CS239044B1 - Isolation and purification of apolipoprotein A-I and apolipoprotein B - Google Patents

Abstract

The purpose of the invention is to improve the process of isolation and purification of plasma proteins, apolipoprotein A-I and apolipoprotein B. The stated purpose is achieved by the proposed technology, which uses physicochemical and immunochemical methods in the following order: ultracentrifugation flotation, chromatography. This process allows simultaneous isolation of both apolipoproteins as antigens in high purity, with minimal requirements for ultracentrifugation flotation.

Description

The invention relates to the isolation and purification of apolipoprotein A-i (Apo A-I) and apolipoprotein Β (Apo B) as antigens.

The starting material for the preparation of both apolipoproteins is blood eor or plasma of normolipidemic individuals.

Mixed plasma from multiple donors may also be used, but must meet the above condition. Plasma obtained by plasmaferesis may also be used.

A number of methods are available in the literature for processing the starting material (Burstein, M. et al., Lipid. Res., 11, 1970, pp. 583-592, Havel, R.3. Et al., 3). Clin. Invest., 34, 1955, pp. 1345-1353, Weisgraber, KH et al., Et al., Res., 21, 1980, pp. 316-325, Cohn, E.3 et al. (Am. Chem. Soc., 68, 1946, e. 459-463).

The first stage of processing consists of isolating the individual fractions of blood lipoproteins. For this purpose, almost exclusively two procedures are used. The first is based on selective precipitation of lipoproteins by polyanions (Burstein,

M. et al., Lipid. Res., 11, 1970, pp. 583-592). Burstein can be isolated from fresh plasma by precipitation with sodium tungstate solution (40 g / l) and manganese chloride at 2.0 mol / l a mixture of lipoproteins and very low density (VLDL and LDL) and high density lipoproteins (HOL) ). The second is the gradual ultracentrifugation flotation of Havel et al. (Havel, R.3. Et al., Clin Invest., 34, 1955, pp. 1345-1353). Help flotation with gradually increasing! specific weights are

- 2,239,044 of starting material, blood plasma or serum. Isolated

VLDL, LDL, and HOL. Exceptionally, another method has been used to prepare some of the above mentioned protein fractions.

The second phase is focused on the isolation of individual apolipoproteins from the delipldated fractions of lipoproteins - their protein components. Gel filtration on dextran gels (Sephadex) or electrolytic focusing is most commonly used for this purpose (Alaupovlc, P. et al., Biochim, Blophye Acta, 260, 1971, 8. 689-707, Curry, MD et al., Clin. Chem., 22, 1976, e. 315-322, Curry * MD et al.,

Clin, Chem., 24, 1978, e 280-286, Forhez, P. et al. Biochem. Biophye., 6, 1982, e. 283-296).

In the isolation of individual fractions of pleemetic or serum lipoproteins, ultracentrifugation flotation is preferred over precipitation methods. It can be used to prepare fractions of lipoproteins whose physico-chemical properties are atherently less than the properties of lipoproteins divided by precipitation. Ultracentrifuge flotation is, however, a method highly demanding in terms of quality technical equipment.

Experience has shown that the most suitable procedure for Isolation and Purification of Apolipoproteins is a combination of ultracentrifugation flotation, delipidation and chromatography. This concept has been used to elaborate the proposed production process for the isolation of Apo A-I and Apo B, but so as to minimize technical equipment requirements while maintaining the required purity of both apolipoproteins as antigens.

The present invention is based on a reproducible manufacturing design

239 044 technology Isolation of apolipoproteins A-I and B and with particular regard to achieving the required purity of the end product using as simple a process as possible ·

The preparation of the plasma as starting material consists in adding the sodium salt of ethylenediaminetetraacetic acid and filtering through an aebeatoceluloa layer (Seitz).

2. For the first ultracentrifugation, the specific gravity of the plasma is adjusted to 1.030-1.050 kg / l with sodium bromide. The ultracentrifugation flotation is run at 750 Hz and a temperature of 5-10 ° C for 18-22 hours. After completion, the supernatant is collected. interlayer and infranatant. The intermediate layer is removed.

