JPH0526879A - Manufacture of carrier for immunoassay - Google Patents

Abstract

PURPOSE:To obtain a carrier with a uniform and high adsorption capability by taking a metal particle subjected to hydrophobic treatment as a magnetic substance into a pellet when polymerization reaction among polystyrene pellet, monomeric styrene, etc., is performed. CONSTITUTION:A first method is to obtain a hydrophobic metal particle by performing hydrophobic treatment of a metal particle of a magnetic substance such as cobalt and ferrite and to obtain a pellet which is approximately 1-3mg and is uniformly heavy by cutting polystyrene with a cutter or pushing it out. Under the presence of monomeric styrene, an initiator of polymerization, and a crosslinking agent, a prepared hydrophobic metal particle and polystyrene pellet are treated in a solvent medium where the hydrophobic metal particle and styrene show dissolution-retardant property, thus enabling a polystyrene particle which contains the hydrophobic metal particle to be prepared. This particle itself can be used as a carrier for immunoassay. A second method is to add an inert additive at the time of polymerization reaction between polystyrene and styrene to further enhance adsorption property of the carrier, perform evaporation treatment using an extracting medium, and provide a number of small holes in the polystyrene particle.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing a carrier for immunoassay, and more particularly to a method for producing a carrier having an excellent ability to adsorb antibodies, antigens and the like as compared with conventional carriers.

[0002]

2. Description of the Related Art Generally, the content of trace substances, such as proteins, contained in biological samples such as serum and urine can be known by carrying out immunoassays using antibodies and antigens.

In such an immunoassay, a bead-shaped water-insoluble carrier may be used to immobilize an antibody or an antigen. In particular, a carrier containing a magnetic substance is used to separate a bound / free substance (B / F) which is used for stirring a solution or performing an immunoassay.
It is useful for separation, washing, etc.).

The carrier containing such a magnetic substance is produced, for example, by producing bead-shaped polystyrene by using a mold and then fusing (thermally fusing) the magnetic substance to the polystyrene (Japanese Patent Laid-Open Publication No. Sho. No. 62-197425), a polystyrene material in which a magnetic substance is kneaded is cut into an appropriate size, pellets are produced, and then the pellets are polymerized with a styrene monomer in an appropriate solvent.

[0005]

2. Description of the Related Art The method of producing beaded polystyrene using a mold and fusing a magnetic substance thereto has a problem that it takes a lot of time and cost to manufacture the mold. . Moreover, since only one type of bead-shaped polystyrene can be produced from one type of mold, there is a problem that when changing the size of the carrier, it is necessary to re-produce from the mold.

The amount of the magnetic substance contained in the carrier is preferably as large as possible in consideration of stirring efficiency and the like, but in the method of cutting the polystyrene material in which the magnetic substance is kneaded into pellets and then subjecting it to the polymerization reaction, If a large amount of magnetic substance is mixed into the polystyrene material, the cutting cutter will be damaged during cutting. Moreover, since the carrier obtained by polymerization has a smooth surface, there is room for improvement in terms of the ability to adsorb antibodies and antigens.

[0007]

In view of the prior art, the inventors of the present invention have proposed a method for producing a carrier containing a magnetic substance and having a high adsorption capacity, in spite of containing a large amount of the magnetic substance. The inventors conducted extensive studies on a method that does not damage the cutter or the like. As a result, they found that it is possible to incorporate this into a carrier during the polymerization reaction of polystyrene pellets and styrene monomer, etc., by using hydrophobically treated metal particles as the magnetic substance, and complete the present invention. Came to.

That is, the present invention provides a step of subjecting metal particles to a hydrophobic treatment, a step of cutting polystyrene into pellets, and the hydrophobized metal particles and polystyrene pellets thus prepared are treated with a styrene monomer, a polymerization initiator and a crosslinker. A method for producing an immunoassay carrier, which comprises a step of preparing polystyrene particles containing hydrophobized metal particles by treating in the presence of an agent in a solvent in which hydrophobized metal particles and styrene are hardly soluble.

