GB2152664A - Magnetic assay reagents - Google Patents

Abstract

The invention provides a method of assaying a ligand in a sample by means of contacting the sample with homogeneous colloidal magnetic particles having a reagent immobilised thereon, forming an immobilised complex on the said colloidal magnetic particles, and subsequently separating the colloidal magnetic particles and immobilised complex from the assay medium. Mechanical agitation of the assay medium is unnecessary.

Description

SPECIFICATION Magnetic assay reagents The present invention relates to methods of assay of one of a pair of specific binding partners and to magnetic reagents for carrying out these methods.

There is today a great need for rapid and accurate methods of assaying biologically active substances (which may be at low concentration), particularly in body fluids such as blood, saliva or urine. A wide variety of medical conditions, such as pregnancy, drug overdose, metabolic birth defects, hormonal disorders and diabetes can be diagnosed using such assay techniques.

Many assay methods rely on the formation of a complex between the specifies under assay (hereinafter called "ligand") and another species to which it will bind specifically (hereinafter called "specific binding partner"). The extent of complex formation is a function of the amount of the ligand present.

The assay of ligand is determined by monitoring the extent of complex formation, for example by the use of chemical or biochemical labels. Several methods of labelling have been employed, for example radioisotopic or enzyme labelling, spin-labelling or labelling employing fluorescent or bioluminescent species.

Thus, by using a labelled species which is capable of complexing either with the ligand or with a specific binding partner of the ligand (in competition with ligand present), a proportion of label will, after the complexing reaction, have entered the complexed phase and a proportion will remain in solution.

By separating the two label-containing phases from each other, the proportion of the label in either may be determined by the usual counting methods and the assay of the ligand thereby determined.

Such separations may be difficult to perform without giving rise to significant errors. It is known that the separation may be facilitated by providing one of the species involved in an immobilised form on a solid support, which may subsequently be removed from the assay medium by e.g. centrifugation, filtration or, if the carrier is magnetic, by magnetic separation.

In known methods of separation employing magnetic particles as a solid support for reagents, the solid phase is removed from the solution after the complexing reaction by the application of a magnetic field. Conventionally used magnetic materials are, however, generally appreciably denser than a typical assay medium (e.g. of the order of 5 g cm-3) and since also such particles have hitherto been relatively large, it has been necessary to agitate the assay medium mechanically (e.g. by stirring or by causing the particles to move by the application of a rotating magnetic field) in order to prevent the particles from sedimenting out of the medium before completion of the complexing reaction. The need for mechanical agitators has inevitably increased the bulk, cost and complexity of the equipment required.

The problem of sedimentation is frequently compounded by the magnetic attractive forces between the particles which can cause agglomeration and flocculation.

Hitherto, the most widely adopted solution to the sedimentation problem has been to use socalled composite particles in which the magnetic material is enclosed by a matrix or coating of non-magnetic polymeric material. In addition to reducing the overall density of the particles, the non-magnetic material also reduces the attractive forces between the particles and may provide a convenient foundation on which to immobilise the reagent molecules. However, such composite particles are difficult to prepare reproducibly and may not completely overcome the problem of rapid sedimentation.

It is one of the objects of the present invention to overcome these problems and to provide a simple assay method employing convenient apparatus and reagents.

In its broadest aspect, the present invention provides a method of assaying a ligand in a sample, which includes the steps of contacting the sample with homogeneous colloidal magnetic particles having a reagent immobilised thereon, whereby an immobilised complex is formed on the said colloidal magnetic particles, and subsequently separating the colloidal magnetic particles and immobilised complex from the assay medium.

Immobilised complex will thus be formed between the immobilised reagent and the labelcontaining species from either the complexed or the non-complexed phase of the assay medium.

The reagent immobilised on the colloidal magnetic particle may be a specific binding partner to the ligand or (in the case of a competitive assay) to the ligand analogue. It may also be a species capable of binding specifically to the specific binding partner. In general, any reagent capable of binding with the label-containing species of either the complexed or the noncomplexed phase of the assay medium may be employed.

The term "colloidal particles" and like expressions used herein shall be taken to include particles capable of forming, in a suitable medium, a system from which the particles will not appreciably sediment out under the effects of gravity within the time taken to perform the assay (typically up to 1 or 2 hours), and thus includes within its scope inter alia particles capable of forming a substantially homogneous sol.

