in speed (running time for ELISA is 6-7 hours, for RNGA 1.5-2.5 hours, for the proposed method 2-7 minutes ), with ease of implementation and cost-effectiveness (Table 3).
References
1. Laptakova M.M., Moryakin A.V., Lavrova N.A. Development and use of an immunoperoxidase test system for indicating tick-borne encephalitis virus. Sat. Current issues in medical biotechnology and applied immunology. - Tomsk. - 1990. – volume 36. – pp. 50-65.
2. Podoplekina L.E., Unger T.N. Diagnosticum erythrocyte to tick-borne encephalitis virus immunoglobulin rat dry for RNGA (erythrocyte diagnosticum to tick-borne encephalitis virus). - VFS 42. - 2874 - 97.
3. Podoplekina L.E., Unger T.N. Experimental production regulations No. 527 - 95 Diagnosticum erythrocyte to tick-borne encephalitis virus immunoglobulin rat dry for RNGA. NIIVS NPO “Virion”.
| Table 1. | |||||
| No. | MATERIAL STUDYED | TBE VIRUS ANTIGEN TITER (unit) | |||
| 1 | TBE vaccine “EnceVir” ser. 10. this year 11.02. (produced by Federal State Unitary Enterprise NPO Virion) | 512 | |||
| 2 | TBE virus antigen (K+) ser. 46 from the erythrocyte diagnostic kit. this year 2001 (produced by FSUE NPO Virion) | 256 | |||
| 3 | Brain antigen of uninfected mice (K-) ser.45 from the erythrocyte diagnostic kit of this year. 2001 (produced by FSUE NPO Virion) | 0 | |||
| 4 | TBE virus antigen (K+) ser. 55 from the ELISA kit this year. 10.30.02 (produced by FSUE NPO Virion) | 3200 | |||
| 5 | Brain antigen of uninfected mice (K-) ser.54 from the ELISA kit this year. 10.30.02 (produced by FSUE NPO Virion) | 0 | |||
| Table 2. | |||||
| No. | MATERIAL STUDYED | TBE VIRUS ANTIGEN TITER (in standard units) |
|||
| ELISA | RNGA | RCOA | |||
| 1 | Combined semi-finished product of TBE vaccine (1) | 32 | <8 | 128 | |
| 2 | TBE vaccine concentrate (1) | 1024 | 64 | 4096 | |
| 3 | Purified TBE vaccine concentrate (1) | 512 | 32 | 2048 | |
| 4 | Semi-finished FE vaccine (1) | 128 | <8 | 512 | |
| 5 | Combined semi-finished product of TBE vaccine (2) | 16 | <8 | 32 | |
| 6 | TBE vaccine concentrate (2) | 1024 | 64 | 2048 | |
| 7 | Purified TBE vaccine concentrate (2) | 512 | 32 | 512 | |
| 8 | Purified TBE vaccine concentrate (3) | 256 | 32 | 1024 | |
| 9 | Semi-finished FE vaccine before sorption | 256 | 32 | 256 | |
| Table 3. Sensitivity of ELISA, RNGA, RCOA for protein E. |
|||||
| No. | MATERIAL STUDYED | PROTEIN E CONTENT (µg/ml) | ELISA (sample units) PROTEIN E (ng) | RNGA (mod. units) | RCOA (unit) |
| PROTEIN E (ng) | PROTEIN E (ng) | ||||
| 1 | Combined semi-finished product of TBE vaccine(1) | 0,06 | <8 | ||
| 2 | TBE vaccine concentrate(1) | 11,93 | |||
| 3 | Purified TBE vaccine concentrate(1) | 8,78 | |||
| 4 | Semi-finished FE vaccine before sorption (1) | 4,13 | <8 | ||
| 5 | TBE vaccine concentrate(2) | 2,48 | |||
| 6 | Purified TBE vaccine concentrate(2) | 5,5 | |||
| 7 | Purified TBE vaccine concentrate(3) | 6,93 | |||
| 8 | Semi-finished FE vaccine before sorption (2) | 7,83 |
1. A tool for rapid diagnosis of tick-borne encephalitis virus antigen, containing a 2% coagglutinating reagent obtained by sensitizing a 10% staphylococcal reagent with highly purified and high-avidity immunoglobulin against tick-borne encephalitis.
2. A method for preparing a means for express diagnostics of the tick-borne encephalitis virus antigen, which consists of sensitization, centrifugation of the prepared suspension, removal of the supernatant, resuspension of the sediment and preparation of the reagent, characterized in that before sensitization to 10% staphylococcal reagent dissolved in distilled deionized water highly purified and highly avid immunoglobulin against tick-borne encephalitis is added, sensitization is carried out for 1-1.5 hours, then the sediment is resuspended in a 0.15 M sodium chloride solution and the resulting coagglutinating reagent is adjusted to 2% content.
