CS237620B3 - Use of blue affinity cellulose sorbent - Google Patents

Abstract

The invention relates to a method of using a blue affinity cellulose sorbent for the separation and isolation of serum albumin from serum. The essence of the invention lies in the use of a blue affinity cellulose sorbent prepared according to Czechoslovak author's certificate No. 225 971 for the separation and isolation of serum albumin from serum by affinity chromatography. The use of a blue cellulose carrier based on macroporous spherical cellulose or their hydroxyalkyl derivatives with covalently bound reactive blue 2 (color index 61211) leads to almost 90% separation and 75 to 90% isolation of albumin contained in serum.

Description

The invention relates to the use of a blue affinity cellulose sorbent for the separation and isolation of serum albumin from serum.

Separation and isolation of albumin from serum by affinity chromatography using a blue affinity sorbent is used for both analytical and preparative purposes in chemistry, biochemistry and aedicine.

The serum contains a mixture of nearly 100 proteins of different structure and function, with albumin being the dominant component with about 50% by weight.

Several methods have been developed for the isolation of albumin, which are based on selective precipitation or chromatographic separations, and often take place in many reaction steps. Today it is not only important to get albumin on a preparative scale. Just as important is the simple, specific and almost quantitative removal of albumin from serum, primarily for medical diagnostic purposes or in obtaining special serum proteins, for example n-fetoprotein.

For both fields of application, affinity chromatography techniques have been developed in recent years which utilize albumin adsorption by a blue dye referred to as reactive blue 2 (color index 61 211) (trade names Cibacron blue F3G-A; Ostazine blue H-3G) bound to a polysaccharide porters. '*

Attempts to replace these carriers with cellulose, which is more economically advantageous than other polysaccharide carriers, have so far been unsuccessful. Compared to agarose carriers, albumin binding to the corresponding cellulose sorbent 10x was less (S. Angal, P.D.G. Dean: Biochem. J.167, 301, (1977) or no binding was found (G. Birkenmeyer, Dizertace, Leipzig 1981).

The existing drawbacks are overcome by a method for separating and isolating serum albumin from the serum according to the invention, which consists in using a blue cellulose sorbent prepared according to U.S. Pat. No. 225,971 for separation and isolation of serum albumin from serum by affinity chromatography.

The binding of albumin to the reactive dye bound to the carrier upon contact with serum is specific, with a binding capacity greater than 10 mg albumin per ml of carrier. The loop then proceeds in a manner known per se.

Adsorption from serum or diluted serum is carried out at pH 5-9, preferably pH 18, at a temperature of from 1 to 40 ° C by column or batch method. Serum proteins not adsorbed are eluted with adsorption buffer.

The albumin desorption is performed with buffers containing 1 to 2 M sodium chloride (KaCl) or 0.2 to 1 M potassium thiocyanate (KSCN).

An advantage of the process according to the invention is that the carrier can be used repeatedly without observable deterioration of the binding capacity. . .

’« Γ *

The use of a carrier based on macroporous spherical cellulose or their hydrophilic derivatives with covalently bound reactive blue 2 (color index 612 11) results in nearly 90% separation and 75 to 90% isolation of serum albumin.

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Compared to other polysaccharide carriers, the blue cellulose carrier based on macroporous spherical cellulose is more efficient and economically advantageous.

Excellent! the mechanical and adsorption characteristics of the carrier will release its carrier on a large scale.

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The process is illustrated in several cases, but the invention is not limited thereto.

Example 1 ml of macroporous spherical blue cellulose (2.9 to 7.3 pmol reactive blue 2 - color index 61 211 (Cibacron blue / g wet carrier) was equilibrated in a column with a diameter of 1.3 µm with 0.05 U Iris buffer (HCl). 0.5 M NaCl pH 8, at room temperature and then loaded with 2 ml of human serum albumin (40 mg / ml buffer), eluting with 25 to 30 ml of buffer, capturing 88 to 94% of albumin. 0.05 M Iris (HCl) pli 3 buffer containing 0.2 to 1 U KSCN (75 to 90% yield).

Example 2 ml of macroporous spherical blue cellulose (2.9 to 7.3 pmol dye / g wet carrier) was equilibrated with 0.05 M Iris (HCl) 0.5 M NaCl pH 8 buffer and applied with 2 ml. sera either directly or diluted with equilibration buffer (1: 5 to 1: 10). The adsorption of albumin is carried out in a column or batch configuration (1 to 5 hours) at room temperature. Unadsorbed serum proteins (30-45% of the total protein content) are eluted with equilibration buffer. 93-96% of the albumin is captured. The elution of adsorbed albumin proceeds as described in Example 1.

Claims (1)

The use of a blue afinantnihoicellulose sorbent prepared according to U.S. Pat. No. 225,971 for the separation and isolation of serum albumin from serum by affinity chromatography.