According to a further aspect of the present invention, we provide colloidal magnetic particles having a reagent immobised thereon for use in a method of assay as herinbefore defined.

The present invention enables the use of so-called composite magnetic particles to be avoided.

The dimensions of the particles will preferably be such that their fundamental kinetic energy, or 'Brownian Motion', is sufficient to compensate for their tendency to sediment under gravity.

In addition, so long as the magnetic moment of each particle is sufficiently small, appropriate surfactants may be used to control agglomeration and flocculation. It is, however, an important feature of the particles that mechanical agitation is not required in order to maintain them in substantially stable suspension in use.

In general the colloidal magnetic particles will be in the size range 10 to 800 nm, preferably from 30 to 400 nm. The specific gravity of the particles will be, for example, up to 8, preferably from 2 to 6.

The term "magnetic particles" and like expressions, used herein, shall be taken to include ferromagnetic and paramagnetic particles, and particles which may become temporarily magnetic in the presence of a magnetic field. Suitable magnetic materials include, for example, metals (e.g. iron, nickel or cobalt), alloys (e.g. magnetic alloys of aluminum, nickel, cobalt and copper), oxides (e.g. Fe304, Y-Fe203, Cr2, CoO, NiO or Mn203), magnetoplumbites and solid solutions, e.g. of magnetite with ferric oxide. The preferred magnetic materials are magnetite (Fe3O4) and haematite (y-Fe2O3).

If desired, the nature of the magnetic material may be altered to suit the circumstances of the assay. For example, in radioimmunoassays it may be advantageous to use magnetic materials which absorb relatively little of the radiation from the label (e.g. materials containing Al, Fe, Co, Sn or Ni atoms).

The separation of the magnetic particles from the assay medium is effected by the application of a magnetic field, for example using a permanent magnet or an electromagnet.

The reagent employed may be one of the reagents conventionally used in immobilised form on solid supports in known assay methods. Immobilisation of the reagent molecules may be achieved by conventional techniques such as, for example, adsorption, covalent bonding or cross-linking, or a combination of these techniques, e.g. adsorption of a chemical with one or more functional groups followed by covalent bonding or cross-linking of the reagent. In general, suitable reagent immobilisation techniques are known in the art, for example some of those described in Chapter 4 of "lmmobilised Enzymes in Analytical and Clinical Chemistry", ed. Carr P.W. and Bowers L.D. (Wiley, New York, (1980)).

The particles will conveniently be substantially spherical in shape, although other shapes may prove advantageous in some circumstances.

Thus, according to a yet further feature of the present invention, we provide a method of preparing colloidal magnetic particles having a reagent immobilised thereon as herein before described, which comprises contacting the particles with the desired reagent, optionally in the presence of one or more binding agents.

This method can be carried out without preliminary formation of a non-magnetic polymeric matrix or coating to the particles.

The magnetic particles may conveniently be stored in a stable form in the presence of a surfactant prior to immobilisation of the reagent. It may be necessary temporarily to destabilise the particles by removing the surfactant to allow immobilisation of the reagent. Destabilisation may, for example, be carried out by washing in an organic solvent such as, for example, acetone or by boiling in distilled water, optionally in the presence of an acid such as, for example, tartaric acid.

The immobilisation of reagent onto the particles is preferably carried out in the presence of a pH buffer.

After immobilisation of the reagent, the particles may be washed to remove or desorb unbound or incompletely bound reagent molecules. The pH of the medium may be varied from the buffered pH in order to facilitate this removal or desorption.

The medium may if necessary subsequently be restabilised into colloidal form by sonication.

Suitable surfactants (e.g. T,ween-20) may be present. In general, the particles will be stored in an aqueous medium.

According to a still further feature of the present invention, we provide colloidal magnetic particles having a reagent immobilised thereon when prepared by a method as described.

The invention will be particularly described hereinafter with reference to an antigen as the ligand. However the invention is not to be taken as being limited to assays of antigens.

Examples of ligands which may be assayed by the method of the invention are given in Table I below, together with an indication of a suitable specific binding partner in each instance.

Suitable reagents which may be immobilised on the colloidal magnetic particles in these assays are not listed in Table I; however, such reagents would be readily apparent to the skilled reader from known assay methods.