For the second ultracentrifugation separation, the specific gravity of the infranatant is adjusted with potassium bromide (230 to 1,050) kg / l. and infranatant.

- The supernatant is used to prepare Apo B,

- Infranatant is used to prepare Apo A-I.

3. Preparation of apolipoprotein A-I.

The specific gravity of the infranatant obtained by the second ultracentrifugation flotation is adjusted to 1.23 kg / l with potassium bromide. The following ultracentrifugation separation is carried out at 750 Hz and at a temperature between 50 ° C and 10 ° C for 40 to 45 hours. After adjusting the specific gravity ee the ultracentrifugation is repeated under the same conditions ·

The supernatant is dialysed against distilled water β by addition of 0.02 mol / l EOTA sodium salt for a period of time

239,044 twice for 4 hours. The dialyzed material is lyophilized.

Delipidation of the lyophilized material is performed in a glass centrifuge tube. To the material was added a mixture of chloroform (2 parts) and ethanol (1 part), and the cuvette was ground for 30 minutes in a mixture of dry ice (CO 2 solid) with ethanol. Occasionally the contents of the cuvette are mixed. Then the cuvette is removed from the coolant and centrifuged at 42 Hz. The solvent is poured off and the extraction is repeated under the same conditions. Ether is then added to the material, mixed well and centrifuged at 42 Hz. This ether extraction is also repeated once more,

The delipidated material was carefully dried with a stream of nitrogen and dissolved in a solution of 2 M acetic acid. Dissolution can be improved by adding urea. The solution obtained is filtered through glass wool and applied to a Sephadex G-100 column (90 x 2.5 cm) which is equilibrated with the same solution. IX and other proteins. The fractions containing Apo A-I combined are concentrated on an Amicon membrane and rechromatographed under the same conditions. The fraction ee obtained is dialyzed against water containing sodium EDTA at a concentration of 0.02 mol / l, at 4 ° C and for 24 hours,

4, Preparation of the apolls room B

The specific gravity of the supernatant obtained by the second ultracentrifugation flotation was adjusted to 1.050 kg / l with potassium bromide. The following ultracentrifugation separation is performed at 750 Hz and a temperature of β to 10 ° C for 15 hours

239,044 to 20 hours. The obtained eupernatant ee dialylated twice for 4 hours against distilled water containing sodium EDTA (0.02 mol / l). The dialysed material ee lyophilized ·

The delipidation of the lyophilized material ee is carried out as in the case of the isolation of apollpoprotein A-I.

Execution example

The procedure for the isolation and purification of the apolloproteins A-I and B and the preparation of the material for isolation is divided into 3 steps of 9 steps.

The process from processing the starting material to step 1.2. allows simultaneous preparation of both antigens. Also, some other steps for antigen isolation (2.2 and 3.2) are common to both antigens, which simplifies the procedure.

0.0. Out! raw material

To isolate apolloproteins A-I and B, we will use remote human plasma from healthy donors. It is preferred to start from 500 ml of plama. Add ED7 sodium salt 0.37 g / l and sodium azide to a final concentration of 1 g / l. Then filter the plasma through an asbeetocellulose layer (Seitz, K5).

1.0. Isolation of plasma lipoproteins

1.1. Ultracentrifugation flotation of plasma lipoproteins

The specific weight of the starting material is adjusted to 1.030 kg / l with solid potassium bromide (KBr). Ultracentrifugation flotation is performed on C H RII S T Omega II (rotor 9710) at 750 Hz and

- 6,239,044 and a temperature of 5 to tallow for 18 to 22 hours. After completion, separate supernatant and intermediate layer from infranatant Supernatant and remove intermediate layer ·

1.2. Classification of plamatic lipoproteins according to their specific weight

The specific gravity of the infranatant obtained in step 1 «2« is adjusted to 1,050 kg / l with solid potassium bromide. · Ultracantrifugation flotation is performed on an ultracentrifuge Chriet Omega II (rotor 9730) at 750 HZ and temperature 5 to lo | ° C for 18 to 22 hours · Separate supernate, intermediate layer and infranatant, supernatant serves as a source of apollpoprotein B, infranatant as a source of apollpoprotein AI, and removes interstitial ea.