3. The method according to claim 2, characterized in that sensitization is carried out at a temperature of 25-30°C.
4. The method according to claim 2, characterized in that an equal amount of 0.1% highly purified and high-avidity immunoglobulin against tick-borne encephalitis is added to a 10% staphylococcal reagent dissolved in distilled deionized water.
5. The method according to claim 2, characterized in that the sediment is resuspended in a solution of 0.15 M sodium chloride, pH 5.4-5.5.
6. A method for express diagnostics of the antigen of the tick-borne encephalitis virus by examining the material, determining the presence and/or quantitative content of the antigen of the tick-borne encephalitis virus by diluting it in a 0.15 M solution of sodium chloride, characterized in that the test material or its dilutions in in an amount of 15-20 µl, add an equal amount of a means for rapid diagnosis of tick-borne encephalitis virus antigen containing a 2% coagglutinating reagent obtained by sensitizing a 10% staphylococcal reagent with highly purified and high-avidity immunoglobulin against tick-borne encephalitis, mix thoroughly by shaking the slide and after 2-7 minutes, the presence and/or amount of tick-borne encephalitis virus antigen is visually determined by the coagglutination phenomenon.
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The invention relates to medicine, namely to medical microbiology, and can be used for serological diagnosis of urogenital chlamydia, and concerns a method for obtaining species-specific antigen C.
DIAGNOSTICS(Greek, diagnostikos capable of recognizing) - suspensions of neutralized microorganisms used as antigens for serological reactions. The danger of working with live cultures, their ability to vary, and the presence of broad antigenic bonds make it advisable to use D. - more standard and homogeneous preparations containing certain antigenic components.
With the help of D., agglutination reactions, passive (indirect) hemagglutination (RPHA), and others are used to identify specific antibodies in the sera of people and animals for the purpose of making a diagnosis and studying the immune state of the body.
D. is especially widely used in laboratory studies for intestinal infections. However, the common antigenic structure of intestinal bacteria determines the presence of cross-reactions and requires differentiated detection of antibodies. For this purpose, selective suppression of individual antigens is carried out: using phenol or formalin, O-agglutinability is suppressed, as suggested by Felix and Olitzki (A. Felix, L. Olitzki, 1928). By treating with alcohol according to the method of Wien and Sontag or by heating according to Kauffmann, O-diagnosticums with inactivated flagellar antigen are obtained. Even more promising is the use of mono diagnosticums, the principle of which was developed by F. G. Bernhof (1944). These drugs contain only one antigenic component, and they interact only with certain specific antibodies.
The possibility of preserving the properties of bacterial D. by their lyophilization has been shown (see).
D., used for serodiagnosis of various infections, are fundamentally similar, however, certain types of these drugs have certain specifics.
It is generally accepted that RPGA is more sensitive and specific than bacterial agglutination. Formalized erythrocytes sensitized with antigens obtained using the Boivin and Westphal methods are used as antigens in RPGA.
It is also possible to use erythrocyte D. to detect antigen in tissues, secretions of patients, extracts of various substances, etc. In these cases, erythrocytes sensitized with antibodies are used - “antibody diagnosticums”.
Viral D. are used mainly in the complement fixation reaction (CRF), RTGA, and neutralization reactions. They are prepared from virus-containing culture liquids treated (inactivated) in various ways.
A brief description of the main D. and the scope of their application is presented in the table.
There are also experimental drugs used in scientific work: erythrocyte colidiagnosticums, diphtheria erythrocyte D., hemagglutinating antigens of mumps viruses, measles hemagglutinating antigen, etc.