Table I Ligand Specific Binding Partner antigen specific antibody antibody antigen hormone hormone receptor hormone receptor hormone polynucleotide complementary polynucleotide strand strand avidin biotin biotin avidin protein A immunoglobulin immunoglobulin protein A lectins specific carbohydrate specific carbohydrate lectins of lectins It will thus be seen that the method of the invention has very broad applicability, but of particular interest are assays of: hormones, including peptide hormones (eg thyroid stimulating hormone (TSH), lutenising hormone (LH), follicle stimulating hormone (FSH), insulin, prolactin and human chorionic gonadotrophin (HCG)) or non-peptide hormones (eg steroid hormones such as cortisol, estradiol, progesterone, testosterone or thyroid hormones such as thyroxine or triiodothyronine), proteins (including carcinoembryonic antigen (CEA) and alphafeto protein (AFP)), drugs (eg digoxin), sugars, toxins and vitamins.

The term "antigen" as used herein will be understood to include both permanently antigenic species (for example, proteins, bacteria, bacteria fragments, cells, cell fragments and viruses) and haptens which may be rendered antigenic under suitable conditions.

It will be understood that the term "antibody" used herein includes within its scope a) any of the various classes or sub-classes of immuno-globulin, e.g. IgG, IgM, derived from any of the animals conventionally used, e.g. sheep, rabbits, goats or mice, b) monoclonal antibodies, c) intact molecules or "fragments" of antibodies, monoclonal or polyclonal, the fragments being those which contain the binding region of the antibody, i.e. fragments devoid of the Fc portion (e.g., Fab, Fab', F(abt)2) or the so-called "half-molecule" fragments obtained by reductive cleavage of the disulphide bonds connecting the heavy chain components in the intact antibody.

In methods of assaying antigens according to the present invention the reagent immobilised on the colloidal magnetic particles may for example be selected from reagents which are capable of binding selectively to the antigen ligand and to a ligand analogue (in the case of competitive assays), or to the antigen ligand or antibodies to the said antigen.

Thus, for example, assays may be carried out in which the immobilised reagent comprises an antibody to the antigen, the antigen sample and a labelled antigen in known amount being added together to compete for binding to the antibody, or the immobilised reagent may comprise an antibody to the primary antibpdy (i.e. it may comprise a second antibody) and be added at the end of the assay to effect magnetic separation. Alternatively, the antigen sample may be added together with a labelled specific antibody (the labelled and immobilised antibodies being directed against different, roomly-spaced determinants of the antigen) so that a proportion of the the labelled antibody will become immobilised, via the antigen.

In a modification of this second method, an additional "sandwich" technique may be employed, in which the immobilised reagent is an antibody which is directed to a third antibody reagent comprising an antibody or a complex of antibodies (e.g. cross-linked or hydridised) to the antigen under assay, the label thereby becoming immobilised via the antigen and the antibody reagent.

Alternatively, the immobilised reagent may interact specifically with a reagent Z provided on this third antibody. Reagent Z may, for example, be such as to render the third antibody antigenic to the immobilised reagent, which in that case will be an antibody reagent raised to reagent Z. In a preferred embodiment, reagent Z will be a hapten, but antigenic substances such as, for example, proteins, cells, virus particles or membranes may also be employed.

When the immobilised reagent is an antibody to reagent Z, Z may be, for example, fluorescein isothiocyanate (FITC), rhodamine isothiocyanate, 2,4-dinitrofluorobenzene, phenyl isothiocyanate or dansyl chloride. The antiserum immobilised on the magnetic particles is an antibody reagent raised to reagent Z and may be prepared in a conventional manner, for example by immunising sheep with FITC conjugated to keyhole limpet haemocyanin. Coupling of the antiserum to the magnetic particles may be effected for example by the adsorption of glutaraldehyde to the particles followed by covalent linking of the antibody or by the reverse, adsorption of antibody to the particles followed by cross-linking. Reagent Z may be incorporated into the antibody by direct reaction, followed by gel filtration, or indirectly using bifunctional reagents or spacer groups.The imobilised reagent and reagent Z may also, for example, be a specific binding protein and the corresponding ligand such as, for example, avidin and biotin which constitute a very rapid, high affinity binding system. The use of such methods of binding offers the great advantage that the reaction can be made very rapid and complete.

It is possible, in certain circumstances, to intwduce reagent Z into an antibody at multiple sites, thus enhancing its reactivity with the magnetic particles.

