CH658728A5 - Process for the production of particulate reagents for immunoassays, and these reagents and their use - Google Patents

Description

The invention relates to a process for the production of particulate, magnetically attractable reagents for immunoassays, and to reagents and immunological determinations produced in this way using these reagents.

As is known, antigens (and haptens), antibodies (and other binding proteins) and other substances, such as drugs, in liquids of, for example, biological origin, can be determined by a competitive binding reaction using a labeled reagent, the determination (the assay) thereby effecting is that one either measures the amount of label that is bound in a complex or that that is unbound in solution. An example of such a method is the known radioimmunoassay, in which a radioactive label is used. Analog techniques are known using other labels such as enzymes and fluorophores.

These procedures generally require either the complexes formed or the free, unbound, labeled reagent to be separated from the rest of the reaction mixture to determine the amount of label in either part. This separation is rarely easy to perform and is therefore a source of error in these assays.

In order to avoid or at least reduce these difficulties, a method according to BE-PS 852 327 uses magnetically attractable particles which have a reagent covalently bound to them on their outer surface. The particles, together with the reaction product between the reagent and a labeled component from the reaction mixture, can be separated cleanly and easily from the rest of the reaction mixture, after which the labeling in this residue or on the particles themselves can be measured.

A limitation or disadvantage of this method, as was previously known, lies in the fact that the production of the magnetic particles carrying the reagent is often laborious and time-consuming. The particles consist of magnetically attractable material embedded in a polymer matrix, for which cellulose is usually used, and in order to bind a particulate reagent to it, the cellulose must first be activated and then reacted with the reagent, normally a bifunctional bridging group is used. The activated cellulose particles which occur as an intermediate product cannot be kept indefinitely, so that the two production steps (activation and subsequent implementation) have to be carried out anew each time.

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A way to avoid these difficulties has now been found. In particular, a storage-stable, reactive magnetic material has been found to which certain reagents can be bound in one step; this material represents an intermediate product of the method according to the invention.

The invention relates to a method for producing a particulate. Magnetically attractable reagent for use in immunoassays, which is characterized. that a) one or more monomers in the presence of magnetically attractable solid particles with immediate formation of a synthetic, water-insoluble polymer matrix. in which the solid particles mentioned are uniformly embedded, polymerized in bulk in the presence of a catalyst and / or initiator, with a 1) the monomer or monomers being selected such that the matrix formed is aldehyde, keto, cyanate, iso- cyanate, thiocyanate, isothiocyanate, halogen-nitro-aromatic, s-triazine, aromatic sulfonyl chloride, isoxazole, aziridine, imino, imide, carboxyl, epoxy or acid anhydride groups or several types of these Contains groups that are free so that they can directly couple with a reagent that can react with them so that the reagent is bound to the matrix, or a2) generates the matrix by polymerization via the ethylenic double bond of monomer which is the or each monomer of one of the monomers acrylonitrile, methacrylonitrile, acryloyl chloride and methacryloyl chloride,

b) after the polymerization, the polymer matrix formed is comminuted into particles, and c) the particulate material obtained is brought into contact with a reagent which, based on the monomer selection according to a1), contains directly in the free coupling groups or directly in the polymer matrix by the monomer polymerization according to a2) introduced into the polymer matrix, having hetero atoms functional groups, and this reaction can take place in order to bind the reagent directly to the polymer matrix.

The invention further relates to the particulate magnetically attractable reagent produced in this way.

The invention finally relates to the use of a particulate reagent according to the invention in an immunoassay for determining a component in a liquid, which is characterized in that the liquid is brought into contact with the reagent particles according to the invention and then these particles are magnetically separated from the liquid .

The individual particles of the reagent according to the invention are referred to below as “reagent particles according to the invention”.

Of the various possible free groups in the polymer matrix formed, the aldehyde and keto groups are preferred.

According to the invention, one or more of the monomers (in the presence of magnetically attractable solid material) are polymerized in this way. that a solid polymer is immediately produced. which can be finely crushed after suitable separation from the reaction mixture and necessary washing. Such polymerization is carried out in the absence of a solvent (i.e., bulk polymerization j using a catalyst or initiator. For example, in the case of acrolein, the monomeric acrolein is mixed with tetramethylene diamine and polymerization rapidly occurs to form a solid product.

In the case of acrylonitrile, ammonium persulfate is also added to accelerate the reaction.

