CH648127A5 - Reagent for the selective adsorption of components in a liquid, and use and preparation thereof - Google Patents

Description

The invention relates to a reagent for the determination of a component of a liquid sample by means of selective adsorption of one or more components contained in the liquid.

As is known, antigens and antibodies (as well as other binding proteins) can be determined with the aid of a competitive binding reaction using a labeled reagent, the determination being carried out by measuring the amount of labeling substance either bound to a complex or remaining unbound in solution. An example of such a method is the known radioimmunoassay technique, in which a ra

dioactive labeling is used. Analog techniques using other labeling substances such as enzymes and fluorophores are also known.

In these methods, it is necessary either to separate the complexes formed or the free, unbound, labeled reactant from the rest of the reaction mixture in order to measure the amount of labeled substance in one of the fractions. This separation step is rarely easy to carry out and is a source of error in such determinations.

In order to avoid or at least reduce this difficulty, the publication DE-OS 27 10 438 dated November 3, 1977 proposed the use of magnetically attractable particles in immunoanalyses which have a reactant bound to them on their peripheral surface. The particles can (together with the reaction product between the reactant and a labeled compound of the reaction mixture) be clearly and simply separated from the rest of the reaction mixture and the labeled substance in this residue or on the particles can then be determined.

A limitation of this method is that the only reagents that can be coupled to the magnetic particles are so far only those substances that can be covalently bound to the polymer matrix of the particles. So far, the particles consist of magnetically attractable material that is embedded in a matrix of a polymer, such as cellulose, the reagent being covalently bound to the cellulose. It has not previously been possible to use reagents that are not bound to the magnetic particles in this way. Among the reagents that could not be used in this way are inert, solid adsorbents such as activated carbon and the like; however, these materials are extremely useful for immunoassays, since they can be used for the adsorption of free, unbound labeled substance from the reaction mixture and can then be separated off from the reaction mixture in the form of solids.

According to the invention, these problems are solved by a reagent for the determination of a constituent of a liquid sample by means of selective adsorption of one or more components contained in the liquid, with particles from a continuous phase made of a water-insoluble polymer matrix, which is a magnetically attractable material and discrete, solid particles contains an adsorbent material that is intended for selective adsorption of at least one of the components contained in the liquid. Thus, the use of magnetically attractable particles (with their inherent advantages in separation) can be combined with the use of adsorbents such as activated carbon and the like (with the inherent advantages in selective adsorption of reactants) by using a magnetically attractable, particulate reagent for use in immunoassays is formed. In contrast to the known reagents, in which a material is covalently bound to a polymer base, the adsorbent is embedded in the polymer matrix. In the following, the magnetically attractable particulate materials, which consist of discrete, magnetically attractable particles and discrete particles of adsorbent, which are distributed in a polymer matrix, are simply referred to as "particles".

The invention furthermore relates to the use of the reagent for determining a constituent in a liquid sample, the sample being mixed with a substance,

3 648127

which reacts with the constituent to be determined contained therein, forms a reaction mixture, adds the reagent for the selective adsorption of a component of the reaction mixture, magnetically separates the reagent from the reaction mixture and analyzes the constituent to be determined by analyzing the separated particulate reagent or the separated reaction mixture determined.

In a preferred use for carrying out the immunoassay of two or more liquid samples to determine different constituents of each of these samples using the continuous flow method, the procedure is that a reaction mixture is formed from each of the liquid samples with a substance which corresponds to those to be determined Constituents of samples i5 reacted, allowing the reaction mixtures of each of the liquid samples to flow sequentially and subdivided from one another along a line, introducing a particulate reagent according to the invention into each of the reaction mixtures while flowing along the line, by the adsorbent being able to dissolve bound or free fraction in each of the reaction mixtures, magnetically separate the reagent from each of the reaction mixtures, and analyze the separated reagent or reaction mixture to determine the component to be analyzed in each of the 25 liquid samples iert.

The matrix can consist of a large number of different materials, such as natural and synthetic polymers, such as polyacrylamide, cellulose, cellulose ester, polyamide, polycarbonate, polyvinyl resins, methyl 30 methacrylates, polystyrene, polystyrene-acrylonitrile, epoxy resins, melamine-formaldehyde, Phenol-formaldehyde, urea-formaldehyde, polyester, polyacrylonitrile, polyacrolein, poly-acetal, cross-linked proteins and the like.

