JPS63183909A - Method for manufacturing magnetically attractive substances for manufacturing analytical reagents - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing magnetically attractable materials, particularly materials suitable for use in making particulate magnetically attractable reagents for analytical purposes.

Antigens (and adherents) in biological ultra-thick fluids, e.g.
It is well known to assay antibodies (and other binding proteins) and other substances such as drugs by competitive binding reactions using labeled reagents. This assay is performed by measuring the amount of label either bound in the complex or remaining unbound in solution. One example of such a method is the well-known radioimmunoassay technique, in which radioactive labels are used. Similar techniques using other labels such as enzymes and fluorophores are known.

These methods either separate the resulting complex from the remainder of the reaction mixture or separate the free, unbound, labeled reactant from the remainder of the reaction mixture, either by removing the label in either part. Generally necessary for measuring quantities. This separation is rarely easily performed and is a source of error in such assays.

With the idea of avoiding or at least reducing this problem, the inventor proposed the use of magnetically attractable particles in immunoassays that have reagents covalently attached to the peripheral surface of the particles. (See Belgian Patent No. 852,327 for details thereof.) The particles (along with the products of the reaction between the labeled components of the reaction mixture and the reagents) are cleaned from the residue of the reaction mixture and Can be easily separated. The label in this residue or on the particles is then measured.

A limitation or disadvantage of this previously proposed method lies in the fact that the preparation of reagent-loaded magnetic particles is often time-consuming and labor intensive. The particles consist of a magnetically attractable material embedded in a polymer matrix, usually cellulose, and in order to bind a specific reagent to it, the cellulose must first be activated and then reacted with the reagent. However, a difunctional crosslinking group is usually used for this purpose. Cellulose particles activated at intermediate stages cannot be stored for any useful time. The result is that two steps in the preparation (activation and subsequent reaction) have to be carried out in each case.

A way around this problem has now been discovered by the inventors. In particular, storage-stable reactive magnetic materials have been devised that are capable of binding certain reagents in essentially one step.

According to one embodiment of the invention, one or more of the above-mentioned solids are added in the presence of said solids so as to directly create a synthetic water-insoluble polymeric matrix with magnetically attractable solids embedded therein. A process for the production of autoreactive magnetically attractable materials comprising the polymerization of more than one monomer, said monomers from which the substrate formed is an aldehyde, ketone, cyanate, isocyanate, thiocyanate, isothiocyanate. , halonitro-aromatic, S-trieno/, aromatic sulfonyl chloride, isoxazole, anolinone, imine, imide, carboxyl, epoxide, and acid anhydride; A method is provided, characterized in that it uses a free group that is capable of binding said reagent to said substance by direct binding to said reagent which is reactive with said substance.

According to another embodiment, the invention includes a magnetically attractable material preferably made in the form of particles.

When the particles of the present invention are contacted with a reagent having a reactive group under appropriate reaction conditions, the reagent becomes directly bound to the particles.

contacting the particles of the invention with a reagent capable of directly reacting with free radicals in the polymer (matrix) substrate of the particles of the invention;
When reacted to bind the reagent to a polymeric substrate, a particulate magnetically attractable reagent for use in immunoassays is obtained.

Particles of the invention having reagents attached to the particles are hereinafter referred to as reagent particles of the invention for the sake of clarity.

Among the various possible free radicals in the polymer matrix produced, aldehyde and ketone groups are preferred.

In a highly preferred embodiment of the invention, polymerization is achieved by polymerization of at least one ethylenically unsaturated monomer containing said groups which remain in the polymer and which can then react directly with the reactive groups of the reagent. A coalescent matrix is created directly.

Among the preferred monomers that can be so polymerized are acrolein, methacrolein, methacrylonitrile,
Acrylonitrile, trimethylketene, acryloyl chloride, methacryloyl chloride, acrylic acid and methacrylic acid.

After suitable separation from the reaction mixture and washing if necessary, a solid polymeric product is produced directly (magnetically attractable) which can be ground to give particles of the invention. It is preferred to polymerize one or more of these monomers (in the presence of solids). Such polymerizations are usually carried out using catalysts and initiators and without any solvents, ie bulk polymerizations. For example, in the case of acrolein, the acrolein monomer is N
, N,N,N'-tetramethylenezamine and the polymerization reaction to form a solid product occurs rapidly. In the case of acrylonitrile, ammonium persulfate is also added to accelerate the reaction.