Furthermore, it is preferred to prepare the polymer matrix by radical polymerization of acrolein in the presence of tetramethylene diamine. If acrolein is polymerized in the presence of FeiCh in this way, a homogeneous mass of bulk polymer and Fe.iO-i is easily formed. This mass can easily be ground into small particles.

The polymer matrix and in particular the free reactive groups in it are selected depending on what kind of reagent is to be coupled to them. In general, the reagent contains free amino or sulfhydryl groups, which is why a polymer matrix containing aldehyde groups is used in these cases. Reagents that contain free amino groups are, for example, proteins, such as antibodies and enzymes, as well as certain antigens, haptens, drugs and antibiotics. Examples of such substances are the thyroid hormones T »and Tj (thyroxine and triizothyronine), the tricyclic antidepressants (nortriptyline, desipramine and protriptylin), the aminoglycoside antibiotics (gentamicin, sisomycin, netilmicin, amikacin and streptomycin. N'eomycinin -mycin), absorbents with different affinity for the isolation or determination of antibodies and other binding proteins, as well as intermediates in the production of first antibodies in the solid phase, which are to be used for the determination of paired hapten (conjugate hapten).

A great advantage of the present invention is that the reagent can be bound to the particles which are obtained in step b) of the process according to the invention in one stage. For example, the reagent can simply be brought into contact with the particles in a medium of a suitable pH. Polymer matrices containing free aldehyde groups are "self-reactive" to free amino groups, and the reagent is thus reacted with the matrix and bound directly to it. The reagent particles formed in this way according to the invention are then ready for use. The self-reactivity of the particles obtained in step b) of the process according to the invention is a very great advantage, since it eliminates the need to use bridging groups (prior art); the overall result is a simpler, faster and more effective process.

The reagent particles according to the invention can have any size, but in general it is expedient that they are 1 to 20 µm in size. Particularly when used in continuous flow processes, the specific weight of the particles should preferably be about 1.4 to 3.2 in order to avoid excessive floating or settling of the particles in the flowing liquid reaction mixture.

The particles obtained in step b) of the process according to the invention (i.e. in the state before the reagent is bound to them) can be stored before they are used. As is clear to the expert. the particles must be kept away from substances with which they could react during the storage period. Alternatively, the solid mass obtained in step a) of the process according to the invention from self-reacting Pohmerisatma-trix containing the magnetic particles can be preserved, and if particles are needed, files of the mass can be comminuted so that they react with a reagent can be. Thus, the reagent parts according to the invention can be made simple and fresh immediately before they are used.

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The reagent particles according to the invention are useful in immunoassays for determining a component of interest in liquids. They can thus be used in manual or automatic continuous flow determinations such as those described in U.S. Patent No. 2,797,149. They can be used there, as described, for example, in BE-PS 852 327.

Such determinations include in particular an analytical method in which a reaction mixture is flowed along a line, which consists of a liquid phase with a liquid sample containing a component to be determined and a solid phase of reagent particles according to the invention that in the "mixture a reaction takes place, whereby a component of the reaction mixture reacts with the solid phase or is selectively adsorbed on it, that one magnetically traps the particles in the line in order to hold them against the flow and in this way the solid phase from the flowing liquid Phase to separate, and that downstream of it the component to be determined in the sample is determined by analysis of the separated solid or liquid phase.

A preferred embodiment of an analysis device for performing this method is described in BE-PS 852 327.

The exact manner in which the reagent particles act in immunoassays depends on the type of reagent, as will be apparent to those skilled in the art. For example, in an immunoassay for an antigen using a labeled antigen and an antibody or other binding protein, the antibody can be the reagent on the reagent particles. Under these circumstances, the reagent forms complexes with both labeled and unlabeled antigen and can be determined based on the label after removal from the reaction mixture, or the remaining reaction mixture can be determined based on the label.

In a preferred embodiment of the use according to the invention, the reaction mixture (as a second reagent) contains a certain amount of a labeled substance which reacts with the component to be determined to form a complex, and the reagent on the reagent particles according to the invention binds to it the excess from the second reagent, after which the separated mixture is analyzed to determine the component to be determined in the sample. In this method, the analysis is carried out either for the complex or for the unreacted excess of the second reagent, as appropriate.

The above examples are for illustration only and the versatility of the reagent particles according to the invention in various different gluing processes is clear to the person skilled in the art.