Table. Brief description of the main diagnosticums and the purpose of their use
|
Diagnosticums |
Brief description |
Purpose of use (serum of the subject is used) |
|
Bacterial diagnosticums |
||
|
Diagnosticums from bacteria of the intestinal family: Shigella Flexner, Sonne, Newcastle; Salmonella typhus (OH, O), paratyphoid A and B, cholera su is, typhimurium, enteritidis |
Microbial suspension (3 billion microbial bodies in 1 ml), inactivated with 0.4-0.5% formaldehyde solution |
Statement of an agglutination reaction to clarify the wedge, diagnosis of the disease |
|
Salmonella O-diagnosticums (2, 4, 7, 8, 9 and 3, 10) |
Microbial suspension containing partial O-antigen (3 billion microbial bodies in 1 ml), obtained from selected strains, inactivated with 15% glycerol solution |
Setting up an agglutination reaction to detect O-antibodies for salmonellosis |
|
Salmonella H-monodiagnosticums (a, b, c, d, eh, c, k, lv, gm, p, st and second phase antigens - 1, 2, 5, 6, 7) |
Microbial suspension containing components of the flagellar antigen of the 1st and 2nd phases (3 billion microbial bodies in 1 ml), obtained from selected strains, inactivated with 0.5% formalin solution |
Performing an agglutination reaction to detect H-antibodies for diagnostic purposes and to establish a history of disease |
|
Vi-diagnosticum |
Microbial suspension (3 billion microbial bodies in 1 ml) from strains containing Vi-factor, treated with 0.4% formalin solution and 0.6% calcium chloride solution |
Performing an agglutination test to detect typhoid bacteria carriage |
|
Brucellosis unified diagnosticum |
A suspension of Brucella in 12% sodium chloride solution, colored blue and inactivated with 0.5% phenol solution |
Staging an agglutination reaction (Wright reaction and Huddleson reaction to identify infected people and animals - see Brucellosis, research methods) |
|
Tularemia diagnosticum |
Microbial suspension (25 billion microbial bodies in 1 ml) of the vaccine strain, inactivated with 0.5% formaldehyde solution |
Setting up volumetric droplet agglutination reaction on glass for serodiagnosis and studying the immune state of vaccinated people |
|
Leptospirosis diagnosticums |
Lyophilized microbial suspension of 11 strains of the most common serotypes |
Statement of RSC to confirm the wedge, diagnosis of the disease |
|
Rickettsial diagnosticums |
Corpuscular antigens - a suspension of rickettsiae grown in the yolk sacs of chicken embryos or pulmonary rickettsial material from infected rodents, treated with ether, celite or differential centrifugation |
Setting up an agglutination reaction for the differential diagnosis of rickettsial diseases |
|
Erythrocyte diagnosticums |
||
|
Erythrocyte diagnosticums from Shigella Flexner - Sonne |
Formalized erythrocytes sensitized with antigens of Boivin, Westphal, etc. |
Statement of RPGA to clarify the clinical diagnosis of dysentery |
|
Erythrocyte Salmonella Vi-diagnosticum |
Formalized erythrocytes sensitized with purified Vi antigen |
Statement of RPGA to detect typhoid bacteria carriage |
|
Erythrocyte Salmonella O-diagnosticums (1, 2, 12; 1, 4, 12; 6, 7; 6, 8; 1, 9, 12; 3, 10 and complex) |
1% suspension of formalinized erythrocytes sensitized with antigens Boivin, Westphal, etc. |
Statement of RPGA to clarify the clinical diagnosis of the disease |
|
Lyophilized tularemia erythrocyte diagnosticum |
Formalized lyophilized red blood cells sensitized with tularemia antigen |
Statement of RPGA to clarify the clinical diagnosis of tularemia, as well as the antibody neutralization reaction to detect: antigen |
|
Viral diagnostics |
||
|
Vaccinia virus antigen |
Dry preparation of live vaccinia virus cultured on the chorion-allantoic membrane of chick embryos |
Statement of RPGA for the detection of antihemagglutinins in patients and vaccinated people |
|
Adenoviral specific antigen |
Prepared by cultivating type 6 virus in a culture of continuous A-1 cells (antigen common to all adenoviruses) |
Testing of RSC to detect complement-fixing antibodies in patient serum |
|
Diagnostics of tick-borne encephalitis and etiologically similar diseases |
Statement of RSC and X-ray to clarify the wedge, diagnosis of the disease |
|
|
Ornithosis antigen |
Dry preparation from boiled virus-containing yolk sacs of chicken embryos, extracted with ether, precipitated with acetone and inactivated with merthiolate |
Statement of RSC for the diagnosis of ornithosis in humans, birds and animals |
|
Dry influenza diagnosticum |
Allantoic fluid of developing chicken embryos infected with one of the strains of influenza virus type A, B or C, inactivated by formalin, merthiolate. Due to the variability of the antigenic structure of the influenza virus, frequent changes in production strains are provided |
Statement of X-ray to clarify the clinical diagnosis of the disease |
|
Parainfluenza diagnosticum type 1, 2, 3 for RTGA, dry |
Culture fluid (human embryonic kidneys) containing one of the parainfluenza virus strains, treated with Tween-80, ether and merthiolate |
Statement of X-ray to clarify the wedge, diagnosis of the disease |
Bibliography: Zuev A. S. Bacterial vi-diagnosticum for identifying chronic carriers of typhoid bacteria, Zhurn, mikr., epid, i imm., No. 2, p. 51, 1959, bibliogr.; Zuev A. S., Novoselova A. I. and Likina I. V. Development of a method for the production of O-diagnosticums and N-monodiagnosticums and their use in the serodiagnosis of salmonellosis, ibid., No. 3, p. 42, 1956; Immunology and prevention of intestinal infections, ed. S. I. Didenko, p. 180, M., 1962; Karalnik B.V. Erythrocyte diagnosticums, M., 1976; Guide to microbiological diagnosis of infectious diseases, ed. K. I. Matveeva, p. 172, M., 1973; Subbotina Yu. L. and dr. Serological diagnosis of salmonellosis and antigenic connections in reactions with erythrocyte and bacterial O-diagnosticums, Zhurn, mikr., epid, i imm., No. 3, p. 19, 1970, bibliogr.