N, N, 1'7. It is a highly preferred feature of the present invention that the polymer matrix is made with the free radicals of acrolein in the presence of N'-tetramethylenezamine. When acrolein is polymerized in this way, for example in the presence of Fe3O4, a homogeneous bulk polymer/FeO composition is easily produced. This composition can then be easily ground to particle size.

The polymeric substrate and particularly the reactive free radicals therein are selected with care to the reagent with which it is to be coupled. Most commonly this reagent contains free amino or sulfhydryl groups, in which case a polymeric substrate containing aldehyde groups is used. Reagents containing free amino groups include proteins such as antibodies and enzymes, certain antigens, butenes, pharmaceuticals, and antibiotics. Examples of such substances are thyroid hormones T4 and T3 (thyroxine and triodothyronine), tricyclic anti-inhibitory drugs (nortriptyline, desipramine d6s ipramine and protriptyline), amino-glycoside antibiotics. ()f′ nontafcin gentamicin
, sysomicin, neomycin, amicasia ami
Kacin.

neomicin, streptomycin, kana
mycin and tobramycin
), various affinity absorbers for the isolation or analysis of antibodies or other bound proteins, and solid phases used for the analysis of conjugated haptens, such as intermediates in the preparation of antibodies.

It is a very advantageous feature of the invention that reagents can be attached to the particles of the invention in essentially one step.

Thus, for example, reagents can simply be brought into contact with particles in a suitable pn medium. Polymeric substrates containing free aldehyde groups are "autoreactive" toward free amine groups, such that the reagent reacts and binds directly to the substrate. The reagent particles of the present invention thus produced can be used at any time. It is a very advantageous feature since according to the invention the need to use bridging groups (in the prior art) is avoided and it is ultimately a simpler, faster and more effective process.

The particles of the present invention and the reagent particles of the present invention may be of any particle size, but it is generally convenient for it to be from 1 to 100 μm. Particularly when used in continuous flow techniques, the specific gravity of the particles should preferably be between about 1.4 and 3.2 to avoid excessive flotation or settling of the particles in the flowing liquid reaction mixture.

The particles of the invention can be stored for a period of time before use (ie, before binding reagents to them).

As is well known to those skilled in the art, during storage the particle should be kept from contact with substances with which it may react. Alternatively, a solid mass of autoreactive polymer matrix containing magnetic particles can be stored and then a portion of this mass can be used for reaction with a reagent if particles are required. Can be crushed to size. In this way, the reagent particles of the invention can be simply prepared fresh immediately before their use.

The reagent particles of the present invention are particularly, but not exclusively, useful for analyzing components of interest in liquids.

i.e., these particles can be used in manual or automated continuous flow assays (see U.S. Pat. No. 2.79 for a general description).
No. 7,149). They are also used, for example, in our Belgian patent no. 852
, No. 327.

The invention thus includes a method for analyzing components of interest in a biological fluid sample, the method comprising the steps of contacting the sample with reagent particles of the invention and separating the particles by magnetic means. This includes:

The invention further provides the steps of: flowing along a conduit a reaction mixture consisting of a liquid phase containing a liquid sample containing a component of interest and a solid phase containing reagent particles of the invention, in which a reaction takes place; One component of the reaction mixture reacts with, or is selectively absorbed by, said solid phase, magnetically holding the particles against the flow in the conduit, thus separating the solid phase from the flowing liquid phase. The liquid phase is then passed downstream, and the method also includes methods consisting of quantifying the components of interest in the sample by analysis of the separated solid or liquid phase.

One preferred form of analytical apparatus for carrying out this method is described in our Belgian Patent No. 852,327.

The precise manner in which reagent particles operate in a biological assay, such as an immunoassay, will vary depending on the nature of the reagent, as will be apparent to those skilled in the art. For example, in immunoassays of antigens using labeled antigens and antibodies or other binding proteins, the antibodies can constitute reagents on reagent particles.

Reagent complexes with both labeled and unlabeled antigen can be assayed against the label under these circumstances and upon removal from the reaction mixture (or against the label from the remaining reaction mixture). ).