The following further examples also serve to illustrate the invention.

example 1

Production of particles containing polyacroline

2 g acrolein and 2 g magnetizable iron (11, HI) oxide (Fe.'Oi) were mixed vigorously at room temperature and the mixture was mixed with 100 (tetramethylene diamine) and stirring continued for about 6 to 7 min, after which polymerization occurred and a homogeneous solid product was obtained This product was ground in an electric coffee grinder (brown) to fine granules further grinding in a fine grinding device (McCrone)

for 30 min gave a stable, self-reacting product with a suitable particle size.

Example 2

Production of particles containing polyacrylonitrile

2.5 ml of acrylonitrile and 2 g of magnetically attractable iron (II, III) oxide were mixed vigorously at room temperature and the whole was mixed with 100 al of tetramethylene diamine. Then 1 ml of a 33% (weight / volume) ammonium persulfate solution was added. Polymerization occurred after about 7 minutes and a homogeneous solid product was obtained. This product was crushed to a particle size of less than 1 mm and air-dried for 72 h at room temperature. The product was further milled in a micro grinder (McCrone). after which stable, self-reacting particles were obtained.

Example 3

Coupling of anti-T-i serum (y-globulin fraction) to particles according to Example 1

1 g of the polyacrolein / FesCU particles according to Example 1 was washed four times with 20 ml of water and then twice with 0.1 M sodium carbonate / bicarbonate buffer of pH 9.0. A suspension of the particles in the buffer was brought to a volume of 14 ml. The coupling was carried out by adding 1 ml of anti-TA serum (y-globulin fraction) to the particle suspension and gently mixing the whole thing at 4 ° C. for 24 h. The particles were then allowed to settle on a multipole ferrite magnet and the supernatant liquid was removed. The reagent particles produced in this way were washed three times with the bicarbonate / carbonate buffer and then twice with 0.05 m phosphate buffer with a pH of 7.4. Finally, the volume of the suspension was brought to 20 ml with phosphate buffer. -

Example 4

Coupling of antidigoxin serum (y-globulin fraction) to particles according to Example 1

The same procedure as described in Example 3 was carried out, with the exception that 1 ml of antidigoxin serum (γ-globule infraction) was used instead of anti-T4 serum.

Example 5

Coupling of anti-HPL (Iactogenic hormone of the human placenta) serum (y-globulin fraction) to particles according to Example 1

As described in Example 3, the difference was that 1 ml of anti-HPL serum (y-globulin fraction) was used instead of the anti-Ti serum.

Example 6

Coupling of antidigoxin serum (y-globulin fraction) to particles according to Example 2

1 g of the particles according to Example 2 was washed four times with 20 ml of water each time and then twice with 0.1 M sodium carbonate / bicarbonate buffer of pH 9.0. The suspension of the particles in the buffer solution was brought to a volume of 14 ml. The coupling was carried out. by adding 1 ml of antidigoxin serum (y-globulin fraction) to the particle suspension and mixing gently at 24 ° C for 24 h. The reagent particles prepared in this way were then allowed to sit on a multipole ferrite magnet after which the supernatant was removed. The particles

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were washed three times with the bicarbonate / carbonate buffer and then twice with 0.05 m phosphate buffer with a pH of 7.4. Finally, the volume of the suspension was made up to 20 ml with phosphate buffer.

Example 7

Manual radioimmunoassay of T4 using the anti-T4 polyacrolein / Fe304 reagent particles

Antibody Dilution Curves: The diluent for all assays was 0.05 m pH 7.4 phosphate buffer. A series of dilutions was prepared from the suspension of the reagent particles: 5.2.5.1.25.0.625.0.312.0156 g of matrix per 100 ml of suspension. A T4-12SI indicator solution was then prepared so that 500 cps in 50 | il was achieved. 8-Aniline-1-naphthalenesulphonic acid was added to the indicator solution in a ratio of 8 ng / ml serum sample. The dilution curves were established by mixing the reagents in the following order and in the following amounts.