L. B. Bogoyavlenskaya.
Contains high titer antibodies against tick-borne encephalitis virus. Obtained from the blood serum of horses hyperimmunized with tick-borne encephalitis virus. It is used for emergency prevention and treatment of patients with tick-borne encephalitis.
FSME-Bulin. Human immunoglobulin against tick-borne encephalitis (Baxter AG, Austria)
Contains antibodies that neutralize the tick-borne encephalitis virus, stabilizer - glycerin, no preservative. It is used for emergency prevention and treatment of patients with tick-borne encephalitis.
Diagnostic drugs
Serums
Anti-smallpox serum
Contains antibodies to variola virus. Used to identify the virus in neutralization reactions.
Anti-rabies fluorescent serum
Contains antibodies to the rabies virus, labeled with fluorochrome. Used to search for pathogens in RIF.
Specific serum against tick-borne encephalitis virus
Contains antibodies to tick-borne encephalitis virus. Used for virus identification in RTGA, RNGA, etc.
Diagnosticums
Smallpox diagnosticum
Contains variola virus antigens. It is used in the serological diagnostic method.
Diagnosticum from tick-borne encephalitis virus
Contains virus antigen, prepared from the brains of mice infected with tick-borne encephalitis virus. It is used to set a reaction in the serological method.
Test system for detecting antibodies to HIV in ELISA
Contains a specific HIV antigen and additional ingredients necessary for ELISA. It is used at the first stage of serological diagnosis of HIV infection.
Test system for immunoblotting for the diagnosis of HIV infection
Contains a complex of HIV fractions (antigens) separated by electrophoresis: gp41, gp120, p24, p31, etc. It is used as a confirmatory test at the final stage of serological diagnosis of HIV infection.
Examples of descriptions of immunobiological drugs:
Precipitating anthrax serum
KP: Serum
DN: At
WITH: Fraction of blood serum of animals hyperimmunized with antigens of the anthrax pathogen, containing precipitins
Pr: Express diagnostics of the pathogen in the test material
SPr: To perform the ring-pricipitation reaction
Luminescent typhoid serum
KP: Serum
DN: At
WITH: Fraction of blood serum of animals hyperimmunized with antigens of the causative agent of typhoid fever, containing antibodies labeled with FITC
Pr: Express diagnosis of typhoid fever
SPr: For staging RIF.
Plague bacteriophage
KP: Bacteriophage
DN: Bacteriophage
WITH: Sterile filtrate of plague pathogen broth culture phagolysate
Pr: Bacteriological diagnostic method
SPr: To perform a reaction of increasing phage titer in order to detect the causative agent of plague in environmental objects
Dysenteric polyvalent bacteriophage in tablets
With acid-resistant coating
KP: Bacteriophage
DN: Bacteriophage
WITH: Sterile filtrate of phagolysate of broth culture of phagolysates of Shigella dysentery Flexner and Sonne
Pr: Emergency prevention and treatment of bacterial dysentery
SPr: Orally
Brucellosis vaccine, therapeutic
KP: Vaccine
C: Killed by Heat Brucella abortus And B. melitensis
Pr: For the treatment of patients with acute, subacute and chronic brucellosis
SPr: Parenterally
Pertussis monovaccine
KP: Vaccine
DN: Ag
WITH: Attenuated strains of the causative agent of whooping cough
Pr: To create active artificial antimicrobial immunity
SPr: Parenterally according to epidemiological indications
Calendar of preventive vaccinations in Russia, 2002 1
| Age | Name of vaccination |
| Newborns for the first time 12 hours | First vaccination - hepatitis B |
| Newborns 3-7 days | Vaccination – tuberculosis |
| 1 month | Second vaccination – hepatitis B 2 |
| 3 months | First vaccination – diphtheria, tetanus, whooping cough and polio |
| 4.5 months | Second vaccination – diphtheria, tetanus, whooping cough, polio |
| 6 months | Third vaccination – diphtheria, tetanus, whooping cough, polio Third vaccination – hepatitis B |
| 12 months | Vaccination – measles, mumps, rubella |
| 18 months | First revaccination – diphtheria, whooping cough, tetanus, polio |
| 20 months | Second revaccination – polio |
| 6 years | Second vaccination – measles, mumps, rubella |
| 7 years | Second revaccination – diphtheria and tetanus First revaccination – tuberculosis 5 |
| 13 years old | Vaccination against viral hepatitis B Vaccination against rubella (girls) 4 |
| 14 years old | Third revaccination – diphtheria and tetanus Revaccination – tuberculosis 6 |
| 16 years old | Third revaccination – polio |
| Adults | Revaccination – diphtheria and tetanus every 10 years after the last revaccination |
Notes:
1 Immunization within the framework of the National Vaccination Calendar is carried out with vaccines of domestic and foreign production, registered and authorized for use in the prescribed manner in accordance with the instructions for their use.
