JPH02184339A - Adsorbent for heparin - Google Patents

Abstract

PURPOSE:To obtain an adsorbent having a great affinity for heparin and used to purify, remove, capture or quantify heparin by reducing fibronectin and alkylating the reduced fibronectin or further supporting the resulting fibronectin deriv. on an insoluble support. CONSTITUTION:Fibronectin is reduced with a reducing agent such as mercapto compd. in a flow of an inert gas and the reduced fibronectin is alkylated with an alkylating agent such as alkyl halide to obtain an adsorbent for heparin. The resulting fibronectin deriv. may be fixed by bonding to a proper insoluble support such as cellulose resin or polyacrylamide resin to obtain a composite body for adsorbing heparin. By using this composite body, heparin especially in blood is removed or quantified. The fibronectin deriv. has an extremely great affinity for heparin.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to an adsorbent used for purification, removal, collection and quantitative determination of heparin.

[Conventional technology]

Heparin is a mucopolysaccharide with an average molecular weight of 5.000 to 30.000, and is known to have anticoagulant effects and lipid serum clarifying effects, and has been used particularly as an anticoagulant in drug therapy. That is, when heparin is administered, heparin quickly binds to antithrombin in the blood, and as a result, exerts an antithrombin effect, stopping the conversion of fibrinogen to fibrin and preventing blood from coagulating. There is. As described above, since heparin is a substance that plays an important role in the blood, measuring and quantifying heparin is of extremely great significance. Recently, FDP has been used as an in vitro clinical diagnosis.
As the need to measure the blood concentration of fibrin (fibrin, a product of fibrinogen degradation) has been recognized, it has been measured using an immunological method; FD is caused by fibrinogen contamination in the sample, which separates into serum in the sample.
It often happens that P is higher than the true value (false positive), and to prevent this, it is strongly desired to remove heparin from the sample.

[Problem to be solved by the invention]

However, since the removal of heparin is insufficient with conventional methods, the present inventors searched for a substance that adsorbs heparin quickly and easily, and as a result of intensive research, they found a substance obtained by reducing and alkylating fibronectin. has a strong affinity for heparin, and it was discovered that the above problems could be solved, leading to the completion of the present invention.

[Failure to solve the problem]

The present invention therefore relates to adsorbents for heparin consisting of reduced and alkylated derivatives of fibronectin.

Furthermore, the present invention also relates to a heparin adsorption complex characterized by comprising a reduced and alkylated derivative of fibronectin and an insoluble support that immobilizes and supports the derivative.

The heparin adsorbent of the present invention can be prepared by reducing and alkylating fibronectin.

Fibronectin is produced by mesenchymal cells such as fibrotic cells and smooth muscle cells, and has a molecular weight of 40o, 000-500.0.
There are two types of 00 glycoproteins: cellular fibronectin, which is distributed on the cell membrane surface and becomes Sr1 of intercellular striations, and plasma fibronectin, which migrates into the blood.The fibronectin used in the present invention is particularly Cellular fibronectin or plasma fibronectin can be used without requiring a specific origin.

Although fibronectin itself has some affinity for heparin, it does not bind completely, and when the ionic strength is increased, it easily separates and is not necessarily suitable as an adsorbent for heparin. However, reducing and alkylating fibronectin significantly increases its affinity for heparin. Fibronectin is Waxda
The method of I et al. [Biochemistry, 7.195
9-1966 (1968)], it can be purified from tissue or plasma by Cohn's fractionation, ammonium sulfate fractionation, DEAE-Sephadex A-50, and gelatin-agarose column. Also, JP-A-58-1212
It is also possible to use the method described in Publication No. 20.

Reduction of purified fibronectin is carried out by treatment with a reducing agent, in particular a mercapto compound capable of reductively cleaving disulfide bonds, such as dithiothreitol, under a stream of inert gas, such as nitrogen. The processing temperature is 3
The temperature is 0 to 60°C, and the treatment time is 20 to 60 minutes.

Before reducing the fibronectin, it is preferred to pre-treat it with chaotropic ions such as guanidine.

The reduced fibronectin is then treated with an alkylating agent, particularly an al-1-halide. The alkyl portion of the halogenated alkyl may be substituted with a functional group such as an amide group. A preferred alkylating agent is iodoacetamide. Alkylation is generally 25-60℃
It is carried out for 1 to 6 hours.

The reduced and alkylated fibronectin derivative thus obtained can be purified by desalting using Sephacryl S-300.

The affinity of the reduced and alkylated derivative of fibronetatin of the present invention for heparin is irreversible, and once it binds to heparin, it does not dissociate with ordinary salts or 1111, and has 1.0.5 mol of NaCl and 8 mol of urea. Dissociation can only be achieved under extremely extreme conditions of coexistence. As described above, the fibronectin derivative of the present invention has an extremely strong affinity for heparin and is therefore suitable for heparin collection.

In addition, a heparin adsorption complex can be prepared by further bonding and immobilizing the above-mentioned fibronectin derivative to a suitable insoluble support (eg, agarose resin, cellulose resin, polyacrylamide resin). This operation is a general one, and can be carried out by referring to, for example, "Immobilized Enzymes" (Kodansha Scientific) and "Experimental and Applied Affinity Chromatography" (i11! Dansha Scientific).

