UA52744U - method for antigen preparation for diagnostics of cattle leucosis in immune diffusion reaction - Google Patents

Abstract

A method for antigen preparation for diagnostics of cattle leucosis in immune diffusion reaction, comprising the cell cultivation of growth medium mix, containing cattle serum, clarification of cultural liquid, and antigen deposition. The cattle blood serum free of γ-globulines, cultural liquid is concentrated by ultrafiltration on columns with hollow fibers. The concentrated cultural liquid is clarified by centrifugation.

Description

A comparative analysis with the closest analogue Example 1. allows us to conclude that the use of y-B5bdm3 virus-culture fluid, initially globulin serum, allows to obtain highly concentrated by the method of ultrafiltration, and only coactive and specific antigen, ultrafiltra- then cleaned of detritus by centrifugation on columns with soft fibers allows to eliminate 5000 rpm. In the cell detritus, which was obtained to reduce the loss of antigen, clarification of the concentrated culture liquid after low-speed centrifugation by centrifugation allows a moderately concentrated virus-culture liquid to avoid a large loss of viruses, control of contamination, specific proteins of bovine VL were not detected in the RID. nation by foreign viruses with the help of PCR to- During the production of VL antigen for concentration, it allows to detect contaminants of viral etiology, virus-culture fluid according to current technology that do not cause cytopathogenic effect in cultures, ultrafiltration on empty cells was used, allows to increase the specific- fibers with a retention threshold of 100 kD many times over . Considering the quality of the antigen. on the fact that the diagnostic value is represented

The method is performed as follows. surface proteins of VL dr 51, dr 30 and internal -

Transplantation of the RI K-VI M cell culture is carried out by p2g4 with a molecular weight of 51 to 24 kD, for once every 7-10 days with a seed concentration of 50 - the concentration of the virus-culture liquid was 10Otys. cells/cm3. Ultrafiltration modules with empty media 199, Igla and serum with 15 kD and 50 kD fibers were tested as a growth medium (experiment). It was established

Cattle purified from immunoglobulins. The nature of the growth was improved, that when obtaining the VL antigen according to the standard and the formation of a monolayer of cells and their quality by an excellent technology, its quantity was determined by viewing the culture under light (335.0 x 7.6 cm) with B0dm3 virus-culture liquid- microscope with activity in KIN 1:1.2 - 1:1.8, at that time until

Control of the sterility of the cell culture RI K- antigen was obtained (450.02417) cm from

VI M and bovine leukemia virus antigen are carried out at an activity of 1:2.0 - 1:3.0 (Table 1). The table for pre-thioglycol medium (TS) and control contains comparative data and evaluations of treatment methods according to using PCR. detection of bovine leukemia virus antigen from culture

The glycoprotein antigen of VL of cattle is obtained from the RIK-VIGM fluid, which was obtained for the virus - culture fluid after its 2-3 times in the medium with native serum. freezing-thawing. Virus-culture Thus, the use of fibers with a smaller liquid is initially concentrated 50 - 100 times at the retention threshold allowed to increase by 3495 ultrafiltration unit with hollow output of antigen VL. fibers with a retention threshold of 10-200kD. Then Example 2. the concentrate is clarified by centrifugation at 5Odm3 virus-culture fluid accumulates - 3000 rpm. during 30 minutes After that, supernals during the cultivation of R-K-VIM cells on the medium are collected, 5095 PEG-higher solution is added to it with 1095 aglobulin bovine blood serum. 6000 (based on the calculation of 200 cm3 of solution per dm3 concentration) and kept for 12-24 hours at the temperature of the antigen production technology, its quantity is 42 b. After settling, the antigen precipitated consisted of (90,055,7) cm? with 5Odm? virus - by centrifugation at 5000 rpm. for 30 - culture liquid with activity in 1:3.0 - 40 minutes. The supernatant is drained and disposed of, and the sediment is 1:4.0, while using the new technology, the antigen VL is collected and resuspended in an equal (experiment) received antigen in the amount (155.0418.9) volume of buffered saline. Anti-cm3 with an activity of 1:4.0 - 1:5.0, which is 7295 more than the gene, is subjected to ultrasound disintegration at 400 W, 22 kHz (Table 2). The table shows comparative data of 100 A for 10 minutes. of methods of obtaining bovine leukemia virus antigen

Cellular detritus, which is obtained during the formation of the RIK-VIM culture fluid, which was obtained from the virus-culture fluid, is collected, dissolved in a medium with aglobulin serum. With a physiological solution in a ratio of 1:1, with the help of this method, the technology has been improved.

UZ-disintegration at 400W, 22kHz, 100A during 1.9 - 1.7 times to increase its output from a unit of volume-minutes. The disintegrated detritus is illuminated by the virus-culture liquid, which increases the effect by centrifugation at 3000 rpm. during ZOkhv. tivity of production.

The supernatant is precipitated with a 50956-th solution of PEG-6000. Thus, the method of obtaining antigen for (based on 200 cm3 of the solution per edum of the concentrate), the diagnosis of bovine leukemia in the reactor is maintained for 12-24 hours at a temperature of 42C. Immunodiffusion (ID) is an economical and effective way to defend the antigen is precipitated by a centrifuge, it allows you to increase the yield of the antigen by inhaling at 5000 rpm. for 30-40 minutes. on 3495 - 72. It is suitable for use in

The supernatant is drained and disposed of, and the sediment is also collected on an industrial scale and allows to obtain an active, specific and stabilizing solution biresuspended in an equal volume of buffered filler. of the VLVRH antigen preparation.

Table 1

The method of obtaining an antigen for the diagnosis of bovine leukemia in the immunodiffusion reaction (ID)

Amount of liquid per Received Activity | Quantity Activity

Method of production of VL antigen: from VL antigen, detritus to concentration, dm cM3 AG in RID | detritus, cm3 RID

According to TU 335.07, |1:1.2- 17.81 240.02523.1

Concentration of the virus-containing liquid, centrifuged at ep. 4. 5000 rpm, deposition of antigen VL 5O 450.0417.3 |1:2.0-1:8.0) 90.0415.2

PEG - 6000

Table 2

The method of obtaining an antigen for the diagnosis of bovine leukemia in the immunodiffusion reaction (ID)

Amount of liquid per Received Activity | Quantity Activity

The method of production of the VL antigen is derived from the VL antigen, with! detritus in concentration, dm cM3 AG in KIN | detritus, cm KIN

According to TU 90.0x25.7 11:3.0 - 1:4.0| 240.02523.1

Concentration of virus-containing liquid, centrifugation at dp. 5000 rpm, deposition of antigen VL 5O 155.0218.9, 114.0 1:50) 90.0х415.2

PEG-6000

Computer layout L. Kupenko Signature Circulation 26 approx.

Ministry of Education and Science of Ukraine

State Department of Intellectual Property, str. Urytskogo, 45, Kyiv, MSP, 03680, Ukraine

SE "Ukrainian Institute of Industrial Property", str. Glazunova, 1, Kyiv - 42, 01601