The present invention also provides a step of hydrophobizing metal particles, a step of cutting polystyrene into pellets, and the hydrophobized metal particles and polystyrene pellets thus prepared are treated with a styrene monomer, a polymerization initiator, and a crosslinker. In the presence of an additive which is inert to the polymerization reaction of the agent and the styrene monomer and has a property of being extracted or evaporated in a later step,
A step of preparing a polystyrene particle containing the hydrophobized metal particles and the additive by treating in a solvent in which the hydrophobized metal particles and styrene have poor solubility, treating the polystyrene particles thus prepared with an extractant, or A method for producing a carrier for immunoassay, which comprises a step of evaporating to extract or evaporate an additive contained therein. Hereinafter, the present invention will be described in detail.

The present invention relates to two processes which consist of different steps. In this specification, for convenience, the first method or the second method will be described below.

The first method is a manufacturing method comprising the following steps. (1) a step of subjecting the metal particles to a hydrophobic treatment, (2) a step of cutting polystyrene into pellets, (3) a step of converting the hydrophobized metal particles and polystyrene pellets prepared in the above (1) and (2) into a styrene monomer. A step of preparing polystyrene particles containing hydrophobized metal particles by treating in the presence of a polymerization initiator and a crosslinking agent in a solvent in which hydrophobized metal particles and styrene hardly dissolve. The metal particles used are not limited as long as they are magnetic substances. For example, cobalt, manganese, nickel, ferrite, iron, or oxides thereof can be exemplified. A hydrophobizing treatment is performed for the purpose of incorporating these metal particles into a carrier in a polymerization step described later. This treatment is carried out using a surface treatment agent such as a silane coupling agent typified by octadecyltriethoxysilane, and the surface treatment agent used may be one that can introduce a hydrophobic group on the surface of the metal particles. There is no particular limitation.

The polystyrene used in the present invention does not need to be composed of polystyrene alone (100% polystyrene), and may be any polystyrene containing about 50% or more polystyrene. The process of cutting this polystyrene into pellets is
It can be easily carried out by using an ordinary cutter or extruding. In order to manufacture a carrier having a uniform size in the manufacturing method of the present invention, polystyrene may be cut into pellets having a uniform weight in this step.

In the present invention, the weight of the pellet is not limited, but in consideration of the efficiency of the subsequent polymerization step, polystyrene is usually cut into pellets of about 0.2 to 200 mg, especially about 1 to 3 mg. .

The order of performing the two steps described above is not particularly limited, and it is sufficient that they are simply performed prior to the next step.

Next, the prepared hydrophobized metal particles and polystyrene pellets are treated in the presence of a styrene monomer, a polymerization initiator and a crosslinking agent in a solvent in which the hydrophobized metal particles and styrene are hardly soluble. Polystyrene particles containing hydrophobized metal particles are prepared. This step is a step of polymerizing the styrene monomer using the pellet as a nucleus, and the hydrophobized metal particles are incorporated during the polymerization.

There is no particular limitation on the polymerization initiator and the crosslinking agent,
Examples thereof include benzoyl peroxide and the like, divinylbenzene and the like, respectively. There is no particular limitation on the solvent in which the hydrophobized metal particles and styrene are hardly soluble, and examples thereof include water and an aqueous solution of polyvinyl alcohol. Further, detailed conditions such as treatment time and treatment temperature may be appropriately determined depending on the size of pellets to be treated, the chemicals used, the type of solvent, and the like. For example, for pellets having a weight in a particularly preferred range in the present invention as described above,
When this step is carried out in an aqueous solution using benzoyl peroxide and divinylbenzene, the treatment may be carried out at a temperature of 50 to 100 ° C. for 15 minutes to 48 hours, preferably 1 to 4 hours. Here, the solvent may be gently stirred.

Through the above steps, the beer containing metal particles is
Molded (almost true spherical) polystyrene particles are produced.
The particles can adsorb antibodies, antigens, etc., and can be used as they are as carriers for immunoassay. However, it is preferably subjected to a step of roughening the surface for the purpose of enhancing its adsorption ability.

The step of roughening the surface can be easily carried out by using an abrasive such as aluminum oxide which can scrape the surface of the polystyrene particles. Specifically, it is possible to exemplify adding an abrasive and polystyrene particles to a solvent in which these are insoluble, such as water or methanol, and stirring. It should be noted that this step may be carried out for about 15 minutes to 24 hours under temperature conditions in which the polystyrene particles are not deformed.