2 Children born to mothers who are carriers of the hepatitis B virus or patients with hepatitis B in the 3rd trimester of pregnancy are vaccinated according to the schedule of 0-1-2-12 months.
3 Vaccination against hepatitis B at 13 years of age is carried out for those who have not previously been vaccinated or who have received only one vaccination.
4 Vaccination against rubella is given to girls at the age of 13 who have not previously been vaccinated or have received only one vaccination.
5 Revaccination with BCG at 7 years of age is carried out for tuberculin-negative children who are not infected with Mycobacterium tuberculosis.
6 Revaccination with BCG at 14 years of age is carried out for tuberculin-negative children who are not infected with Mycobacterium tuberculosis and who have not received vaccination at 7 years of age.
7 Preventive vaccines (except BCG) used within the framework of the National Calendar can be administered simultaneously (or with an interval of 1 month) with different syringes into different parts of the body.
8 If the start date for vaccinations is not met, the latter are carried out according to the schemes provided for in this calendar and instructions for the use of drugs.
Bibliography
1. Guide for extracurricular work of students on immunological drugs: textbook. allowance / L.A. Levanova, V.A. Gromova, I.E. Filippova, E.V. Surikova, L.P. Osipova, Yu.V. Zakharova, V.P. Kovtun, T.E. Garanina. – Kemerovo: Kemerovo State Medical Academy, 2010 – 107 p.
2. Guide to practical training in medical microbiology, virology and immunology” / Edited by Academician of the Russian Academy of Medical Sciences O.V. Bukharin. M.: Medicine; Ural Branch of the Russian Academy of Sciences, 2002 – 340 p.
3. Handbook on the use of bacterial and viral drugs (edited by S.G. Dzagurov and F.F. Rezepov). – M.: Medicine, 1975.
4. Borisov, L.B. Guide to laboratory classes in medical microbiology, virology and immunology: Textbook. allowance. / L.B. Borisov, B.N. Kozmin-Sokolov, I.S. Freidlin. – M.: Medicine, 1993. – 240 p.
5. Clinical and immunological effectiveness of immunobiological drugs: (reference book) T.I. Abakumov et al., ed. M.P. Kostinova and N.A. Ozeretsky - M.: Miklos, 2008 - 256 p.
The invention relates to the field of medicine and veterinary medicine. The method involves ultrasonic treatment of a virus-containing suspension from various, including avid, strains of tick-borne encephalitis virus. In this case, they are treated with ultrasound at a frequency of 22 kHz for 1 minute, repeating this procedure three times with an interval of 2 minutes, using an UZDN-1 or UZDN-A apparatus. The method makes it possible to increase the diagnostic effectiveness of the resulting drug, which is expressed in an increase in the titers of antibodies detected in the blood sera of patients, diagnostic increases in antibody titers in the hemagglutination inhibition reaction and the reliability of the frequency of confirmation of the clinical diagnosis of “tick-borne encephalitis” up to 33%. The invention can be used in the production of diagnostic drugs and to improve the efficiency of serological diagnosis of tick-borne encephalitis. 1 salary files, 4 tables.
The invention relates to medicine and veterinary medicine, namely to virology, and can be used in the production of diagnostic drugs and to increase the efficiency of serological diagnosis of viral infections, in particular tick-borne encephalitis (TBE).