Maximum binding curve:

(I) Serum T4 free 50 (il

(II) T4-'- 5I indicator solution (in the diluent for the assay) 50 y. 1

(III) Anti-T4 reagent particle suspension (Example 3)

for the dilutions listed above 50 (il

Top standard binding curve:

(I) Upper standard in T4-free serum (400 nmol / 1) 50 p.1

(II) T4-i: 5I indicator solution 50 | il

(III) Anti-T4 reagent particle suspension (Example 3) 50 p.1

Non-specific binding curve:

(I) 20-fold concentration of the upper standard

(8 ii.Mol / 1) in T4-free serum 50 y. 1

(II) T4 - ': 5I indicator solution 50jil

(III) Anti-T4 reagent particle suspension (Example 3) 50 [il

The tubes were incubated at room temperature overnight. The solid phase particles were allowed to sit on a ferrite magnet and the supernatant solution was discarded. The particles were washed three times with the diluent intended for the assay and finally counted with a Wilj apparatus. The results are shown in Fig. 1.

In the attached figures 1, 2 and 3 the curve of the maximum binding is designated I, II means the curve of the upper standard binding and III is the curve of the non-specific binding.

(III) Antidigoxin reagent particle suspension

(Example 4) 50 p.1

Upper standard binding curve:

(I) Upper standard 8 nmol / 1 in plasma 200 (il 5 (II) digoxin-l25I indicator solution 50 | il

(III) Antidigoxin reagent suspension

(Example 4) 50 jxl

Non-specific binding curve:

10 (I) 20 times the upper standard concentration

(160 nmol / 1) in plasma 200 p.1

(II) Digoxin 125I indicator solution 50 p.1

(III) Antidigoxin reagent particle suspension

(Example 4) 50 µl

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The tubes were incubated at room temperature overnight. The solid phase particles were allowed to sit on a ferrite magnet and the supernatant was discarded. The particles were washed three times with the diluent used for the 20 assay and finally counted using a Wilj apparatus. The results are shown in Fig. 2.

Example 9

is a manual radioimmunoassay of HPL (human placenta lactogenic hormone) using the anti-HPL polyacrolein / Fe304 reagent particles

Antibody Dilution Curves: A dilution series was made from the antibody reagent suspension as follows, 5, 2.5, 1.25, 0.625, 0.313 g particles per 100 ml suspension. HPL-125I indicator solution was made to achieve 500 cps in 50 | il. The dilution curves were established by mixing the reagents in the following order and in the following amounts:

Maximum binding curve:

(I) men's serum 50 y. 1

(II) HPL-'25I indicator solution 50 jj.1 40 (III) anti-HPL reagent particle suspension at the dilutions indicated above 50p.l

Upper standard binding curve:

(I) Upper standard (12 (ig / ml) 50 p.1

45 (II) HPL-I25I indicator solution 50 p.1

(III) Anti-HPL reagent particle suspension 50 (j.1

Non-specific binding curve:

(I) 20 times the upper standard concentration (240 µg / ml) 50 years 1

50 (11) HPL-l2SI solution '50 p.1

(III) Anti-HPL reagent particle suspension 50 years 1

Example 8

Manual digoxin radioimmunoassay using the antidigoxin-polyacrolein / Fe304 reagent particles. Antibody dilution curves: A series of dilutions was made with the antibody reagent particle suspension as follows: 5, 2.5, 1.25, 0.625, 0.312, 0.156, 0.078 g particles per 100 ml suspension. A digoxin 1: 5I indicator solution was also prepared so that 500 cps were achieved in 50 p.1. The dilution curves were prepared by mixing the reagents together in the following order and in the following canteens.

Maximum binding curve:

(I) plasma

(II) Digoxin I25I indicator solution

200 ul 50 (il

The tubes were incubated at room temperature overnight. The solid phase particles were allowed to sit on a ferrite magnet and the supernatant was discarded. The particles were washed three times with the diluent used for the assay and finally counted with a Wilj device. The results are shown in FIG. 3. The antibody dilution curves 6 "of FIGS. 1 to 3 show that the antisera towards Tj, HPL and digoxin retain their immunoreactivity when they have been covalently bound to the polyacrolein.

Example 10

65 Automatic radioimmunoassay of HPL (lactogenic hormone of human placenta) using the anti-HPL polyacrolein / Fe30-i reagent particles

The reagents specified in Example 9 were in

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an automatic device with continuous flow (BE-PS 852327) was used, wherein an on-line incubation of the flowing discrete assay mixture was used for 10 min and then a magnetic separation of the reagent particles from the mixture. The separated particles, which now carried the antibody-bound fraction of the lactogenic hormone of human placenta, were then passed through a gamma counter, after which the counts were recorded. These data were used to produce a standard curve from which the unknown concentrations of HPL in the samples to be examined were found by interpolation.

