SU649435A1 - Method of preparing erythrocyte - Google Patents

Description

one

FIELD OF THE INVENTION The invention relates to the field of medical criminality, namely to the introduction of viral preparations.

A method of preparing erythrocytes for the adsorption of the influenza virus by washing the chicken erythrophytes with physiological saline followed by formalization, routine laundering and prepared erythrocyte susiese is known.

However, the known snozob does not provide a high adsorption-eluting erythrocyte capacity.

The aim of the invention is to increase the adsorption-eluting ability of red blood cells.

This goal is achieved by the fact that formally erythrocytes are washed twice with a borate-phosphate buffer solution of sodium chloride at pH 9.0-9.2 and once with a phosphate buffer solution of sodium chloride at pH 5.5-5.7 and the suspension of erythrocytes is prepared in the same way. solution.

The method is described as follows.

Fresh washed chicken erythrocytes, diluted 1: 1 with saline, are incubated with a 20% volume of formalin solution for 3 days in a conventional refrigerator. Then they are applied to washing them in a laboratory using a CLR-refrigeration centrifuge in 250 ml glass centrifuge cups. Centrifugation at all stages is carried out with

speed of 1000 rpm no more than 4-5 minutes. These conditions are chosen in order to prevent the formalinized erythrocytes from destroying cells and other impurities from falling into the sediment.

long and high-speed centrifugation and capable of contaminating vaccinated material at the stage of the eluted process.

The sequence of washing the forma l1 erythrocytes is as follows.

After removal of formalin, the erprocytes are washed twice with physiological saline pp 7.0. Then they are centrifuged twice in n; fir-tree borate-phosphate

sodium chloride solution pH 9.0. In this case, in the washing liquid, the method of protein distribution according to Lowry reveals from 0.8 to 1.1 µg of it in 1 ml, whereas after washing the erythrocytes with a usual physiological solution of pH 7.0, protein impurities are not discharged.

Next, the precipitate of formalinized erythrocytes is washed in a CLR-1 centrifuge once with phosphate buffer solution

sodium chloride RP 5.6 and resuspendyd them in equal quantities of the same solution to p. 50 50% by volume of suspension. To obtain 1 and purified and conceptual vaccinated material, for every 100 ml of allangoic fluid, 6 ml of 507 ° C erythrovdts are added and left to adsorb the virus in the fridge for overnight. The next day, the precipitate of formalinized erythrocytes loaded with virus was centrifuged at CLR-1 at 1000 rpm for 5 minutes, then once and spayed with physiological solution pH 7.0 cooled to 4-6 ° C and put into a water bath for elute virus At 38 ° C in physiological solution, the volume of which is 10 times less than the volume of the initial allantoic fluid (tenfold eluate). After 4 hours, the suspension of erythrocytes in a water bath is centrifuged at CLR-1 or 2000 rpm for 15–20 minutes, the supernatant (1 eluate) is drained and the elution cycle is repeated for 3 hours in the same volume of saline. The degree of elution is determined by the haemagglutination reaction (DSA) with a 1% suspension of overweight chicken erntrocytes on the leech eye glands. The volume is reactive; each well is 1 ml. For comparison, the adsorption process and two elution cycles of the same virus are carried out with formalized erythrocytes prepared using a well-known method (without washing them with alkaline and acidic buffer solutions). As shown by the results of the experiments, when using purified formalin erntrocytes prepared according to the proposed method, the desorption process of the virus develops faster. So, already after 2 hours, virus A (Victory) 75 was eluted in a water bath 75 from 81% to 81% in physiological saline as compared with the hemagglutinating activity in the allantoic fluid, taking into account small losses after adsorption of the virus erythrocytes. When using formalized erythrocytes, irifted in a well-known way, eluted from 22 to 30% during this period. After 4 hours (1 elution cycle), in the nerve case, 83 to 85% were released, and the second, from 40 to 43%. In the end, in two elution cycles (7 hours of incubation in water; 1st bath), purified formalin-nizirovanny erythrocytes released about 7–89% of the adsorption of the Rivine virus, and erythrocytes prepared using the well-known method — 43-46% compared to the heme: - 1G1 luginii of alaggoisii liquid, calculated on the basis of the volume of the material, 1a. In order to remove the remaining virus in the second case, the third and fourth ethanol was required to elute the virus, i.e., additional curing in the water bath for 6-7 hours, which lengthened the process of preparation of the purified virus-containing material, leading to loss of hemagglutination and its activity on stages. Using the proposed method provides zkorenie. In order to reduce the technological process of manufacturing the inactivated flu vaccine, as well as reducing the hemagglutinating activity of the viral erythrocytes, as well as reducing the hemagglutinating activity during the purification of the virus by the method of adsorbing erythrocytes to the hemgglutinating activity of the virus in the eryos pro;: zvitetve vaccine gitammy with low reproduction capacity and low yield of hemaglutus - nins. The form for preparing the erythrocytes for the adsorption of the influenza virus by washing the erythrocytes of chickens with physiological saline followed by formalization, rewashing and preparing erythrocyte suspensions, is also invented by us, and it was created by using a cell that was used for the synthesis of the erythrocytes and for the purpose of gaining the use of erythrocytes. Formalized red blood cells are washed twice with sodium phosphate borate buffer solution at pH 2 and once with sodium chloride chloride phosphate buffer solution at pH 5.5-5.7 and erythro suspension Oocytes are prepared 15 of the same solution. Sources of information taken into account in the examination 1. Reglameit anti-influenza serum production with sulfonamides. LenNIIVS them. Mechnikov, 1966.