IT8922425A1 - COLUMN OF IMMUNOAFFINITY FOR THE SIMULTANEOUS EXTRACTION OF MORE ANABOLIC HORMONES FROM BIOLOGICAL LIQUIDS AND PROCEDURE FOR ITS PREPARATION. - Google Patents

Description

DESCRIPTION

TO . Technical field

The present invention? relative to an immunoaffinity column? for the simultaneous extraction of pi? residues of anabolic hormones from biological liquids or from homogenized biological tissues digested with proteolytic enzymes and a procedure for its preparation.

The column according to the invention allows to separate from biological liquids (urine, saliva, serum, blood, sweat, tears) or from homogenized biological tissues digested with proteolytic enzymes any residual anabolic hormones that have been ingested by the animal or by the man from whom these biological liquids come.

The residues of hormones blocked by the column are eluted with a solvent and subjected to quantitative analysis with the usual means of analytical investigation such as gas chromatography (GC), in particular with chemical ionization detectors with negative ions (NICI), or high chromatography pressure with capillary columns (HPLC) and mass spectrometry (MS). B. Anterior Technique

The traditional techniques for the extraction of hormones from biological matrices are based on the use of liquid / liquid extraction techniques with the use of appropriate organic solvents, such as ethyl ether and chloroform, and of columns that are used only once and have stationary phases of Alumina or Octadecisilane (C18), such as those marketed under the SEP-PAK brand by Millipore-Waters.

Said purification techniques are rather laborious and require considerable experience on the part of the operator to obtain significant samples. Furthermore, the simple liquid / liquid extraction does not allow to obtain sufficiently pure samples, ie exempt? from substances that are present in biological liquids and that interfere with the residues of hormones that are to be analyzed in the subsequent phases of GC or MS analysis.

Recently other techniques have been proposed, based on immunoaffinity. (IAC), to obtain samples of residual hormones pi? pure and therefore more? easily quantitatively determinable in the subsequent phases of GC or MS analysis.

In particular we can recall the works published by L.A. Van Ginkel and others in Journal of Chromatography, 489, (1989) .95/104 and 111/120.

Is the IAC technique based on specificity? of the antigen / antibody bond, which allows the separation of the residues of a hormonal nature contained in the biological liquid under examination from the other substances present that interfere in the subsequent phases of analytical demination.

C. Technical problem

The use of anabolic hormones in both the veterinary and human fields to increase muscle mass, despite being prohibited by law in many countries, is increasingly profitable. It is necessary to have survey systems that give reliable results and are easy to use even by not excessively specialized personnel.

In particular ? It is necessary to have systems for isolating residues of anabolic hormones from biological liquids or biological tissues that contain them in very low concentrations (nanograms / ml) and which may have different chemical structures.

Said isolation systems must guarantee a safe separation of the products sought while being fast and easy to use so that even technicians with little specialized experience can obtain reliable results.

D. Detailed description of the invention Yes? now found that? Is it possible to prepare and use chromatographic columns based on immunoaffinity techniques? to separate specifically and simultaneously pi? residues of anabolic hormones from liquids or biological tissues that contain them. According to a fundamental characteristic of the present invention, the immunoaffinity column? ? consists of a container equipped with a filter at its end? lower, said filter having a supporting function for the gel that is inside said container, said gel being formed by at least two different gel fractions having a single polysaccharide-type matrix, each fraction being conjugated with an antibody capable of binding to one of the hormones that you intend to isolate or to pi? hormones having a similar similar structure and therefore capable of interacting with the same antibody.

The container ? built in non-reactive material, typically neutral glass or polymeric material, preferably of the polyolefin type.

The preparation of the gel fractions present inside the immunoaffinity column? is an integral part of the present invention and consists in conjugating the specific immunoglobulins of each substance to be isolated with a gel of a polysaccharide by incubating an aqueous solution of immunoglobulins with an aqueous suspension of the polysaccharide gel for a period of time between 3 and 12 hours at a temperature of 4 to 25 ° C.