Diluent buffer

0.05 M pH 7.4 phosphate buffer containing 0.25% Tween 20.0.1% sodium azide and 0.25% bovine serum albumin.

HPL standards (50 jxl)

Pure male placenta lactogenic hormone was spiked into normal male serum to give concentrations of 0.1.2.4.6.8.10.12 µg / ml.

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HPL-l25I indicator (50 ^ 1)

The indicator stock solution prepared was diluted with buffer solution, so that a total count of 1700 per 50 1 and 1 second was achieved.

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Anti-HPL polyacrolein / FeßO-t particle suspension (50 fil)

This suspension was used in a concentration of 0.625 mg per 50 and 1 buffer dilution. The standard curve obtained by plotting the counts in the form of js Ci / Co% against the logarithm of the concentrations of the standards is shown in FIG. 4. Thus, Figure 4 shows the standard curve for the human placenta lactogen hormone (HPL) assay in a continuous flow automatic device.

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Claims (10)

658 728 PATENT CLAIMS 1. A process for the preparation of a particulate, magnetically attractable reagent for use in immunoassays, characterized in that a) one or more monomers in the presence of magnetically attractable solid particles with immediate formation of a synthetic, water-insoluble polymer matrix. in which the solid particles mentioned are embedded uniformly, polymerized in bulk in the presence of a catalyst and / or initiator, al) the monomer or monomers being selected such that the matrix formed is aldehyde, keto, cyanate and isocyanate -, thiocyanate, isothiocyanate, halogen-nitro-aromatic tables. contains s-triazine, aromatic sulfonyl chloride, isoxazole, aziridine, imino, imide, carboxyl, epoxy or acid anhydride groups or several types of these groups which are free so that they can couple directly with a reagent , which is able to react with them so that the reagent is bound to the matrix, or a2) generates the matrix by polymerization via the ethylenic double bond of monomer, in which the or each monomer one of the monomers acrylonitrile, methacrylonitrile, acryloyl chloride and methacryloyl chloride is b) after the polymerization, the polymer matrix formed is comminuted into particles, and c) the particulate material obtained is brought into contact with a reagent which is directly in contact with the free coupling groups contained in the polymer matrix on the basis of the monomer selection in accordance with a1) or directly with the groups which can be obtained by the Monomer polymerization according to a2) introduced into the polymer matrix, having hetero atoms functional group, and this reaction can take place in order to bind the reagent directly to the polymer matrix. 2. The method according to claim 1, characterized in that the or each monomer from acrylonitrile, methacrylonitrile, acryloyl chloride, methacryloyl chloride, acrolein, methacrolein, dimethyl ketene, acrylic acid or methacrylic acid is selected and the monomer or monomers are polymerized via the ethylenic double bond. 3. The method according to claim 1, characterized in that in step b) the polymer matrix is comminuted into particles of a size of 1 to 20 (in and a specific weight of 1.4 to 3.2. 4. The method according to claim 2, characterized in that acrolein or acrylonitrile is polymerized in bulk. 5. The method according to claim 4, characterized in that tetrameth-ylenediamine is used as catalyst or initiator. Process according to claim 1, characterized in that the particulate material to be reacted with the reagent contains free aldehyde groups and that the reagent used is one which contains free amino or sulfhydryl groups and is a protein, antigen, hapten or enzyme. 7. Particulate, magnetically attractable reagent for use in immunoassays, produced by the method according to one of claims 1 to 6. 8. Suspension of a reagent according to claim 7 in an aqueous liquid. Use of a reagent according to claim 7, in an immunoassay for determining a constituent in a liquid, characterized in that the liquid is brought into contact with the particulate reagent according to claim 7 and then the particulate reagent is magnetically separated from the liquid. 10. Use according to claim 9, characterized in that a reaction mixture of a liquid phase which contains a sample of the liquid and a solid phase which contains particulate reagent according to claim 7 is allowed to flow along a line, the mixture between the Reagent and one or more other constituents of the mixture a reaction takes place, through which the component is selectively bound to the particles, that one magnetically captures the particles in the line in order to hold them against the flow and in this way the solid phase of the flowing separate liquid phase, and that downstream of it the component to be determined in the sample is determined by analysis of the separated solid phase or liquid phase.