The matrix serves to keep the magnetic and absorbing particles in a uniform particle shape, without one of the components being chemically bound to the matrix. Since some, if not most, of the adsorbent particles are completely enclosed within the matrix and are not partially exposed on the surface of the matrix, the matrix is preferably porous to such an extent that it is sufficient to allow the reaction mixture to occur. The porosity of the matrix can advantageously be controlled in order to allow only selected dissolved components to enter, for example small antigens, but 45 non-large antibodies; this so-called gel filtration effect is itself known. Polyacrylamide is a particularly suitable polymer for the production of matrices with a selected pore size.

Suitable magnetically attractable materials are those described in the above-mentioned publication DE-OS 27 10 438.

The type of adsorbent can vary within wide limits depending on the end use of the particles according to the invention. Activated carbon is most preferred, but other materials such as talc, ion exchange resins, bleaching earth, silicon dioxide, dioxides of zirconium, aluminum and titanium, porous glass, zeolites, finely divided natural and synthetic polymers, polymerized first or second antibodies or can also be used Enzymes, cell 60 surface antigens and receptors, subcellular particles, viruses and bacterial cells can be used.

The action of the adsorbent is to adsorb or chemically bind part of the reaction mixture used for the immunoanalysis so that this part of the mixture with the particles of the invention is removed. For example, when analyzing a specific antigen, a reaction mixture is formed that contains the sample, an amount of labeled antigen and a

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contains an antibody. Complexes are used in the mixture for analyzing a liquid with regard to a

between the antibody and the antigen and between the tuning component is characterized in that the antibody and the labeled antigen are formed. The one

Mixtures are then added to particles according to the invention (a) a mixture of the liquid sample and a reagent

sets, and these can, for example, form the free, unbound 5 that selectively reacts with the component to be determined in the antigen (both labeled and unlabeled) of liquid;

adsorb. Separation of the particles by a magnetic (b) the mixture flows along a line;

see case causes only the complexes to remain in solution- (c) particles according to the invention remain in the mixture, after which either the solution or the ones which contain particles as a particulate adsorbent contain a material on their content of labeled antigen io that a component of the implementation mixture can be examined se-hin. adsorbed;

It is a considerable advantage of the present invention (d) to magnetically trap the particles in the conduit so that the described particles are nonspecific rather than against the flow, thereby separating them from the immunospecific and therefore separating a wide range of application / reaction mixtures ; and own area. For example, a particle adsorbs the constituent to be determined by analyzing the particles according to the invention, which contains particles separated from activated carbon as adsorbent and / or the separated urea, many different small antigens. Determined in this mixture.

The particles according to the invention can be used for immune purposes. In a preferred embodiment of this method

analyzes of many different antigens are used, rens the reagent is a predetermined amount of a sub-

This is of great advantage, since it eliminates the need to stamp the component to be determined with training.

is avoided, a manure of a complex reacts for each different immunoassay, the particles being provided according to another reagent on the magnetic particles, the invention deals with the excess reagent in which it was previously necessary. The field of application of the mixture binds and either the separated liquid particle according to the invention naturally varies with the separated or the separated particle to determine the interest

used adsorbent, but for materials such as the active ingredient of the sample.

charcoal and other similar solid adsorbents, it can be The particles of the reagent can be made

be highly significant. by using the particulate adsorbent and

The particles are particularly suitable for radioimmunoparticle-shaped, magnetically attractable material in an assay. That for a specific immunoanalysis or other polymer or in a mixture that forms a polymer

Analysis suitable particulate adsorbent is the 30 det, dispersed. In the case of a polyacrylamide matrix

Those skilled in the art are either known or can routinely use the two particulate materials, for example with a

experiments are determined. Some examples of certain nem or more liquid acrylamide monomers and a

Uses are as follows: activated carbon for the analyzer selected amount of a crosslinking agent, such as

steroids, thyroid hormones T4 and T3, ACTH, for example methylene-bis- (acrylamide).

giotensin-II, insulin and glucagon, human wax- 35 The polymerization is carried out using ammonium

tumors hormone, vitamin B12, folate and others; Talcum or umpersulphate initiated and the solid mass obtained dry

Silicon dioxide in analyzes of ACTH, growth hormone, net, and ground to the desired particle size

Parathroid hormone, insulin and calcitonin as well as vitamin seven. This can vary within wide limits, but is for

BI2; Anion exchange resins in the analysis of insulin and most immune analyzes have a particle size in the range

Growth hormone, digoxin, cyclic AMP, cyclic - »o from 1 to 20 jxm generally satisfactory.