By using the heparin adsorption complex of the present invention, heparin in body fluids, particularly thin fluids, can be removed from solutions and quantified.

〔Example〕

EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited thereto.

Example 1 Production of reduced/al;)-lated fibronectin (1) Both fractions of fibronectin obtained by ethanol fractionation from pooled normal adult plasma.
■ Add 10 times the volume of 55 mmol citrate buffer (pH 6).
, 4) was added and dissolved.

Next, it was rapidly heated to 50°C and kept at that temperature for 30 minutes. The resulting precipitate was removed by centrifugation gjlf to obtain a crude plasma fibronectin. This coarse side 100m
g was applied to a 2 x 80 cm Sephas 4B (manufactured by Almasia, code No. 1.7-0120-01.) column, and the absorption at 2801 m was measured using a Boehringer Fibronecdin measurement kit (Product No. 40121.).
8) Molecular weight cutoff 300.000-500.000
fractions were collected. Next, divide the collected amount into 2.
5 x 5 cm gelatin-agarose (Nukuma G
-5384) Both fractions eluted with 4 mol of urea were collected and dialyzed against 50 mmol of phosphoric acid solution (p++ 7.0) to remove urea. To the solution, ammonium sulfate was added to a maximum concentration of 20% while cooling to 4°C, stirring was continued for 60 minutes, centrifuged at 10,000 G for 5 minutes, and a supernatant was collected. Ammonium sulfate was added to this solution. was added at 4°C to a final concentration of 40%, stirred for 60 minutes, centrifuged at 1.0000G for 5 minutes, collected a white pellet-like precipitate, and dissolved in the minimum amount of the above phosphate buffer. Ammonia sulfate was removed by fermentation overnight in a 0.5 mol Tris-HCl buffer (pH 8.1) containing 2 mmol of EDT, and 60 mg of purified fibronectin was obtained.

(2) Reduction and alurination of fibronectin The above purified fibronectin solution was prepared to a concentration of 10 mg/me, 6M guanidine was added, and the pH was adjusted to 8.1. 5 g of this solution was allowed to stand at 50° C. for 30 minutes under a nitrogen stream, then 54 mg of dithiothreitol was added, and the mixture was further reacted at 50° C. for 4 hours under a nitrogen stream. Further, 130 mg of iodoacetamide was added under a nitrogen stream, and the reaction was carried out in the dark for 20 minutes. The resulting product was 3.5 x 80 c
m of Sephacryl S-300 Carano, 0. ]5
l) Desalt using hydrochloric acid buffer (pH 7,4) and reduce the elution amount from 230 to 2.
Fractions of 70 mff were collected to obtain 5.5 mg of reduced/alkylated fibronectin.

The recovery rate from killed fibronectin was still less than 1%. Finally, 50 mIJ mole) IJ suth-acid buffer (p
+17.4), the recovery rate from fibronectin was 101%, and the recovery rate from reduced/alkylated fibronectin was 99%.

Table-1 1.5X 5 cm Heparin-Sepharose CL6B
(Manufactured by FARMA, CO-1' No. 17-0690
-02) 10 mg each of fibronectin and reduced/alkylated fibronectin were added to Carat, and eluted with 50 mmol Tris-HCl buffer (pH 7,4). As shown in Table 1, the recovery rate of fibronectin was 14%, while the recovery rate of reduced and alkylated fibronectin was 1% or less. Same (0.
In 50 mmol Tris-HCl buffer (pH 7.4) containing 5 mol NaCβ, the recovery rate from fibronectin was 98%, reduced Al/Q) Example 3 Sepharose/I B-Fibronectin CN
2 ml of Br-activated Sepharose 4B (manufactured by Pharmacia, Coat No. 17-0430-Ol, 1)
After washing with mmol hydrochloric acid, 0 containing 0.5 molar NaCR
．． 1 molar sodium bicarbonate buffer (p++ 8.3
> I put it in. 10 mg of reduced/alkylated fibronectin, which had been dialyzed in advance with the above buffer, was added and reacted at room temperature for 3 hours.
．． 5% l) IJ sous-acid buffer (pit 8.2
) and then the reaction gel was washed and filtered through a glass filter to obtain Sepharose 4I3-reduced/alkylated fibronecdin complex 19. This Sepharose 4F3-reduced and alkylated fibronectin complex Kel (0.1 mβ) was mixed with 50 mmol of IJ-hydrochloric acid buffer (containing 20 units of heparin (Novoindas) manufactured by IJ-Inc.) and 0.5 mol of NaCβ. p++ 7.
4) After mixing with 1,9me and gently inverting for 60 seconds, the mixture was centrifuged (5000 rpm) to obtain a supernatant.

When the heparin activity in this -1-clear liquid was measured using Test Team Heparin manufactured by Daiichi Chemical Co., Ltd., heparin was found to be 0.2
It was below the unit. Therefore, more than 99% of the above returns and
It was found that it was adsorbed to alkylated fibronectin.

Claims (1)

[Scope of Claims] 1. An adsorbent for heparin comprising reduced fibronectin and its alkylated derivative. 2. A complex for adsorbing heparin, comprising a reduced fibronectin and alkylated derivative thereof, and an insoluble support that immobilizes and supports the derivative.