In a further method, the immobilised reagent may be capable of binding specfically to uncomplexed labelled antibody, in which case removal of the particles from the assay medium will remove the uncomplexed labelled antibody. In such a case, the immobilised reagent may, for example, comprise a hapten, a reagent capable of binding to a reagent Z provided on a hapten or protein which will bind to the Fc portion of the antibody.

Conveniently, the label used may be an anaiytically indicatable atom or group such as a radioacive atom or group e.g. 1251, or other methods of labelling known in the art may be used, e.g. fluorimetric labelling or enzyme labelling, which may be direct or indirect, covalent or noncovalent.

The reagent may be immobilised on the colloidal magnetic particles by generally nonimmunochemical binding (e.g. adsorption, cross-linking, covalent bonding or a combination of these techniques).

As mentioned above, the use of the colloidal magnetic particles according to the invention provides methods of assay which employ simpler reagents and more convenient apparatus than known methods.

In a still further feature of the present invention, we provide kits of reagents for carrying out the assays of the invention. Suitable kits may comprise a suspension of colloidal magnetic particles according to the invention having immobilised thereon a suitable reagent for the assay to be performed, and optionally other components (e.g. labelled species).

The following non-limiting Examples are intended to illustrate the invention more fully: Example 1 Radioimmunoassay for Cortisol A. Preparation of anti-cortisol colloidal magnetic microparticles 3 ml of a colloidal suspension (0.5 g/ml) of magnetic particles (approximately 30 nm in diameter) in surfactant solution was destabilised by boiling in 1 50 ml distilled water for 5 hours.

The particles were then washed six times with 100 ml distilled water each time and then adjusted to a volume of 10 ml in sodium phosphate buffer 0.05M, pH7.4.

30 yl of a 50% solution of glutaraldehyde was added to 3 ml of the destabilised particle suspension and mixed for 30 minutes at room temperature (20"C). 0.2 ml of sheep anti-cortisol antiserum was then added and the suspension mixed for a further 60 minutes at room temperature. The particles were then washed twice with 10 ml sodium bicarbonate buffer 0.1 M, pH 8.6, twice with sodium acetate buffer 0.1 M, pH 4.0, then four times with sodium phosphate buffer 0.05M, pH 7.4, containing 0.25% (w/v) bovine serium albumin, 0.25% (v/v) Tween-20 and 0.1 % (w/v) sodium azide. The suspension was sonicated for 30 seconds and then diluted in the same buffer to give a concentration of approximately 5.6 mg/ml.

B. Radioimmunoassay for cortisol Duplicate samples were run in which 100 ILl of '251-labelled cortisol reagent (Serono Diagnostics, code 32392) was added to 50 pal of specimen (serum), followed by the addition of 100 IL1 of anti-cortisol colloidal magnetic microparticles. After mixing, the tubes were incubated for 30 minutes without further agitation. The magnetic microparticles were separated magnetically, the liquid removed by decantation and the resultant pellet counted for bound '251-labelled cortisol. An example dose response curve obtained using pre-calibrated standards (Serono Diagnostics, code 52392) is shown in Fig. 1. Counts per minute bound are plotted on the vertical axis and cortisol concentration on the horizontal axis.

Example 2 Immunoradiometric Assay for THS A. Preparation of anti-fluorescein isothiocyanate colloidal magnetic microparticles 3 ml of a colloidal suspension (0.5 g/ml) of magnetic particles (approximately 30 nm in diameter) in surfactant solution was destabilised by boiling in 1 50 ml distilled water for 5 hours.

The particles were then washed six times with 100 ml distilled water each time and then adjusted to a volume of 18 ml in sodium phosphate buffer (0.05M, pH 7.4).

100 it1 of a 25% solution of glutaraldehyde was added to 2.4 ml of the destablised particle suspension and mixed at room temperature for 60 minutes. The particles were then washed twice with 10 ml sodium phosphate buffer (0.05M, pH 7.4) and the volume adjusted to 2.5 ml.

250 yl of sheep anti-FITC antiserum was added and the suspension mixed for 1 8 hours at 4"C.

The suspension was washed twice with 10 ml sodium bicarbonate buffer (0.1 M, pH 8.6), twice with 10 ml sodium acetate buffer (0.1 M, pH 4.0), twice with 10 ml sodium phosphate buffer (0.05M, pH 7.4) and then finally twice with 10 ml sodium phosphate buffer (0.05M, pH 7.4), containing 0.25% (w/v) bovine serum albumin, 0.1% (w/v) sodium azide, 0.8% (w/v) Methocel E50 and 0.25% (v/v) Titon X-100, before being adjusted to a concentration of 5 mg/ml in the same buffer.