GMP, insulin, angiotensin-I and thyroxine (T4); polymeric The weight ratio of the particulate material in the first or second antibodies for adsorption of anti-matrix can vary within wide limits as desired,

genes or first antibodies or for the production of Thy- The invention is based below on examples of roxin and cortisol, etc. free sera; Cell surface antigens explained in more detail.

and receptors and subcellular particles for separation «

purposes and bacterial cells as well as viruses for the detection of antibacterial and antiviral antibodies in a sample. Example I

The second antibodies mentioned are those which immuno-produce particles for a reagent according to the invention.

logically with a first antibody instead of a tapping containing activated carbon.

or protein reacted. Cell surface antigens and -re- so A stock suspension of 5 g activated carbon of the zeptors was described in «Hudson and Hay, Practical Im- name NORIT OL (BDH) in 200 ml of an analysis

Munology, Blackwell Scientific Publications », page 41 ff. Diluent (0.05 m phosphate buffer with pH 7.4)

The particles can be produced for individual (manual) analyzes. 152 ml of the stock suspension were mixed with 3.8 g or in automated systems after the continuous acrylamide, 0.2 g N, N-methylenebisacrylamide, 3.8 g iron (III)

Flow principle are used, as they are added, for example, in 55 oxide (Fe304, Fisons and 1 ml of N, N, N ', N'-tetramethyldiamine from the above-mentioned publication DE-OS 27 10 438.

are written. Particularly in the systems of continuous 1.2 g of ammonium persulphate, the specific weight of the analytical diluent had to be converted into that of the particles according to the invention with vigorous stirring in 2 ml of the flow-through principle. This was followed by an exothermic reaction, which came close to the formation of the mixture, so that no eo of 13.2 g of a homogeneous gel (dry yield) resulted in inadmissible floating or settling and the gel was broken up into narrow pieces of about 0.25 cm3 cut in suspension in the reaction mixture and dried for 24 hours in a vacuum desiccator, wherever possible. In general, specific weights are satisfactory after the hard, dry granules obtained in a Kaf of about 1.4 to 3.2. An exemplary embodiment of a grinding mill (Braun) comminuted and dispersed at 2.0 g each in 30 ml water game of the present invention relates to a use. Thereafter, the suspension was used for 20 minutes in the reagent for immunoanalysis, as described in the McCrone fine grinding device mentioned above and in which the patent specification was used, in which particles of the gel were used after washing four times with the dilution type described in the analysis. This preferred crosslinking agent was resuspended in 2 ml portions in 30 ml. 0.4 ml of

This suspension per test tube was used for the separation step.

Example 2

Antibody dilution curves (Fig. 1)

A series of dilutions of antiserum was made to double the previous dilution, and aliquots of 50 µl were duplicated at 1: 400 to 1: 25600 dilutions into tubes. A solution of 125I-labeled digoxin was prepared by diluting a stock solution in 1: 8 analytical diluent. 100 nl of the digoxin labeled with 125 I was introduced into each tube. Thereafter, 200 μl of digoxin-free plasma was introduced into each tube, after which the tubes were incubated overnight.

Thereafter, 400 µl of the particle suspension prepared according to Example 1 was added to each tube. After 10 minutes of incubation, 2 ml of analytical diluent was added and the particles were allowed to settle quickly by placing the tubes on a multipolar ferrite plate magnet (Magnet Applications Ltd.). The supernatant became aspirated and the particles washed once with 2 ml analytical diluent and then the 125I activity measured in a gamma counter The dilution curve obtained is shown in Fig. 1 together with that obtained using the conventional method with free activated carbon that the new material according to the invention has similar adsorption properties as free activated carbon.

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Example 3

Standard curves

Antiserum was diluted with analytical diluent (1: 6400). Standards of digoxin (0.5.1.0.2.0.3.0, 5 4.0 and 6.0 ng / ml "pooled" serum) were introduced into test tubes at 200 fai each, followed by 100 in each tube jj.1 digoxin labeled with 125I were added. The whole was mixed with 50 | xl antiserum solution. The tubes were incubated at room temperature for 30 min, after which 400 μl of the particle suspension prepared according to Example 1 were added. After 10 minutes, the particulate material was separated in a magnetic field as described in Example 2. The standard curve obtained, together with that obtained using the conventional Abis separation technique with free activated carbon, is shown in Fig. 2, and it is found that the new material has analog adsorption properties as the old one.

20 Example 4

The utility of the particulate material was confirmed by the following experiment.

Sera from 25 patients were analyzed twice using both the conventional method and the method according to the invention. An acceptable correlation was achieved between the two methods (r = 0.959, Fig. 3). Statistically, the slope of the line was not significantly different from 1.0 and the intercept was not significantly different from 0.0.