There is a known method for producing the TBE diagnosticum, characterized in that in order to increase the specific activity of the diagnosticum, an avid strain of the TBE virus 4072 is used, and to inactivate the virus, betapropiolactone is added to the resulting virus-containing suspension to a 0.1% concentration and kept for 24 hours at a temperature 4°C, and after centrifugation, inactivation continues for another 3 days (see A.S. No. 1378112 A1, A61K 39/12, G01N 33/53, 1987 / Kokorev V.S., Mansurov P.G., Zlobin V .I., Subbotina L.S., Gaidamovich S.Ya., Melnikova E.E. / Application No. 4044449/28-13, dated 02/11/1986).
The disadvantage of this method of obtaining an antigen is the inclusion in the technological process of a highly active chemical reagent betapropiolactone, which negatively affects the antigenic determinants of the virus when inactivating infectious activity. In this regard, the quality of antigenic preparations, even when using avid variants of the virus, decreases.
There is also a known method in which avid variants of the virus - strains 4072 or 3745 - are used as a source of TBE virus antigen, and inactivation of infectious activity is carried out with methylglyoxal at a concentration of 0.1-0.05% at 4-8°C for 24 -72 hours (see A.C. No. 1169219 A, A61K 39/12, C12R 1/91, 1985 / Gizatullina N.K., Ravilov A.E., Kolotvinov S.V., Kokorev V.S./ Application No. 3509839 /28-13, dated 05.11.1982).
The disadvantage of this method is similar to that stated above. In addition, in both cases, the time to obtain an antigenic drug and the costs of its production increase with the insufficiently high efficiency and sensitivity of the drugs.
There is also a known method for producing a vaccine, during the production of which the inactivation of the virus in the culture liquid is carried out by irradiation, while cobalt-60 is used as a source of gamma emitter at a dose of 15-20 kGy for 2 hours at a temperature of 19-21 ° C (See Patent RF No. 2181295, A61K 39/12, 39/245. Publ. 20.04.2002.
The main disadvantage of this method is the special processing conditions that require radiation protection and specially trained, highly qualified personnel.
There is also a known method of increasing the antigenicity and immunogenicity of a biomaterial by exposing it to laser radiation with a wavelength from 9 to 11 microns and a power density of 0.1-1.0 kW/cm 2, conducted on a jet of biomaterial suspension with a diameter of 0.5-15 mm, at jet outflow speed from 0.05 m/s to 10 m/s (see RF Patent No. 2209085, A61K 41/00, 39/00. Publ. 07.27.2003. Bulletin No. 21).
With good results of such processing, some complexity of the laser processing process is the need for laser installations, which require special premises and additional measures to protect operating personnel, which increases the cost of obtaining drugs.
The objective of the invention is to exclude toxic chemical reagents from the technological cycle for obtaining a diagnosticum for tick-borne encephalitis and at the same time increasing the sensitivity and specificity of the diagnosticums while reducing the time and money spent on obtaining the drug with expanding the range of diagnosticums, in particular tick-borne encephalitis, hypersensitivity.
The problem is solved by the fact that when preparing a drug for processing a virus-containing suspension, it is exposed to ultrasonic radiation (US) at a frequency of 22 kHz for 1 minute, repeating this procedure three times with an interval of 2 minutes, while the ultrasonic treatment is carried out on devices of the UZDN-1 type or UZDN-A using a universal emitter at 22 kHz, and blood serum samples from patients with tick-borne encephalitis are subjected to anti-inhibitory treatment with kaolin at pH 7.4, and the following strains of tick-borne encephalitis virus are preferably used: reference strain Sofin, avid strains 4072 and 3445, non-avid strain 80, and processing is carried out at an ambient temperature of 25-30°C.
The method is carried out as follows.
Antigen preparation:
Use: 1. Author's strain of the TBE virus 4072 / Kokorev B.C. Melnikova E.E., Kolotvinov S.V. and others/, deposited in the GKV under No. 633. Strain 4072 was isolated in 1966 from the blood of a patient, highly sensitive to the neutralizing effect of specific antibodies (according to the results of cross-kinetic RTGA), avid, which differs from the reference strain Sofin;
2. The author's strain of the TBE virus 3745 /Kolotvinov S.V., Kokorev V.S., Melnikova E.E., etc./, deposited in the State Book of Vacancies under No. 636. The strain was isolated from the blood of a person without clinical manifestations of the disease, previously attacked by ticks, highly sensitive to neutralizing effect of specific antibodies, aviden;
3. Author's strain of TBE virus 80 / Kokorev V.S., Melnikova E.E., Kolotvinov S.V. and others/, deposited in the State Bill under No. 634. The strain was isolated from Ixodes persulcatus ticks, is little sensitive to the neutralizing effect of specific antibodies, and is non-avid;
4. Reference strain Sofiin of the TBE virus, obtained from the GKV Institute of Virology named after. D.I. Ivanovsky RAMS.
