GB2238050A - Immunoaffinity column for simultaneous extraction of several anabolic hormone residues - Google Patents

Abstract

A column for the simultaneous extraction of several anabolic hormone residues from biological liquids or biological tissue homogenates digested with proteolytic enzymes contains two or more gel fractions having a single polysaccharide matrix but each conjugated with a specific immunoglobulin for one of the hormones to be isolated. Conjugation may be effected by incubating an aqueous solution of said immunoglobulin with an aqueous suspension of a respective gel fraction for a time of between 3 and 12 hours at a temperature of between 4 and 25 DEG C.

Description

IMMUNOAFFINITY COLUMN FOR THE SIMULTANEOUS EXTRACTION OF SEVERAL ANABOLIC HORMONE RESIDUES FROM BIOLOGICAL LIQUIDS AND THE METHOD FOR ITS PREPARATION DESCRIPTION A. Technical sector This invention relates to an immunoaffinity column for the simultaneous extraction of several anabolic hormone residues from biological liquids or biological tissue homogenates digested with proteolytic enzymes, and a method for its preparation.

With the column according to the invention, biological liquids (urine, saliva, serum, blood, perspiration, tears) or biological tissue homogenates digested with proteolytic enzymes can be treated to separate any residues of anabolic hormones which have been ingested by the animal or human being from which said biological liquids originate.

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

B. Prior art The traditional methods for extracting hormones from biological matrices are based on the use of liquid/liquid extraction using appropriate organic solvents such as ethyl ether and chloroform, and columns for once-only use comprising alumina or octadecyl-silane (C18) as stationary phases, such as the Millipore Waters type marketed under the name of SEP-PAK.

These purification methods are rather laborious and require considerable operator experience to obtain significant samples. In addition, simple liquid/liquid extraction does not enable sufficiently pure samples to be obtained, ie samples free of substances present in the biological liquids which could interfere with the hormone residues to be analyzed during the subsequent CG or MS analysis.

Other methods have therefore been recently proposed based on immunoaffinity (IAC) to obtain hormone residue samples which are purer and therefore more easily determinable quantitatively during subsequent GC or MS analysis.

In this respect, reference can be made to works published by L.A. Van Ginkel and others in Journal of Chromatography 489 (1989), 95/104 and 111/120.

The IAC technique is based on the specificity of the antigen/ antibody bond, which enables the hormone residues contained in the biological liquid under examination to be separated from other substances present which could interfere with the subsequent analytical determination.

C. Technical problem Although the use of anabolic hormones in the veterinary and human fields for muscle potentiation is prohibited by law in many countries, their very use creates an increasing requirement for investigative systems which give reliable results and are simple to use even by personnel who are not excessively specialized.

A particular requirement is for systems for isolating anabolic hormone residues from biological liquids or biological tissues which contain them in very low concentrations (nanograms/ml) and which can have various chemical structures.

Said isolation systems must ensure reliable separation of the required products while on the other hand being quick and simple to use so that even technicians of little specialist experience can obtain reliable results.

D. Detailed description of the invention It has now been found possible to prepare and use chromatographic columns based on immunoaffinity techniques for the specific simultaneous separation of several anabolic hormones from biological liquids or tissues which contain them.

According to one basic characteristic of the present invention, the immunoaffinity column is a container provided with a filter at its lower end, said filter acting as a support for the gel contained in said container, said gel being formed from at least two different gel fractions having a single polysaccharide matrix, each fraction being conjugated with an antibody able to bind to one of the hormones to be isolated or to several hormones of similar structure and therefore able to interact with the same antibody.

The container is constructed of non-reactive material, typically neutral glass or polymeric material, preferably polyolefinic.

The preparation of the gel fractions present within the immuno-affinity column forms an integral part of the present invention and consists of conjugating the specific immunoglobulins of each substance to be isolated with a polysaccharide gel by incubating an aqueous solution of immunoglobulins with an aqueous suspension of the polysaccharide gel for a time of between 3 and 12 hours at a temperature of between 4 and 25 C.

The IgG immunoglobulins required for preparing the individual gel fractions for placing in the column are obtained from rabbit serum to which there have been previously administered the hormones or other anabolic substances of hormone type which are to be subsequently isolated and purified by the immunoaffinity column.

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

The quantity of each gel fraction conjugated with a specific antibody for a determined hormone is typically between 100 and 300 ul.

A smaller gel quantity does not ensure sufficient separation within a reasonable percolation time of the hormone contained in the biological liquid or tissue undergoing purification, whereas a larger quantity is superfluous considering the high selectivity and blocking capacity of gels coupled to specific antibodies.

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

As the reaction rates of the various hormones present in the biological liquid to be treated with the immunoaffinity columns of the present invention can differ from each other, the column is calibrated with known contents of hormones to be isolated.