2.0. Isolation and purification of apollpoprotein A-I

2.1. Ultracentrifugation flotation

The specific gravity of the infranatant obtained in step 1.2. adjust with solid potassium bromide to 1.230 kg / l. The ultracentrifugation flotation is performed again on the ultracantrifusion Chriet Omega II (rotor 9730) at 750 Hz and at a temperature of β to IC for 40 to 45 hours. Measure the specific gravity of the obtained euparnatant, adjust it to 1,230 kg / l with solid potassium bromide if necessary and repeat the ultracentrifugation flotation under the same conditions.

2.2. Dialysis and lyophilization

The supernatant obtained in step 2.1. dialysis is carried out against the end of the distilled water by the addition of sodium> 239 044 eol EDTA at a concentration of 0.02 mol / l at 4 to 8 ° C for 4 hours. Then replace the dialysis solution and repeat the dialysis under the same conditions. The material obtained is lyophilized.

2.3. Oellpidace

Delipidaei of the lyophilized material of step

2.2. performed in a 100 ml glass centrifuge tube. Add 2 parts chloroform and 1 part ethanol 50 ml. Place the cuvette for 30 minutes in a mixture of solid C0 ₂ and ethanol. Stir the cuvette regularly. Centrifuge at 42 Hz at room temperature for 10 minutes, discard the solvent and repeat the extraction under the same conditions. 30 ml of ether are added to the material so obtained. Mix well by centrifugation at 42 Hz and room temperature for 10 minutes, decant the ether and repeat the extraction under the same conditions. Finish the obtained material with a low current dual.

2.4. Chromatography

The material obtained in the previous step (2.3) was subdivided on a Sephadex G-100 column of dimensions

2.5 x 90 cm in 2 M-cyeelines with acetic acid. Before application to the column, dissolve the material in 8 ml of acetic acid of the same concentration. If it is difficult to dissolve, add a small amount of urea, p.a.

After dissolution and before application to the column, filter the solution through a cotton swab. Wash the column with 2 Mrottok acetic acid. Collect fractions after 15 minutes

- 8 239 044 at a flow rate of 20 ml / h. The individual fractions are measured on a spectrophotometer at 280 nm for obeah proteins. The fractions corresponding to each peak are pooled and after concentration on an Amlcon membrane, rechromatographed under the same conditions. The resulting material was flushed against the stem of the volume of distilled water with the addition of sodium EDTA (0.02 mol / l) in ice for 24 hours. Then we determine in the obtained material immunochemically the obeah apolipoprotein A-I. Then lyophilize the material ·

3.0. Isolation and purification of apolipoprotein B

3.1. Ultracentrifugation flotation

The specific weight of the eupernatant obtained in step 1.2. read again, adjust as necessary with solid potassium bromide to 1,050 kg / l and repeat ultracentrifuge flotation under the conditions given in the same step (1.2,).

3.2. Oialysis and lyophilization

The supernatant obtained by ultracentrifugation flotation in the previous step (3.1) was plotted against the volume of distilled water with the addition of sodium EDTA at a concentration of 0.02 mol / l at 4 hours. Then we repeat the dialysis under the same conditions.

In the obtained material, the content of Apo B is determined by immunochemistry and then lyophilized.

Claims (1)

SUBJECT OF THE INVENTION 239 044 Method for the isolation and purification of apolipoprotein A1 and apolipoprotein 3, characterized in that antigens are isolated from human plasma to which an alkali metal bromide or chloride is added to achieve a specific weight of 1.006 kg / l by subjecting the plasma to ultracentrifugation flotation, repeating the procedure with increasing salt concentration by using a specific gravity range of 1.030-1.050 kg / l for the isolation of apolipoprotein 3 and a specific gravity range of 1.050-1.230 kg / l for the isolation of apolipoprotein A1 and subjecting the appropriate fractions to column chromatography. using dextran gels.