Hemagglutinating antigens of these viruses are obtained by borate-salt extraction with treatment with protamine sulfate. To do this, prepare a 10 or 20% suspension from the brain of newborn white mice in a borate buffer solution pH 9.0-9.3 in the cold, centrifuge in the cold for 1 hour at 10,000 rpm. A solution of protamine sulfate of appropriate concentration is added to the supernatant liquid in an amount of 0.1 volume, i.e. per 10 ml add 1 ml of protamine sulfate solution. A solution of protamine sulfate is prepared in saline at a concentration of 50 mg/ml or 25 mg/ml for 20% and 10% brain suspension, respectively. The mixture is placed in an ice bath for 30 minutes, stirring occasionally, and then centrifuged for 15 minutes at 2500 rpm in the cold. The clear supernatant is the antigen.
Preparation of blood serum samples.
Removal of serum inhibitors with kaolin at pH 9.0 and removal of normal serum agglutinins is carried out according to generally accepted methods. Treatment of blood serum samples with a kaolin suspension at pH 7.4 is carried out as follows. To 0.2 ml of the test sample add 0.8 ml of a borate buffer solution with pH 7.4 and 1 ml of a 25% kaolin suspension prepared in a borate buffer solution with pH 7.4. The resulting mixture is shaken for 25 minutes and then centrifuged at 1000 rpm for 30 minutes. Add 2 drops (0.05 ml) of washed goose red blood cells to the supernatant (to remove normal serum agglutinins) and shake periodically for 25 minutes. The supernatant obtained after repeated centrifugation is adjusted to pH 9.0 with NaOH solution.
New proposed operation - Treatment of antigenic preparation - diagnosticum with ultrasound.
Ultrasound treatment of the antigen preparation is carried out using an UZDN-1 or UZDN-A apparatus (output power 400 W) at a frequency of 22 kHz for 1 minute, repeating this procedure three times with an interval of 2 minutes, at an ambient temperature of 25-30°C. The treated material is centrifuged at 12,000 rpm for 15 minutes.
Technological parameters of the proposed ultrasonic treatment method: ultrasonic frequency 22 kHz; ultrasound processing time - within 1 minute; repeatability of ultrasound treatment - 3 cycles, medium temperature and centrifugation modes after ultrasound - were determined and justified practically in the laboratory based on the results of numerous studies, taking into account the analysis and review of available sources, conducted in the laboratory of vector-borne viral infections and tick-borne encephalitis of the Ekaterinburg Scientific Research Institute Research Institute of Viral Infections" and laboratories of the Department of Microbiology and Virology of the Federal State Educational Institution of Higher Professional Education "Ural State Agricultural Academy" Yekaterinburg.
Example 1. Using the proposed method, hemagglutinating antigens are obtained from the reference strain Sofiin and the avid strain 4072, which are used in the study of paired blood serum samples from patients with tick-borne encephalitis.
Ultrasound treatment of virus-containing suspensions changes not only and not so much their hemagglutinating activity (hemagglutinin titers, as a rule, increase by 2 times), but also the activity in the hemagglutination inhibition reaction (HAI): the number of so-called seronegative samples is reduced, the increase in antibody titers is higher than with using a drug untreated with ultrasound (Table 1 and Table 2), which is a non-obvious effect of the proposed method with ultrasound treatment of the drug instead of introducing chemical preservatives - stabilizers.
Thus, from tables 1 and 2 it is clear that when using ultrasound antigens, the diagnostic value of RTGA increases, namely: the frequency of confirmation of the clinical diagnosis of “tick-borne encephalitis” increases up to 33% by a 2-4-fold increase in antibody titer compared to the reaction, delivered with unprocessed ultrasound diagnosticum.
| Table 1 | |||
| Study of blood serum samples from patients with TBE in the RTGA with diagnosticum from the reference strain Sofin, treated with ultrasound | |||
| Antihemagglutinin titers in RTGA with ultrasound diagnosticum from the Sofiin strain | |||
| Unprocessed drug | Ultrasound-treated preparation | ||
| 4501 | 1 | 1:20 | 1:320 |
| 2 | 1:20 | 1:1280 | |
| X-ov | 1 | 1:160 | 1:320 |
| 2 | 1:160 | 1:640 | |
| I-ev | 1 | 1:80 | 1:160 |
| 2 | 1:80 | 1:320 | |
| 4512 | 1 | 1:20 | 1:40 |
| 2 | 1:40 | 1:80 | |
| 4271 | 1 | 1:160 | 1:320 |
| 2 | 1:320 | 1:1280 |
| Table 2 | |||
| Study of blood serum samples from patients with TBE in the RTGA with diagnosticum from avid strain 4072, treated with ultrasound | |||
| Paired samples of patient blood serum | Antihemagglutinin titers in RTGA with ultrasound diagnosticum from strain 4072 | ||
| Unprocessed drug | Ultrasound-treated preparation | ||
| 4501 | 1 | 1:320 | 1:320 |
| 2 | 1:640 | 1:1280 | |
| X-ov | 1 | 1:640 | 1:640 |
| 2 | 1:640 | 1:1280 |
Example 2. Diagnosticums prepared in a similar way are used in RTGA when studying blood serum samples that have been pre-treated with anti-inhibitor treatment with kaolin at pH 7.4. The diagnostic increase in antibody titers increases 8-32 times (Table 3).