The IgG immunoglobulins necessary to prepare the single gel fractions to be introduced into the column are obtained from the serum of rabbits to which the hormones or other hormonal anabolic substances which are subsequently intended to be subsequently isolated and purified with the immuno-activity column have been administered. .

The column of immunoaffinit? according to the present invention it typically has a diameter between 0.5 and 1 cm and a height between 10 and 15 cm.

The quantity of each fraction of gel conjugated with a specific antibody for a given hormone? typically between 100 and 300? l.

A quantity? lower gel does not allow to ensure sufficient separation of the hormone contained in the liquid or biological tissue subjected to purification in reasonable percolation times, while a higher quantity is useless considering the great selectivity? and capacity? blocking of gels coupled with specific antibodies.

The various fractions of each gel are mixed together in suspension and the suspension of the selected mixture is then introduced into the column.

Given that the speeds? reaction of the various hormones present in the biological liquid to be treated with the columns of immunoaffinit? of the present invention can be different from each other, the columns are calibrated with mixtures having a known content of hormones to be isolated.

One then proceeds to ensure a sufficiently long contact time between the liquid under examination and the gel of the immunoaffinity column. to ensure a quantitative recovery of all the hormones present or, alternatively, the quantities vary? relative of the various gels present in the column in order to compensate with a greater volume of a gel the lower speed? of coupling of the hormones present in the solution.

According to a preferred embodiment of the invention, the immunoaffinity column is is marketed in the form of a kit suitable for treating urine or serum samples and which includes: a) a column in polyolefin material of the capacity? of 20 ml in total, containing agarose gel on which the anti-hormone antibodies related to the hormone residues to be isolated and identified have been immobilized;

b) a bottle containing a buffer solution for preserving the gel at 4? C without loss of its capacity? binder;

c) a bottle containing a buffer solution to be used for washing the contents of the column after the elution of the sample to be examined; d) a bottle containing a buffer solution to dilute the sample to be examined and digest it with the? -glucuronidase enzyme; e) a bottle containing an enzyme solution? glucuronidase to be used for the treatment of the sample to be examined after its dilution with the buffer referred to in point d); f) a bottle containing an extraction buffer to be used to dilute the digested samples with? glucuronidase before loading them into the immunoaffinity column;

g) a detailed description of the procedure for using the various components of the kit.

In this embodiment, the use of the column to obtain the separation of the residues from the biological liquids or tissues containing them? greatly facilitated and can? be easily performed safely even by technicians without a long specialist preparation.

EXAMPLE

A) Preparation of the gel conjugated with immunoglobulins. A lyophilized polysaccharide gel marketed by Pharmacia under the name of Sepharose CNBr-4B is hydrated and resuspended by means of 50 volumes of a 1 mM HCl solution (solution A).

About 3.5 ml of gel are obtained from 1 g of Sepharose CNBr-4B powder.

The gel thus obtained is placed in a polypropylene column having 0.5 cm in diameter and 10 cm in height and washed with 10 volumes of buffer solution consisting of 10 mM phosphate, 140 mM NaCl, and having pH 6.5 (buffer B ), checking that the pH remains at 6.5.

The purified IgG are diluted in cold buffer B until an immunoglobulin concentration of 2 mg / ml of gel is obtained in two volumes of buffer for each volume of gel.

The resulting suspension is incubated at a temperature of from 4 to 25 ° C for a period of time from 3 to 12 hours while maintaining under stirring.

At the end of the incubation the suspension is allowed to settle and the supernatant is recovered, checking the residual content of immunoglobulins that have not bound to the gel.

The incubation operation is repeated if the quantity? of immunoglobulins conjugated to the gel is less than 70% of those placed in incubation.

The conjugated gel is washed with 5 volumes of buffer B and then treated with 2 volumes of an aqueous solution of glycine 0.2 M at pH 8 (solution C) at a temperature of 4 to 25 ° C for a period of time 3 to 12 hours.