In one preferred method of the invention, the reaction mixture consists of a predetermined amount of a labeled substance that reacts with the component of interest forming a complex with that component as a second reagent, and in this case the reagent of the invention The reagent on the particles is combined with an excess of the second reagent, and the separated mixture is also analyzed to quantify the components of interest in the sample.

In this method, analysis is performed on either the complex or excess unreacted second reagent, as appropriate. These examples are given by way of illustration only; the versatility of the reagent particles of the invention in many different assay methods will be apparent to those skilled in the art.

The following examples are presented by way of illustration only, in order that the invention may be more fully understood.

Example 1 Preparation of particles according to the invention consisting of polyacrolein (2t) and magnetizable iron oxide Fe3O4 (2f
) were mixed vigorously at room temperature. N, N.

N',N'-tetramethylenezamine (100 μt) was added and stirring continued for approximately 6-7 minutes. After this time polymerization occurred to produce a homogeneous solid material. The product is Br
Grind into fine granules in an aun electric coffee grinder. Further milling for 30 minutes in a McCrone micronizer yielded a stable autoreactive product of appropriate particle size.

Example 2 Preparation of particles according to the invention consisting of polyacrylonitrile Acrylonitrile (2.5 mg) and magnetically attractable iron oxide (21) were mixed vigorously at room temperature. N, N, N,! (-tetramethylenediamine (1
00 μt) was added. Solution of ammonium persulfate (33
Weight/volume: 1-) was added. After about 7 minutes, the polymerization reaction occurred, producing a homogeneous solid material. This product was ground to a particle size of 1111 or less and air-dried at room temperature for 72 hours. The product was further milled in a MeCrone micronizer to obtain stable autoreactive particles of the invention.

Example 3 Coupling of anti-T4 serum (r-globulin fraction) to particles of the invention Polyacrolein/FeO particles (1?) as described in Example 1
) was washed 4 times with water (20 mg), then sodium carbonate/
Wash twice with 0.1 M sodium bicarbonate buffer (pH 9,0). The suspension of particles in buffer has a volume of 14 ml. Anti-T4 serum (γ-globulin fraction IWi
Coupling was performed by adding t) to the particle suspension and gently mixing for 24 hours at 4°C. The particles were then settled onto a multipolar ferrite magnet and surface floaters were removed. The reagent particles thus generated are converted into hydrogen carbonate/
Washed three times with carbonate buffer and twice with 0.05M phosphate buffer (pH 7.4), and finally the volume of the suspension was brought up to 20- with phosphate buffer.

Example 4 Binding of anti-Digoxin serum (γ-globulin fraction) to particles of the invention
The same procedure as in Example 3 was followed except that anti-sogoxin serum (gamma globulin fraction) 1-1 was used instead of 4 serum.

Example 5 Binding of anti-human placental lactodan serum (γ-globulin fraction) to particles of the invention Anti-human placental lactodan serum (γ-globulin fraction) was used instead of anti-T4 serum.
γ-Globulin fraction) 1ffi7! The same method as in Example 3 was followed except using .

Example 6 Binding of anti-sogoxin serum (γ-globulin fraction) to particles of the invention One ton of particles described in Example 2 was mixed with 20 m7 of water! 4 times, then 0
．． 1 M sodium carbonate/sodium bicarbonate buffer (
Washed twice with pH 19,0). The suspension of particles in buffer had a volume of 14-. Binding was performed by adding anti-sopxin serum (r-globulin fraction) 1- to the particle suspension and mixing gently for 24 hours at 4°C. The reagent particles thus produced were then precipitated onto a multipolar ferrite and surface flotsam was removed. The particles were washed three times with bicarbonate/carbonate buffer and then 0.0
Washed twice with 5 M phosphate buffer (pH 7,4). Finally, the volume of the suspension was made up to 20 liters with phosphate buffer.

