SE447339B - PROCEDURE FOR SEPARATION OF ERYTROCYT STRAMA - Google Patents

Description

N '-1 Kf) Blood plasma from animal species is treated with an aqueous solution of acrinol, containing from about 0.1% to about 5% of 2-ethoxy-6,9-æuidindümnn-lædntenmr nedan below referred to acrinol, for the precipitation of blood proteins other than gamma globulin. These proteins include albumin, alpha- and beta-globulin, etc. The plasma is at its normal pH of about 7-8, but it can be adjusted to about 7.6 by the addition of a suitable reagent, such as 1N sodium hydroxide, 0.8M sodium bicarbonate or 1M sodium carbonate or 1N HCl. or acetic acid. The precipitation usually takes place at around 25 ° C, although higher and lower temperatures can be used. From 3.5 to 5 volumes, preferably about 4 volumes of 4% aqueous acrinol solution are used for each volume of plasma.

The precipitate is allowed to be completed by sedimentation and flocculation after the liquid has been allowed to stand, preferably at a temperature of about 4 DEG C. from about 15 minutes until overnight and longer.

The precipitate is separated from the supernatant containing gamma globulin, preferably by filtration, decantation or centrifugation. The precipitate containing acrinol-insoluble blood plasma protein is washed with a 0.1-5% aqueous solution of acrinol. The washed precipitate is then transferred to a solution of gamma globulin activated against antigen from a heterologous source and stirred for a few minutes to several hours at about 4 ° C to about 40 ° C, preferably about 25 ° C, so that the antiplasmic protein the antibodies in the gamma-globulin solution are absorbed on the solid plasma protein. A sufficient amount of gamma-globulin separating agent is kept in suspension so that a multiphase absorption state is present. The solid plasma protein with absorbed antiplasmic protein antibodies is separated, e.g. by decantation, filtration or centrifugation. The absorption process can be repeated as many times as necessary to reduce the amount of contaminated antibodies to the desired level. The amount of solid plasma protein obtained from 0.01 to 5 or more liters of plasma was used to absorb the antiplasmic protein antibodies from heterologously reacted antihuman lymphocyte gamma globulin obtained from one liter of plasma.

This method of absorption has the additional advantage when acrinol is used as a gamma-globulin separating agent, that hepatitis virus appears to precipitate together with plasma protein, whereby the risk of hepatitis virus contamination of the final product is reduced.

The production of stromat for contact with the gamma globulin takes place in a manner known per se, with the exception of a new improvement of one of the isolation steps, which is described below. After dissolution of the erythrocytes and before the gamma globulin is brought into contact with the stroma, the menstrual cavity must be separated from the stroma as efficiently as possible.

The addition of small amounts of acrinol to the dissolved erythrocytes facilitates the separation of the stroma from the menstrual cycle and Centrifugations at relatively low speeds of 2000 rpm for 10-15 minutes are sufficient for separation compared to one or more washes and centrifugations at 3000 rpm. per minute for more than 30 minutes, previously used. Satisfactory separation has in reality often been obtained by the present improvement only by allowing the material to separate overnight in the cold, for example followed by decantation. Stromat is satisfactorily free of hemoglobin when the characteristic color of the suspended fluid has completely disappeared or been determined spectrophotometrically at 555 nanometers. The amount of acrinol used varies from about 1 to about 5 g / l of collected erythrocytes, preferably about 3-5 g / l.

The preparatory steps in the production of stroma are known per se. To the erythrocyte precipitate is added an equal volume of 0.9% NaCl solution. The suspension is then mixed and centrifuged, followed by removal of the yellow film. The washed erythrocytes are then dissolved by a standardized lysis procedure, for example digitonin, water, freezing and thawing. Stromat is separated from the suspension by means of acrinol in the manner previously shown. It can further be pointed out that the choice of an acrinol solution of suitable tonicity enables the erythrocyte stroma to be dissolved and separated with a single reagent, thus avoiding a separate lysis step.

Stromat is then added to the solution of gamma globulin, which has been reacted with heterologous antigen so that absorption of the anti-erythrocyte antibodies by stroma can be obtained. The absorption is performed as many times as necessary to absorb the anti-erythrocyte antibodies. The amount of stroma obtained from 0.01 to 5 or more liters of collected erythrocytes was used to absorb the anti-erythrocyte antibodies from heterologously reacted gamma globulin, obtained from 1 liter of plasma. Multiphase absorption is maintained with an appropriate amount of gamma-globulin separating agents.