The treatment with gliein? essential to block all sites of the gel that remained active after conjugation with immunoglobulins.

The gel is subjected to 3 washing cycles, each cycle comprising a first wash with 5 volumes of buffer solution consisting of 0.1 M sodium acetate, 0.5 M NaCl and having pH 4 (buffer D) and a second wash with 5 volumes of buffer B.

At the end of the washing cycles, a final washing is carried out with a buffer solution consisting of 10 mM phosphate, 140 mM NaCl and having pH 7 (buffer E), to which 2 mM of phenyl-methylsulfones1-fluoride (PMSF) and 3 are added. mM sodium azide checking that the final pH is 7.4.

The immunoaffinity gel? it is stored in the same liquid used for the final washing at a temperature of 4 ° C.

For long-term storage (over a year), an equal quantity is added to the aforementioned liquid. of glycerol and can be stored at -20 ° C.

Using the method described above, immunoaffinity gels were prepared. for zeranol, dienestrol, hexestrol, diethylstilbestrol, 17 -? - estradiol, 19-nortestosterone, clenbutenol and trenbolone.

B) Use of the immunoaffinity column.

An immunoaffinity column, prepared as described in point A) above and containing 150? L of immunoaffinity gel? capable of binding diethylstilbestrol and similar products dienestrol and esestrol and 150? l of immunoaffinity gel? capable of binding zeranol,? was used to treat urine samples from calves experimentally treated with these hormones.

0.5 ml of urine and 1.5 ml of a hydrolysis buffer solution (0.5 M acetate, 3 mM sodium azide, pH 4.5) diluted 1 to 10 and 25? enzyme solution (S-glucuronidase (equal to 2,500 I.U.).

The solution obtained is incubated at 37 ° C for at least two hours.

The column of immunoaffinit? it is washed with 10 ml of an acetone: water mixture (95: 5). 2 ml of distilled water and 2 ml of a buffer solution (0.05 M phosphate, PH 7.4).

The sample contained in the tube is diluted with 4 ml of a buffer solution (1 M phosphate, 3 mM sodium azide, pH 7.4) diluted 1 in 10, centrifuged for 10 minutes at 2,500 rpm and loaded into the immunoaffinity column.

The column is closed and rotated on a tube rotator for 30 minutes. At the end, the liquid is allowed to flow out through the lower filter and washed with 5 ml of a washing buffer solution (1 M phosphate, 1% Tween 20, 3 mM sodium azide, pH 7.4) diluted 1 to 10 and subsequently with 5 ml of distilled water. The washing with the buffer solution and distilled water is repeated.

The content of the column is treated twice more with 5 ml of distilled water and after having drained it well, the elution is carried out with 1 ml of an acetone: water solution (95: 5) and then with 0.5 ml of the same. solution of what is retained by the immunoaffinity column.

The two eluates are combined, the solvent is evaporated in a stream of hot air at 60? C and the residue? treated for 30 minutes at 60? C with 50? L of a solution in acetonitrile (1:20 v / v) of pentafluorobenzylbromide (PFBBr) and 50? L of a solution of? 0? in anhydrous ethanol (8 mg / ml). After evaporation of the solvent, the residue was redissolved in 30 µl of N, 0-bis- (trimethyl-silyl) -trifluoroacetamide (BSTFA), the resulting solution was heated to 60? C for 30 minutes and then injected into an HP 5890 gas chromatograph equipped with a capillary column (0.32 mm in diameter, 25 m in length, type CP Be 5 CB from Chrompack Italia, helium carrier gas at 30 kPa) using as a detector a VG TS-250 mass spectrometer with chemical ionization with negative ions (NICI). The capabilities extraction of the column against the various hormone residues was previously determined by calibration tests with purified hormones under the same conditions used for the treatment of urine samples and was found to be:

80% for trans-diethylstilbestrol, 73% for cisdietylstilbestrol, 53% for dienestrol, 68% for hexestrol, 73% for? '? - zeranol and 50% for? -Zeranol.

These values are sufficient for both qualitative and quantitative determinations.