Example 7 Manual radioimmunoassay antibody dilution curve for T4 using anti-T4 polyacrolein/Fe5o4 reagent particles: Assay diluent for all assays is 0.05
M phosphate buffer (pf 17.4). A series of doubling dilutions were made from the suspension of reagent particles. 5.2.5.1.25.0.625.0 per 1oo of suspension.
-T4- which contains the substrate of 312.0.1569
A 125 etraser solution was made to give 500 cps at QT. 8-anilino-1-naphthalene sulfonic acid was added to the tracer solution at a rate of 8 μf per serum sample. A dilution curve was created by mixing the reagents in the following order and amounts:

Best binding curve: (il T4-free serum 50 μtf
iiiT4- I) Racer solution
50μt (in assay diluent) +iii+anti-T4 reagent particle suspension (Example 3)
50 μt (at dilutions outlined above) Apex Standard Binding Curve: til T4-'25I) Racer Solution
50μ) anti-T4 reagent particle suspension (Example 3)
) 50□ non-specific binding curve: fill T4-I) Racer solution
50μ anti-T4 reagent particle suspension (Example 3)
The 50 μt tubes were incubated at room temperature overnight. The particles of the solid phase settled on the ferrite magnet and the surface floaters were discarded. The particles were washed three times with assay diluent and finally Wi
Counted on l j . The results are shown in FIG.

In Figures 1 to 3, the vertical axis r%BJ represents the bond count, that is, the ratio between the counts for bound substances and the total count, and the horizontal axis rQS
P J represents solid phase weight (mg).

Example 8 Manual radioimmunoassay for digoxin using anti-sogoxypolyacrolein/Fe3O4 reagent particles Antibody dilution curve =
A series of doubling dilutions were made from the suspension of reagent particles.

5 per 100 d of suspension, 2.5.1.25.0.6
25.0.312.0.156 and 0.0789 particles. Digoxin-125, tracer solution was made to give 500 cps at 50 μt.

A dilution curve was created by mixing the reagents in the following order of amounts:

Highest binding curve: (1) Sogoxin-125 tracer solution hitting 200μ of plasma
50μ forward anti-nogoxin reagent particle suspension (Example 4)
50μ to the apex standard Binding curve: (i) Apex standard 8nmkL-' in plasma
200 pL (11) digoxin 125I tracer solution 50μt (III anti-digoxin reagent particle suspension (Example 4) 50μ) Non-specific binding curve: (:) Standard point standard concentration in plasma (160nmL) 20
0 aL (11) digoxin-■ tracer solution
50μt0111 anti-digoxidigoxin reagent particle suspension 50μ was incubated in a test tube overnight at room temperature. The solid phase particles were settled onto the ferrite magnet and the surface floaters were discarded. Particles were washed three times with assay diluent and finally counted on the Wil j. The results are shown in FIG.

Example 9 Anti-Human Placental Lactodan (Anti-HPL) Manual % Formula for HPL Using Polyacrolein/Fe3O4 Reagent Particles Antibody Dilution Curve: A series of doubling dilutions are made from a suspension of reagent particles. It was done. 5.2.5.1.2 per 100 suspension
5.0.625.0.313f particles.

The HPL-I tracer solution was made up to 500 cps in 50μ. A dilution curve was created by mixing the reagents in the following order of amounts:

Best binding curve: (1) Male human serum Sekiμt+ii
+) IPL-I tracer solution shameμ
ttin <at dilutions outlined above) anti-HPL reagent
Particle suspension vertex standard binding curve: (1) Vertex standard (12 μtml)
Hit 50μ HPL-125I) racer solution
Non-specific binding curve of anti-HPL reagent particle suspension: (i+20x apex standard concentration (240μ
Hit 50μ HPL-125I) racer solution
Group μt+m+anti-HPL reagent particle suspension
Keep the sample μ in the test tube at room temperature overnight.
Ta. The solid phase particles were settled onto the ferrite magnet and the surface floaters were discarded. Particles were washed three times with assay diluent and finally counted on a Wi lj. The result is the third
As shown in the figure. The antibody dilution curves shown in Figures 1-3 are T
4. Antisera against HPL and digoxin show that it maintains its immunoreactivity when covalently bound to polyacrolein.

Example 10 Automated radioimmunoassay of HPL using anti-HPL polyacrolein/Fe3O4 reagent particles The reagents characterized in Example 9 (Belgian Patent No. 8523
No. 27) in an automatic continuous flow apparatus. This device utilizes on-line incubation of each incoming assay mixture for 10 minutes, followed by magnetic separation of reagent particles from the mixture. This separated particle now has a fraction of HPL bound to the antibody, which is then run through a gamma counter and the count is recorded. These data are used to draw a standard curve from which the unknown concentration of HPL in the test sample is found by interpolation.