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Claims (14)

648127 PATENT CLAIMS 1. reagent for the determination of a constituent of a liquid sample by means of selective adsorption of one or more components contained in the liquid, with particles from a continuous phase from a water-insoluble polymer matrix which contains a magnetically attractable material and discrete, solid particles from an adsorbing material, which is intended for selective adsorption of at least one of the components contained in the liquid. 2. Reagent according to claim 1, characterized in that the polymer matrix consists of an acrylic polymer. 3. Reagent according to claim 1 or 2, characterized in that it has a specific weight of 1.4 to 3.2. 4. Reagent according to one of the preceding claims, characterized in that the particles have a size of 1 to 20 (im. 5. Reagent according to one of the preceding claims, characterized in that the particles are suspended in water. 6. Reagent according to one of the preceding claims, characterized in that the solid particles made of an adsorbing material made of activated carbon, talc, ion exchange resins, bleaching earth, silicon dioxide, oxides of zircon, aluminum or titanium, porous glass, zeolites, natural or synthetic polymers , polymerized first or second antibodies or enzymes, cell surface antigens or receptors, subcellular particles, viruses or bacterial cells. 7. Use of the reagent according to one of claims 1 to 4 and 6 for determining a component of a liquid sample, characterized in that a reaction mixture is formed from the sample with a substance which reacts with the component to be determined which it contains, adding the reagent for the selective adsorption of a component of the reaction mixture, magnetically separating the reagent from the reaction mixture and determining the constituent to be determined by analyzing the separated particulate reagent or the separated reaction mixture. 8. Use according to claim 7, characterized in that a) a mixture is formed from the liquid sample and a substance which reacts with the constituent of the sample to be determined, b) the mixture flows along a line, c) the mixture is mixed with the reagent, which consists of a number of particles of a material which selectively adsorbs a component from the reaction mixture, d) magnetically captures the particles in the line in order to hold them against the flow and thereby separate them from the reaction mixture, and e) the constituent to be determined is determined by analysis of the separated particles and / or the separated reaction mixture. 9. Use according to claim 7 or 8, characterized in that the reaction mixture chosen is one which contains a known amount of the substance and in which the substance reacts with the constituent to be determined to form a complex and the reagent with the excess of not reacted substance enters into a bond. 10. Use according to one of claims 7 to 9, characterized in that one selects a substance that bears a radioactive label. 11. Use according to one of claims 7 to 10 in the immunoassay according to the principle of continuous flow, characterized in that a mixture is formed from the sample and a substance which reacts with the constituent to be determined, the mixture along a line Line flows into the flowing mixture, the reagent introduces, wherein the reagent selectively adsorbs either the bound or the free fraction of the mixture, magnetically separates the reagent from the mixture while flowing along the line and the separated reagent or the separated mixture Determination of the component analyzed. 12. Use according to claim 7 in immunoassay, characterized in that a) a mixture of a liquid phase consisting of a 15 liquid sample and at least one reagent, and forms a solid phase consisting of a reagent according to one of claims 1 to 4, b) the mixture is reacted, thereby forming a reaction mixture which is at least as a constituent 2o contains a reaction product and some unreacted reagent and unreacted sample, at least one of the constituents being adsorbed on the reagent and the specific weight of the reagent being close to that of the reaction mixture, so that the reagent remains in suspension in the reaction mixture, c) magnetically separating the solid phase with the component adsorbed on it from the reaction mixture, and d) either the component adsorbed on the separated solid phase or one in the reaction mixture 3o remaining component determined. 13. Use of the reagent according to one of claims 1 to 4 and 6 for immunoanalysis of two or more liquid samples for determining different constituents of each of these samples, characterized in that 35 a reaction mixture is formed from each of the liquid samples with a substance which reacts with the constituent to be determined in them, the reaction mixture of each of the liquid samples is passed in succession and separately along a line, into each of the reaction mixtures during the flow along the line the reagent introduces by the adsorbent being able to dissolve the bound or free fraction in each of the reaction mixtures, magnetically separate the reagent from each of the reaction mixtures, and analyze the separated reagent or reaction mixture to determine the component of interest in each of the liquid samples . 14. A method for producing a reagent according to claim 1, characterized in that a magnetically attractable material and discrete, solid particles of such an adsorbent material are uniformly dispersed in a polymeric matrix, the matrix is cured to form a hard solid and the solid to required particle size is ground. 55