It determined? experimentally also the specificity? of the column against the aforementioned hormones, by adding to the calibration samples of natural hormones such as estrone, estriol and 17? estradiol which were not retained by the column. In the immunoaffinity column, after washing with the acetone: water solution (95: 5) used for elution and with distilled water, 5 ml of a storage buffer solution (0.5 M phosphate, sodium azide 3 m ?, pH 7, H) diluted 1 to 10 and the column was stored at 4 ° C until its next use.

The same column? has been used for over 100 separations and purifications of urine samples with no apparent loss of capacity? of separation and purification.

Claims (11)

CLAIMS 1. Column of immunoaffinity? for the simultaneous extraction of pi? residues of anabolic hormones from biological liquids or from biological tissues homogenized and digested with proteolytic enzymes consisting of a container equipped with a filter at its end? lower, said filter having a supporting function for the gel that is inside said container, said gel being formed by at least two different gel fractions having a single polysaccharide-type matrix, each fraction being conjugated with an antibody capable of binding to one of the hormones that you intend to isolate or to pi? hormones having a similar similar structure and therefore capable of interacting with the same antibody. 2. Column as claimed in 1. characterized in that said container? built in neutral glass or polymeric material. 3. Column as claimed in 2. characterized in that said polymeric material? polypropylene. 4. Column as claimed in 1. characterized in that it has a diameter of between 0.5 and 1.5 cm and a height of between 10 and 15 cm. 5. Column as claimed in 1. characterized by the fact that the quantity? of each gel fraction conjugated with an antibody for a given hormone? between 100 and 300? l. 6. Column as claimed in 1. characterized in that the immunoaffinity gels? it contains are selected from the group of gels coupled with specific antibodies for zeranol, dienestrol, esestrol, diethylstilbestrol, 17 -? - estradiol, 19-nortestosterone, clenbutenol, trenbolone. 7. Column as claimed in 1. characterized by the fact that the quantities? of the various gels conjugated with different immunoglobulins present in said column are in relative ratios such as to compensate for the possible lower speed? coupling of one gel with respect to another. 8. Procedure for preparing an immunoaffinity column? for the simultaneous extraction of pi? residues of anabolic hormones from biological liquids or from biological tissues homogenized and digested with proteolytic enzymes consisting in * placing in a container, equipped with a filter at its end? lower, two or more? gels having a single polysaccharide-type matrix but each of which? obtained by conjugating a polysaccharide gel with a specific immunoglobulin of each substance to be isolated by incubating an aqueous solution of said immunoglobulin with an aqueous suspension of said gel for a period of time between 3 and 12 hours at a temperature 4 to 25? C. 9? Procedure for the preparation of an immunoaffinity column as claimed in 8. wherein said immunoglobulins are obtained from the serum of rabbits to which the hormones or other anabolic substances of the hormonal type which are subsequently intended to be subsequently isolated and purified with the immunoactivity column have been previously administered. 10. Kit for the simultaneous extraction of pi? residues of anabolic hormones from biological liquids or from homogenized biological tissues digested with proteolytic enzymes including an immunoaffinity column as claimed in 1. 11. Kit as claimed in 10. comprising: a) a column in polyolefin material of the capacity? of 20 ml in total, containing agarose gel on which the anti-hormone antibodies related to the hormone residues to be isolated and identified have been immobilized; b) a bottle containing a buffer solution for preserving the gel at 4? C without loss of its capacity? binder; c) a bottle containing a buffer solution to be used for washing the contents of the column after the elution of the sample to be examined; d) a bottle containing a buffer solution to dilute the sample to be examined and digest it with the? -glucuronidase enzyme; e) a bottle containing an enzyme solution? glucuronidase to be used for the treatment of the sample to be examined after its dilution with the buffer referred to in point d); f) a bottle containing an extraction buffer to be used to dilute the digested samples with? glucuronidase before loading them into the immunoaffinity column; g) a detailed description of the procedure for using the various components of the kit.