The second method is a manufacturing method comprising the following steps. (1) a step of subjecting the metal particles to a hydrophobic treatment, (2) a step of cutting polystyrene into pellets, (3) a step of converting the hydrophobized metal particles and polystyrene pellets prepared in the above (1) and (2) into a styrene monomer. In the presence of an additive which is inert to the polymerization reaction of the polymerization initiator, the cross-linking agent and the styrene monomer and has the property of being extracted or evaporated in the step (4) described later, the hydrophobized metal particles and styrene are difficult to be present. A step of preparing polystyrene particles containing hydrophobized metal particles and an additive by treating in a solvent exhibiting solubility, (4) treating the polystyrene particles prepared by the above (3) with an extractant or evaporating The step of extracting or evaporating the additives contained therein.

The steps (1) and (2) in the second method are the same as those in the first method described above. However, the step (3) in the second method is different in that an additive is used in addition to the same polymerization initiator and crosslinking agent as in the first method. The additive is a substance which is inert to the polymerization reaction in this step and is extracted or evaporated in the step (4). "Inert for the polymerization reaction" means not participating in the polymerization reaction of the styrene monomer. Examples of the additive include isoamyl alcohol, toluene and the like.

Through the above steps, bead-shaped (substantially spherical) polystyrene particles containing metal particles and additives are produced. Therefore, the step (4) is subsequently carried out for the purpose of extracting or evaporating the additive contained in the particles to produce a carrier having innumerable pores on the surface and having an increased adsorptivity for antibodies and the like.

The details of this treatment are not limited as long as the used additive can be extracted or evaporated from the inside of the produced polystyrene particles, and it may be appropriately determined and carried out in relation to the additive. . For example, when the above-mentioned isoamyl alcohol, toluene or the like is used as an additive, the evaporation treatment can be exemplified. This evaporation treatment can be carried out, for example, by leaving the polystyrene particles under a reduced pressure condition or under a high temperature condition of 40 to 100 ° C.

[0023]

EXAMPLES Examples will be described below to explain the present invention in more detail, but the present invention is not limited to these examples.

Example 1 Reacting Ferrite Hydrophobized Ferrite (manufactured by Toso Co., Ltd.) 1000 g, toluene (manufactured by Nacalai Tesque Co., Ltd.) 3.4 L, and octadecyltriethoxysilane (manufactured by Shin-Etsu Chemical Co., Ltd.) 120 g The mixture was added to the container, and the mixture was heated and stirred under reflux of toluene for 3 hours. Then, the reaction product was filtered and dried to obtain a surface-hydrophobicized ferrite.

Example 2 Production of Polystyrene Particles Containing Ferrite (First Production Method) 25 g of polyvinyl alcohol (Wako Pure Chemical Industries, Ltd.)
Manufactured) was dissolved in 500 mL of ion-exchanged water and heated to 80 ° C. On the other hand, polystyrene (manufactured by Asahi Kasei Co., Ltd.) was cut into 1.3 to 1.5 mg using a cutting machine to form polystyrene pellets, and a total amount of 5 g was added to the polyvinyl solution.

After stirring for 1 hour, 5 g of the hydrophobized ferrite obtained in Example 1, 35 g of styrene monomer (manufactured by Wako Pure Chemical Industries, Ltd.), 15 g of divinylbenzene (manufactured by Tokyo Chemical Industry Co., Ltd.), peroxide 0.67 g of benzoyl (manufactured by Nacalai Tesque, Inc.) was added, and the polymerization reaction was carried out while stirring for 3 hours.

After the completion of the reaction, the produced almost spherical polystyrene particles (carrier) were recovered, washed with water and dried.

Example 3 Surface Roughening of Polystyrene Particles 5 g of the polystyrene particles obtained in Example 2 were added together with 1 g of aluminum oxide (manufactured by Nacalai Tesque, Inc.) to 100 mL of ion-exchanged water and stirred at 25 ° C. While doing 3
After standing for a period of time, polystyrene particles having a roughened surface (carrier) were obtained.

Example 4 Production of Polystyrene Particles Containing Ferrite (Second Production Method) 25 g of polyvinyl alcohol (Wako Pure Chemical Industries, Ltd.)
Manufactured) was dissolved in 500 mL of ion-exchanged water and heated to 80 ° C. On the other hand, polystyrene (manufactured by Asahi Kasei Co., Ltd.) was cut into 1.3 to 1.5 mg using a cutting machine to form polystyrene pellets, and a total amount of 5 g was added to the polyvinyl solution.