Stroma absorption can be performed before, during or after plasma protein absorption.

The following examples illustrate the process of the present invention. The examples are not intended to be limiting but serve only as an example of the method according to the invention.

Example 1 Preparation of anti-human lymphocyte-gamma-globulin Human lymphocytes were prepared in a suitable known manner and a horse was injected with these lymphocytes. When the desired antibody titer was reached, the horse was drained and the serum was separated from the whole blood and the pH of the serum was adjusted to about 7.6 by the addition of 0.8M sodium bicarbonate. An aqueous solution containing 0.4% acrinol was slowly added to the serum with stirring to precipitate blood proteins other than gamma globulin. An excess of acrinol solution was used to ensure complete precipitation. The precipitate was allowed to settle overnight at a temperature of about 4 ° C. The supernatant containing gamma globulin was decanted from the precipitate.

Absorption of Human Anti-Plasma Protein Antibodies by Health Serum in a Properly Known Way To an equine anti-human lymphocyte gamma globulin solution prepared according to Example 1, an equal volume of human whole was added. serum. This health space inactivated the antibodies to human blood protein in the equine anti-lymphocyte-gamma globulin. However, the human gamma globulin is mixed with the equine anti-human lymphocyte gamma globulin and these two can only be separated with great difficulty, if they can be separated at all. ëæm. P61 ä Absorption of human plasma antiplasmic protein antibodies A sufficient amount of an aqueous solution containing about 0.4% 447 339 acrinol was slowly added to human plasma, the pH of which was adjusted to about 7%. 6 with 0.8M sodium bicarbonate, while stirring to obtain a precipitate of acrinol-insoluble proteins. The resulting precipitate was separated from the supernatant by centrifugation. Solid plasma protein obtained from 0.2 l of human plasma was added to a solution of? ALG, obtained from 1 liter of plasma prepared as in Example 1. The resulting suspension was stirred continuously for 3 hours at room temperature and cooled and stored at 4 ° C over at night. The supernatant containing ALG, purified from contaminating human anti-plasma protein antibodies, was then separated by centrifugation at 2000 rpm for 10 seconds. The ALG thus obtained was essentially uncontaminated with harmful human blood plasma proteins or soluble antigen-antibody complexes.

Eeßsiesli Absorption of human anti-erythrocyte antibodies in a manner known per se To a volume of collected human erythrocytes was added an equal volume of 0.92 M aqueous NaCl solution. This solution was mixed and centrifuged at 3000 rpm for 30 minutes at ZÜC and the supernatant and yellow membrane were discarded. To each liter of collected erythrocytes was added 10 volumes of distilled water and the cells were dissolved after standing for 30 minutes. Stromat was washed and centrifuged 10 times at 3000 rpm to reduce the hemoglobin to a satisfactory level. A certain amount of stroma was lost during the washing process. Stroma from 0.1 volume of collected erythrocytes was added to ALG obtained from 1 liter of plasma prepared according to Example 1 and stirred for 3 hours at 2 ° C. Stromat was separated from the ALG solution by centrifugation and the supernatant was treated with additional stroma twice. anti-erythrocytitis to an acceptable level. êäeiuß fl l- 5 Absorption of human anti-erythrocyte antibodies from ALG with current prepared by the addition of acrinol The procedure of Example 4 was repeated to the point where the erythrocytes were dissolved with distilled water. At this point it was diluted

Claims (2)

1. 447 339 the dissolved erythrocyte solution with 2% volume of water based on the original erythrocyte volume. 5 grams of acrinol for each liter of erythrocyte originally collected was added. This suspension was stirred for 1 hour at room temperature and placed in a refrigerator overnight. The suspension was centrifuged at 2000 rpm for 10 minutes and the ALG solution was separated from the stromat. Two additional stroma absorbances were performed as above to reduce the anti-erythrocyte titer to a desired level. Process for the separation of erythrocyte stroma from lysis medium and hemoglobin, characterized in that a suspension of erythrocytes which have undergone a lysis procedure is added with 2. -ethoxy-6,9-acridinediamine lactate monohydrate and the subsequent erythrocyte stromat from the suspension.