Diluent buffer 0.05M phosphate buffer, pH 7.4, 0.2
Contains 20.0% sodium azide and 0.25% bovine serum albumin.

HPL standard (50 μt) Normal male serum mixed with pure HPL to a concentration of 0.1.2
．． 4.6.8.10 and 12μ are now given to hawks.

HPLI Tracer (50μt) Prepared stock tracer was diluted in buffer to 50μt.
It was made to give a total count of 1700 cpa per hit.

Anti-HPL Polyacrolay//Fe3O4 Particle Suspension (50μt) This was used at a concentration of 0.62519 diluent buffer per 50μt. Ci/C versus logarithm of standard concentration.

A standard curve obtained by plotting the counts as a sample is shown in FIG. Samples of unknown concentration can be read from this standard curve in the usual manner.

where rcoJFi is the highest number of counts to be obtained (i.e. the count if no binding occurs), and C1
is the count value when using standard i.

[Brief explanation of the drawing]

FIG. 1 is a curve diagram showing the results of a manual radioimmunoassay for T4 using the anti-T44 liacrolein/Fe3O4 reagent particles described in Example 7 of the Examples. Curve I represents the highest bonding curve, ■ represents the vertex standard bonding curve, and m represents the unspecified bonding curve. Figure WJ2 is a curvature diagram showing the results of a manual radioimmunoassay for sogoxin using the anti-sogoxin polyacrolein/Fe3O4 reagent particles also described in Example 8. Curve I represents the highest binding curve, ■ represents the vertex standard binding curve, and m represents the unspecified binding curve, respectively. FIG. 3 is a curve diagram showing the results of a manual radioimmunoassay of HPL using anti-HPL polyacrolein/Fe3O4 reagent particles also described in Example 9. song! IFi maximum binding curve, rl represents the vertex standard binding curve, and ■ represents the unspecified binding curve, respectively. FIG. 4 shows the anti-HPL described in Example 10, t? I.J.
Figure 2 shows a standard curve for the assay of human placenta rough (HPL) in an automated continuous flow device using acrolein/Fe 304 reagent particles. Figure 1 Figure 2 Figure 3

Claims (7)

[Claims] (1) from polymerizing one or more monomers in the presence of a magnetically attractable solid to directly create a synthetic water-insoluble polymeric matrix having said solid embedded therein; A process for producing an autoreactive magnetically attractable substance, wherein the matrix substrate formed therefrom as monomers is an aldehyde, ketone, cyanate, isocyanate, thiocyanate, isothiocyanate, halo-nitroaromatic, S-triazine, aromatic one or more groups selected from the group sulfonyl chlorides, isoxazoles, aziridines, imines, imides, carboxyls, epoxides and acid anhydrides, which are capable of directly bonding to the group or to reagents reactive with them. Therefore, the manufacturing method described above is characterized in that a substance containing a free group capable of binding the reagent to the substance is used. (2) the monomer or each monomer is an ethylenically unsaturated monomer containing one or more ethylene groups, and the polymerization is carried out via ethylenic unsaturation and this group is present in the polymer matrix substrate thus produced; The method described in the preceding paragraph (1) in which the remains as is. (3) The monomer or each monomer is selected from the group consisting of acrolein, methacrolein, methacrylonitrile, acrylonitrile, dimethylketene, acryloyl chloride, methacryloyl chloride, acrylic acid and methacrylic acid. Method. (4) The method according to item (2) above, wherein the polymerization is bulk polymerization carried out in the presence of a catalyst and/or a reaction initiator. (5) Acrolein or acrylonitrile with N, N,
The method according to the above item (4), wherein bulk polymerization is carried out in the presence of N',N'-tetramethylenediamine. (6) The method according to any one of (1) to (5) above, wherein the polymer matrix substrate thus produced after polymerization is pulverized to produce particles. (7) The particles have a size of 1 to 20 microns and 1.4 to 3.
The method described in the preceding paragraph (6) having a specific gravity of 2.