After stirring for 1 hour, 5 g of the hydrophobized ferrite obtained in Example 1, 35 g of styrene monomer (manufactured by Wako Pure Chemical Industries, Ltd.), 15 g of divinylbenzene (manufactured by Tokyo Chemical Industry Co., Ltd.), and peroxide. 0.67 g of benzoyl (manufactured by Nacalai Tesque, Inc.) and 25 g of isoamyl alcohol (manufactured by Wako Pure Chemical Industries, Ltd.) were added, and a polymerization reaction was carried out while stirring for 3 hours.

After completion of the reaction, the produced almost spherical polystyrene particles were recovered, washed with water and dried.

Example 5 Extraction of Isoamyl Alcohol in Polystyrene Particles In a round-bottomed flask, the polystyrene particles containing isoamyl alcohol in the inside obtained in Example 4 were placed, and under reduced pressure conditions (0.1 atm), The mixture was allowed to stand for 1 hour at 80 ° C. with gentle stirring to evaporate the isoamyl alcohol to obtain polystyrene particles (support) having innumerable pores on the surface.

Example 6 Enzyme Immunoassay of Human Immunoglobulin M-1 Polystyrene particles (carrier) obtained in Example 2 or Example 3
Was used for immunoassay. 1g carrier and 0.2mg
Anti-human IgM antibody (manufactured by Biosystem)
BS solution (Phosphate Buffer Sal
ine, pH 7.4) and mixed at room temperature for 24 hours to adsorb the antibody.

The carrier was recovered from the solution, washed three times with a PBS solution, added to a PBS solution containing 1% by weight bovine serum albumin (BSA), and subjected to blocking treatment.

Human IgM was treated with 0, 100, 200, 40
150μ of test solution containing 0,800ng / mL concentration
L was mixed with 5 of the antibody-adsorbed carriers thus obtained, and the mixture was left at 37 ° C. for 1 hour to carry out an immunoreaction. At this time, an operation of gently stirring the carrier using a magnet was performed.

After completion of the immune reaction, the carrier is recovered from the solution,
The plate was washed 3 times with a PBS solution containing 0.05% Tween 20, 150 μL of an anti-human IgM antibody (TAGO) conjugated with alkaline phosphatase (ALP) was added, and the mixture was allowed to stand at 37 ° C. for 1 hour. At this time, an operation of gently stirring the carrier using a magnet was performed.

The carrier was recovered from the solution and washed 3 times with PBS solution. Then, PNPP (1 mM
100 μL of pH 10 solution containing para-nitrophenyl phosphoric acid) was added and left at 37 ° C. for 1 hour. 100μ
After stopping the enzymatic reaction by adding L 1N-NaOH,
The absorbance at 405 nm was measured.

The results are shown in FIG. According to FIG. 1, in the immunoassay using the carrier obtained in Example 2 or Example 3, the measured signal (absorbance) increases depending on the concentration of human IgM used as an antigen. It will be appreciated that carriers can also be used for immunoassays. It can also be seen that the measured signal is larger in the carrier whose surface is roughened (the carrier of Example 3) than in the carrier whose surface is not roughened (the carrier of Example 2). This is
It is shown that the surface roughening treatment facilitates binding of the antibody to the carrier surface.

Example 7 Enzyme Immunoassay for Human Immunoglobulin M-2 Using the polystyrene particles (carrier) obtained in Example 4, immunoassay was performed. 1 g carrier and 0.2 mg anti-human Ig
M antibody (manufactured by Biosystem) in PBS solution (Ph
Osphate Buffer Saline, pH
The mixture was mixed in 7.4) and left at room temperature for 24 hours to adsorb the antibody.

The carrier was recovered from the solution, washed three times with a PBS solution, added to a PBS solution containing 1% by weight bovine serum albumin (BSA), and subjected to blocking treatment.

Human IgM was treated with 0, 100, 200, 40
150μ of test solution containing 0,800ng / mL concentration
L was mixed with 5 of the antibody-adsorbed carriers thus obtained, and the mixture was left at 37 ° C. for 1 hour to carry out an immunoreaction. At this time, an operation of gently stirring the carrier using a magnet was performed.