The contact time between the liquid under examination and the gel of the immunoaffinity column is made long enough to ensure quantitative recovery of all the hormones present, or alternatively the relative quantities of the various gels present in the column are varied so as to compensate for any lower binding rate of the hormones present in the solution by means of a greater gel volume.

According to a preferred embodiment of the invention the immunoaffinity column is marketed in the form of a kit suitable for treating urine or serum samples and comprises: a) a column of polyolefinic material of total capacity 20 ml containing agarose gel on which the antihormone antibodies relative to the hormone residues to be isolated and identified have been immobilized; b) a bottle containing buffer solution for preserving the gel at 4 G without loss of its binding capacity; c) a bottle containing a buffer solution to be used for washing the column contents after eluting the sample to be examined; d) a bottle containing a buffer solution for diluting the sample to be examined and for digesting it with the ss-glucuronidase enzyme; e) a bottle containing a solution of -glucuronidase enzyme to be used for treating the sample to be examined after it has been diluted with the buffer of item d); f) a bottle containing an extraction buffer to be used to dilute the samples digested with the #-glucuronidase before feeding them into the immunoaffinity column; g) a detailed description of the procedure for using the various kit components.

In this embodiment the use of the column for separating the residues from the biological liquids or tissues which contain them is greatly facilitated and is well within the capacity of a technician without lengthy specialist experience.

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 in 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 obtained is fed into a polypropylene column of 0.5 cm diameter and 10 cm height, and washed with 10 volumes of pH 6.5 buffer solution in the form of 10 mM phosphate and 140 mM NaCl (buffer B), checking that the pH remains at 6.5.

The purified IgGs are diluted in cold buffer B to an immuno-globulin concentration of 2 mg/ml of gel in two volumes of buffer per volume of gel.

The suspension obtained is incubated at a temperature of between 4 and 250C for a time of between 3 and 12 hours while under stirring.

After incubation the suspension is allowed to sediment and the supernatant is recovered, checking the residual immunoglobulin contents which have not bonded to the gel.

The incubation operation is repeated if the quantity of immunoglobulins which have conjugated with the gel is less than 70 of the quantity incubated.

The conjugated gel is washed with 5 volumes of buffer B and then treated with 2 volumes of an aqueous 0.2 M glycine solution of pH 8 (solution C) at a temperature of between 4 and 250C for a time of between 3 and 12 hours.

The treatment with glycine is essential to block all those sites of the gel which have remained active after conjugation with the immunoglobulins.

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

After the wash cycles a final wash is carried out with a pH 7 buffer solution formed from 10 mM phosphate and 140 mM NaCl (buffer E) to which 2 mM of phenyl-methylsulphonyl-fluoride (PMSF) and 3 mM of sodium azide are added, checking that the final pH is 7.4.

The immunoaffinity gel is preserved in the same liquid used for the final wash at a temperature of 4 C.

For lengthy storage (exceeding one year) an equal quantity of glycerol is added to said liquid, storage then being carried out at #200 C.

Using the aforesaid method, immunoaffinity gels were prepared for zeranol, dienestrol, hexestrol, diethylstilbestrol, 17#-estradiol, 19nortestosterone, clenbuterol and trenbolone.

B) Use of the immunoaffinity column An immunoaffinity column prepared as described under section A) and containing 150 ul of immunoaffinity gel able to bind diethylstilbestrol and the similar products dienestrol and hexestrol and 150 ul of immunoaffinity column gel able to bind zeranol was used to treat urine samples from calves which had been treated experimentally with said hormones.

0.5 ml of urine, 1.5 ml of a buffer hydrolysis solution (0.5 M acetate and 3 mM sodium azide, pH 4.5) diluted 1:10 and 25 ul of a solution of ss-glucuronidase enzyme (equivalent to 2500 U.I) in a glass test-tube are mixed.

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

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

The sample contained in the test tube is diluted with 4 ml of a buffer solution (1M phosphate, 3 mM sodium azide, pH 7.4) diluted 1:10, centrifuged for 10 minutes at 2500 r.p.m. and fed into the immunoaffinity column.

The column is closed and rotated on a test-tube rotator for 30 minutes.

The liquid is then allowed to flow through the lower filter and it is washed with 5 ml of a buffer wash solution (1 M phosphate, 1% Tween 20, 3 mM sodium azide, pH 7.4) diluted 1:10, and then with 5 ml of distilled water.

The column contents are washed a further two times with 5 ml of distilled water, and after allowing the column to drain, the column immunoaffinity retained material is eluted with 1 ml of an acetone:water (95:5) solution and then with 0.5 ml of the same solution.