The drugs and methods developed and used in diagnostic practice make it possible to confirm the clinical diagnosis of “tick-borne encephalitis” in a timely manner and in almost every specific case without the use of expensive and scarce serological tests.
| Table 3 | ||||
| Dependence of RTGA results on the method of anti-inhibitory treatment of the patient’s blood serum and ultrasound treatment of the hemagglutinating antigen of the TBE virus (Sofin strain) | ||||
| No. of paired blood serum sample | pH of the environment during anti-inhibitory treatment of serum | Antihemagglutinin titers in reaction with diagnosticum | ||
| Unprocessed | Ultrasonicated | |||
| 4968 | 1 | 1:40 | 1:80 | |
| 2 | 9,0 | 1:40 | 1:80 | |
| 1 | 1:20 | 1:40 | ||
| 2 | 7,4 | 1:320 | 1:1280 | |
| 4569 | 1 | 0 | 1:20 | |
| 2 | 9,0 | 1:40 | 1:40 | |
| 1 | 1:10 | 1:20 | ||
| 2 | 74 | 1:40 | 1:160 |
Example 3. According to the proposed method, using ultrasonic treatment, the hemagglutinating antigen of the TBE virus from the non-avid strain 80 was processed in the specified parameters. According to the cross-sectional RTGA data, it was noted that the avidity of the non-avid strain increases to the level of the avid one (Table 4). This phenomenon (unobvious effect) was revealed for the first time in the history of studying the avidity of viral and bacterial antigens in practice and from available sources.
The proposed method with ultrasonic treatment of the drug in the specified parameters makes it possible to use almost any strain of TBE virus for diagnostic purposes without labor-intensive searches for natural avid variants of the pathogen, which is the novelty of the method.
| Table 4 | ||||
| The influence of ultrasound treatment on the activity of an antigenic preparation from avid and non-avid variants of tick-borne encephalitis virus in reaction with antibodies | ||||
| TBE virus strain | 3745 /avidny/ | 80 /unavidious/ | ||
| Antigenic drug | raw | sonicated | raw | sonicated |
| Result of RTGA with immune rabbit serum to the Sofiin strain | 1:1280 | 1:2560 | 1:320 | 1:2560 |
When ultrasound influences viruses in the specified parameters, large macromolecular complexes are torn off or released, which, with further sonification, “loose” with exposure of a larger number of active antigenic groups (unmasking or descreening of antigenic determinants). It is possible that ultrasonic treatment of virus-containing substrates can also increase the effectiveness of vaccine preparations if the immunogenic and preventive effect of the latter is determined by the activity of the same antigenic determinants as in the preparation of tick-borne encephalitis diagnosticum.
The proposed method for obtaining a diagnosticum for tick-borne encephalitis can find wide practical and scientific application in medicine and veterinary medicine, expanding the range of necessary effective diagnosticums for tick-borne encephalitis with minimal labor costs and using common equipment.
FORMULA OF THE INVENTION
1. A method for obtaining a diagnosticum of tick-borne encephalitis, including the preparation of a virus-containing suspension in a buffer solution in the cold, anti-inhibitory treatment with kaolin, centrifugation and separation of the supernatant followed by physical impact on it for activation, characterized in that the virus-containing suspension from strains of the tick-borne encephalitis virus is treated with ultrasound at frequency of 22 kHz for 1 minute, repeating the procedure three times with an interval of 2 minutes, while ultrasonic treatment of the virus-containing suspension is carried out on UZDN-1 or UZDN-A devices using a universal emitter at a suspension temperature of 25-30 ° C.
2. The method of obtaining a diagnosticum of tick-borne encephalitis according to claim 1, characterized in that the following strains of the tick-borne encephalitis virus are used: the reference strain Sofin, avid strains 4072 and 3445, non-avid strain 80.