After completion of the immune reaction, the carrier is recovered from the solution,
The plate was washed 3 times with a PBS solution containing 0.05% Tween 20, 150 μL of an anti-human IgM antibody (TAGO) conjugated with alkaline phosphatase (ALP) was added, and the mixture was allowed to stand at 37 ° C. for 1 hour. At this time, an operation of gently stirring the carrier using a magnet was performed.

The carrier was recovered from the solution and washed 3 times with PBS solution. Then, PNPP (1 mM
100 μL of pH 10 solution containing para-nitrophenyl phosphoric acid) was added and left at 37 ° C. for 1 hour. 100μ
After stopping the enzymatic reaction by adding L 1N-NaOH,
The absorbance at 405 nm was measured.

The results are shown in FIG. According to FIG. 2, in the immunoassay using the carrier obtained in Example 4, the measured signal (absorbance) increases depending on the concentration of human IgM used as an antigen, and any carrier is immunoassayed. It can be used for.

[0045]

INDUSTRIAL APPLICABILITY The present invention is a method for producing an immunoassay carrier having metal particles near the surface thereof without forming a mold or the like. Moreover, in the production method of the present invention, polystyrene pellets are also used as the core, and when the styrene monomer is polymerized into a bead form, the metal particles are incorporated therein, so that the metal particles are not included. Even when polystyrene pellets are used, a carrier containing metal particles can be finally produced. Therefore, when trying to produce a carrier containing a large amount of metal particles, it is possible to use polystyrene pellets as cores that do not contain metal particles or that contain only a small amount of metal particles that are unlikely to damage the cutting cutter. it can. In particular, when polystyrene pellets containing no metal particles are used, the step of mixing metal particles with polystyrene can be omitted, and the cutter will not be damaged during pellet production.

In the present invention, the amount of metal particles contained in the carrier and the type of metal particles themselves can be easily changed. Therefore,
If metal particles having a high specific gravity are used, it is possible to produce a carrier having a high specific gravity as needed. Moreover, in the present invention, since the metal particles are incorporated during the polymerization, a carrier in which the metal particles are uniformly distributed over the entire carrier is manufactured.

The carrier produced according to the present invention is a carrier having a large number of pores on the surface thereof, or a surface of which the surface is subsequently roughened. The carrier whose surface is processed in this way has a significantly improved adsorption ability for antibodies, antigens and the like, and is a suitable carrier for performing immunoassay.

[Brief description of drawings]

FIG. 1 shows the results of Example 6 of the present invention. In the figure, the horizontal axis represents the concentration of human IgM used as an antigen for immunoassay, and the vertical axis represents the absorbance at 405 nm. White circles show the results for the carrier obtained in Example 3 whose surface was roughened, and black circles show the results for the carrier obtained in Example 2.

FIG. 2 shows the results of Example 7 of the present invention. In the figure, the horizontal axis represents the concentration of human IgM used as an antigen for immunoassay, and the vertical axis represents the absorbance at 405 nm.

Claims (4)

[Claims] 1. A method for producing a carrier for immunoassay, which comprises the following steps. (1) a step of hydrophobizing the metal particles, (2) a step of cutting polystyrene into pellets, (3) a styrene monomer containing the hydrophobized metal particles and polystyrene pellets prepared in (1) and (2) above. A step of preparing polystyrene particles containing hydrophobized metal particles by treating in the presence of a polymerization initiator and a crosslinking agent in a solvent in which hydrophobized metal particles and styrene hardly dissolve. 2. The method for producing a carrier for immunoassay according to claim 1, further comprising subjecting the obtained polystyrene particles to a treatment step of roughening the surface thereof. 3. A method for producing a carrier for immunoassay, which comprises the following steps. (1) a step of hydrophobizing the metal particles, (2) a step of cutting polystyrene into pellets, (3) a styrene monomer containing the hydrophobized metal particles and polystyrene pellets prepared in (1) and (2) above. In the presence of an additive which is inert to the polymerization reaction of the polymerization initiator, the cross-linking agent and the styrene monomer and has the property of being extracted or evaporated in the step (4) described later, the hydrophobized metal particles and styrene are difficult to be present. A step of treating in a solvent exhibiting solubility to prepare polystyrene particles containing hydrophobized metal particles and an additive, (4) treating the polystyrene particles prepared in (3) above with an extractant or evaporating The step of extracting or evaporating the additives contained therein. 4. The method for producing a carrier for immunoassay according to claim 3, wherein the additive is isoamyl alcohol or toluene.