The two eluates are pooled, the solvent is evaporated in a warm air stream at 60 C and the residue is treated for 30 minutes at 60 0C with 50 ul of a solution of pentafluorobenzylbromide (PFBBr) in acetonitrile (1:20 v/v) and 50 ,ul of a solution of KOH in anhydrous ethanol (8 mg/ml). After evaporating the solvent the residue was redissolved in 30 ul of N,O-bis-(trimethylsilyl)-trifluoroacetamide (BSTFA), the solution obtained was heated to 600C for 30 minutes and then injected into a HP 5890 gas chromatograph fitted with a capillary column (0.32 mm diameter, 25 m length, type CP Sil 5 CB of Chrompack Italia, transport gas helium at 30 kPa) using as detector a VG TS-250 negative ion chemical ionization (NICI) mass spectrometer.

The extraction capacity of the column for the various hormone residues was previously determined by calibration tests with hormones purified under the same conditions as used for treating the urine samples, and was found to be: 80X for trans-diethylstilbestrol, 73% for cis-diethylstilbestrol, 53% for dienestrol, 68% for hexestrol, 73% for a-zeranol and 50% for ss- zeranol.

Said values are sufficient both for qualitative and for quantitative determinations.

The specificity of the column for the stated hormones was also determined experimentally by adding to the calibration samples natural hormones such as estrone, estriol and l7#-estradiol, it being found that these were not retained by the column.

After washing the immunoaffinity column with the acetone:water (95:5) solution used for the elution and with distilled water, 5 ml of a buffer preservation solution (0.5 M phosphate. 3 mM sodium azide, ph 7.4) diluted 1:10 were added and the column was stored at 40C until next used.

The same column was used for more than 100 urine sample separations and purifications without apparent loss of separation or purification capacity.

Claims (11)

1. An immunoaffinity column for the simultaneous extraction of several anabolic hormone residues from biological liquids or biological tissue homogenates digested with proteolytic enzymes, consisting of a container provided with a filter at its lower end, said filter acting as a support for the gel contained in said container, said gel being formed from at least two different gel fractions having a single polysaccharide matrix, each fraction being conjugated with an antibody able to bind to one of the hormones to be isolated or to several hormones of similar structure and therefore able to interact with the same antibody.

2. A column as claimed in claim 1, characterised in that said container is constructed of neutral glass or polymeric material.

3. A column as claimed in claim 2, characterised in that said polymeric material is polypropylene.

4. A column as claimed in claim 1, characterised by having a diameter of between 0.5 and 1.5 cm and a height of between 10 and 15 cm.

5. A column as claimed in claim 1, characterised in that the quantity of each gel fraction conjugated with an antibody for a determined hormone is between 100 and 300 pl.

6. A column as claimed in claim 1, characterised in that the immunoaffinity gels contained in it are chosen from the group consisting of gels bonded to antibodies specific for zeranol, dienestrol, hexestrol, diethglstilbestrol, 17#-estradiol, 19nortestosterone, clenbuterol and trenbolone.

7. A column as claimed in claim 1, characterised in that the relative quantities of the various gels conjugated with different immunoglobulins present in said column are in such mutual proportions as to compensate for any lower binding rate of one gel relative to another.

8. A method for preparing an immunoaffinity column for the simultaneous extraction of several anabolic hormone residues from biological liquids or biological tissue homogenates digested with proteolytic enzymes, consisting of introducing, into a container provided with a filter at its lower end, two or more gels which have a single polysaccharide matrix, but have each been 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 time of between 3 and 12 hours at a temperature of between 4 and 250C.

9. A method for preparing an immunoaffinity column as claimed in claim 8, wherein said immunoglobulins are obtained from the serum of rabbits which have previously been administered with the hormones or other anabolic substances of hormone type which are to be subsequently isolated and purified by the immunoaffinity column.

10. A kit for the for the simultaneous extraction of several anabolic hormone residues from biological liquids or biological tissue homogenates digested with proteolytic enzymes, comprising an immunoaffinity column as claimed in claim 1.

11. A kit as claimed in claim 10, comprising: a) a column of polyolefinic material of total capacity 20 ml containing agarose gel on which the antihormone antibodies relative to the hormone residues to be isolated and identified have been immobilized; b) a bottle containing buffer solution for preserving the gel at 4 C without loss of its binding capacity; c) a bottle containing a buffer solution to be used for washing the column contents after eluting the sample to be examined; d) a bottle containing a buffer solution for diluting the sample to be examined and for digesting it with the #-glucuronidase enzyme; e) a bottle containing a solution of #-glucuronidase enzyme to be used for treating the sample to be examined after it has been diluted with the buffer of item d); f) a bottle containing an extraction buffer to be used to dilute the samples digested with the 5-glucuronidase before feeding them into the immunoaffinity column; g) a detailed description of the procedure for using the various kit components.