B. Immunoradiometric Assay for TSH Duplicate samples were run in which 200 jul of specimen (serum) was mixed with 100 it1 of a combined reagent containing '251- and FlTC-labelled monoclonal antibodies to TSH (Serono Diagnostics, code 32732) and incubated for 2 hours at room temperature. 200 yl of anti-FITC magnetic microparticles prepared as described above was added and incubated for 10 minutes after mixing. The solid phase particles were separated magnetically, the liquid removed by decantation and the particles were washed by the addition of 0.5 ml sodium phosphate buffer 0.01M, pH 7.4) containing 0.9% (w/v) sodium chloride and 0.1% (w/v) sodium azide.

Following this, the particles were again separated magnetically, the wash liquid removed by decantation and the resultant pellet counted for bound l251-labeled antibody. An example dose response curve obtained using serum based standards (Serono Diagnostics, code 42732) calibrated against the 2nd International Reference Preparation of human TSH (80/558) is shown in Fig. 2. Counts per min bound are plotted along the vertical axis and micro international units human TSH/ml are plotted along the horizontal axis

Claims (23)

1. A method of assaying a ligand in a sample which includes the steps of contacting the sample with homogeneous colloidal magnetic particles having a reagent immobilised thereon, whereby an immobilised complex is formed on the said colloidal magnetic particles, and subsequently separating the colloidal magnetic particles and immobilised complex from the assay medium.

2. A method as claimed in claim 1 wherein the said colloidal magnetic particles are in the size range 10 nm to 800 nm.

3. A method as claimed in claim 2 wherein the said colloidal magnetic particles are in the size range 30 nm to 400 nm.

4. A method as claimed in any of claims 1 to 3 wherein the said colloidal magnetic particles are of specific gravity up to 8.

5. A method as claimed in claim 4 wherein the said colloidal magnetic particles are of specific gravity between 2 and 6.

6. A method as claimed in any one of the preceding claims wherein the said colloidal magnetic particles comprise magnetite or haematite.

7. A method as claimed in any one of the preceding claims wherein a surfactant is present in the assay medium.

8. A method of assaying a ligand in a sample substantially as herein described.

9. A method of assaying a ligand in a sample substantially as herein described in Example 1 or Exampe 2.

10. Colloidal magnetic particles having a reagent immobilised thereon for use in a method of assay as claimed in any one of the preceding claims.

11. A method of preparing colloidal magnetic particles as claimed in claim 10 which comprises contacting magnetic particles with the desired reagent.

1 2. A method as claimed in claim 11 wherein the said magnetic particles are contacted with the desired reagent in the presence of one or more binding agents.

1 3. A method as claimed in claim 11 or claim 1 2 wherein magnetic particles are employed which have been stored in a stable colloidal form in the presence of a surfactant.

1 4. A method as claimed in claim 1 3 wherein said magnetic particles prior to contact with the desired reagent are destabilised by washing in an organic solvent or by boiling in distilled water.

1 5. A method as claimed in claim 14 wherein said magnetic particles are destabilised in the presence of an acid.

1 6. A method as claimed in any one of claims 11 to 1 5 wherein non-colloidal magnetic particles having an immobilized reagent are initially obtained and are subsequently converted to colloidal form by sonication.

17. A method as claimed in claim 1 6 wherein sonication is carried out in the presence of a surfactant.

1 8. A method as claimed in any one of claims 11 to 1 7 wherein the said magnetic particles are contacted with the desired reagent in the presence of a pH buffer.

1 9. A method as claimed in any one of claims 11 to 18 wherein following immobilisation of the reagent, unbound or incompletely bound reagent molecules are removed.

20. A method of preparing coiloidal magnetic particles having a reagent immobilised thereon substantially as herein described.

21. A method of preparing colloidal magnetic particles having a reagent immobilised thereon substantially as herein described in Example 1 or Example 2.

22. Colloidal magnetic particles having a reagent immobilised thereon whenever prepared by a method as claimed in any one of claims 11 to 21.

23. A kit of reagents for carrying out a method as claimed in any one of claims 1 to 9.