JP2570408B2 - Carrier for immobilizing antigen, antibody or biologically active substance - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a carrier on which an antigen, an antibody or a physiologically active substance used for measuring an immune reaction is immobilized. More specifically, for example, a human body fluid is utilized by utilizing an immune reaction. The present invention relates to a carrier used for an immunological diagnostic method for quantitatively and selectively measuring a trace substance in the substance.

(Background of the Invention and Prior Art) As a method for detecting or measuring the concentration of a trace substance contained in a body fluid, particularly in serum or urine, an immunoassay has conventionally been widely used. The reaction between an antigen and an antibody is known to be highly specific and has a characteristic that occurs even at an extremely low concentration, and by utilizing this characteristic to quantitatively detect the antigen-antibody reaction, the concentration of the antigen or antibody can be reduced. It is an excellent method for measuring.

However, in such a measuring method, the operation of separating the bound B form and the free form F (B / F separation) is generally complicated, and is a great obstacle in performing a rapid and accurate immunoassay. It is a factor.

Here, the outline of the immunoassay to which the present invention is applied will be described below using a heterogeneous sandwich immunoassay as a representative example. This heterogeneous sandwich immunoassay is first performed on a substance to be quantified (hereinafter referred to as “antigen”). The antibody is immobilized by adsorption or chemical bonding (hereinafter, the immobilized antibody is referred to as “immobilized antibody”), and an antibody that recognizes an epitope of an antigen different from the immobilized antibody is labeled with a radioisotope, an enzyme, or the like. (Hereinafter, this labeled antibody is referred to as "labeled antibody")
Prepare

Next, a test solution such as serum or urine is added to a solution containing the two of the immobilized antibody and the labeled antibody, and the antigen contained in the test solution and the labeled antibody are bound to the immobilized antibody. Let it be captured.

Here, since the amount of the labeled antibody-antigen-immobilized antibody conjugate immobilized on the carrier is proportional to the concentration of the antigen contained in the test solution, the labeled antibody captured on the carrier via the immobilized carrier is used. After washing, the target antigen concentration can be measured by measuring the amount of the label.

The above is the basic principle of the heterogeneous sandwich immunoassay. At present, various other measuring methods have been devised and widely used based on the basic principle.

By the way, even when performing an immunoassay using such a basic principle, in order to obtain excellent measurement results, there are various conditions for selecting a material that matches the measurement mode of the immunoassay, There are some conditions that are required no matter what.

For example, one of the substrate conditions required depending on the former measurement mode is the selection of the material and form of the carrier substrate, which is important depending on the measurement mode. For example, in the method of JP-A-61-219863 for performing simultaneous immunoassay of multiple items, a paper disk such as cellulose is used to realize the method. Also, JP-A-61-228354 and JP-A-59-228354
No. 90056 discloses an immunoassay method utilizing the capillary transfer phenomenon of an antigen using a capillary-containing porous carrier to which an antibody is bound. In this method, chromatographic paper, cellulose acetate is used as a carrier substrate. It is said that Rishika fine powder uniformly embedded in the membrane and matrix can be used. Also known is a measurement method for increasing sensitivity by using a magnetically responsive carrier and using stirring by the action of an oscillating magnetic field,
As such an example, for example, Japanese Patent Application Laid-Open No. 61-167866 discloses one in which a magnetically responsive powder such as ferrite is supported on the surface of a thermoplastic resin and the surface is coated with a polymer.

As described above, in selecting a carrier substrate used for immobilizing an antibody, a suitable material and form are often determined depending on a measurement mode.

In addition to the conditions determined depending on the measurement mode as described above, there are also conditions generally determined as a substrate of a carrier for immobilizing antibodies. Examples of such conditions include the following (1) to (1). (4) is illustrated.

(1) Ability to immobilize antibodies (2) Low non-specific adsorption of labeled antibodies, etc. (3) Easy washing (4) Chemically and physically stable In other words, the above conditions mean that the antibody or the like can be immobilized by any method, and that the carrier substrate is required to be a stable substance during and after the immobilization operation.

There are various known methods for immobilizing antibodies.For example, a cellulose sheet such as chromatographic paper is activated with CNBr, and the most typical method is to introduce an active group capable of reacting with an antibody. Known as the method. There is also known a method in which a silanol group of glass is chemically modified with a silane coupling agent on a glass carrier substrate, and an antibody is immobilized via the sensitive group.

However, the former method has a problem that the CNBr used is highly toxic. In addition, the latter method has a problem that the process is complicated and cannot be applied to a fragile substrate such as glass fiber paper.

A further known method for immobilizing an antibody is a method using adsorption. For example, polystyrene is known as having a high antibody-adsorbing property. However, the shape of this polystyrene that can be processed is limited. For example, it is not practical to produce a paper-like or sponge-like thing with polystyrene.

As described above, the carrier for immunoassay has an important role in performing the measurement. However, the material and morphology of the carrier are limited, depending on the type of carrier that can function depending on the measurement format and conditions. At present, it is used that is considerably limited.

(Problems to be Solved by the Invention) The present invention has been made to solve the above problems, and one of the objects thereof is to immobilize an antigen, an antibody or a biologically active substance to perform an immunological reaction. It is an object of the present invention to provide a carrier for solid phase immobilization of an antibody or the like having excellent properties for use in the above.

Another object of the present invention is to provide a carrier for immobilization of an antibody or the like which can be easily produced and can be provided easily and at low cost.

(Means for Solving the Problems) The features of the present invention made to achieve the above object are as follows.
Antigens, antibodies or biologically active substances (hereinafter referred to as "antibodies")
Is provided with a coating layer made of polyparaxylylene or a derivative thereof (hereinafter collectively referred to as "polyparaxylylene etc.") to provide an immobilized surface for antibodies and the like. .

 Hereinafter, the present invention will be described specifically.

As the polyparaxylylene or the like in the above configuration, those having the following general formula can be used, (However, in the above formula, n is 500 or more on average, and R ₁ and R ₂ each represent hydrogen or halogen, and may be the same or different.) Specific examples include the following.
Ie The coating of the carrier substrate surface with polyparaxylylene etc.
For example, the method described in (J. Polum Sci., Vol. 22, pp. 475 to 491) can be used. Specifically, for example, using a device of "Parylene vapor deposition device model 1010" (manufactured by Union Carbide Co., Ltd.) described in the above document, first, a base material, and a dimer as a raw material such as polyparaxylylene The dimer is heated and sublimated when the internal pressure of the device reaches a predetermined decompressed state (for example, 0.1 torr). By heating to about 700 ° C before reaching the surface, it is thermally decomposed and cleaved to produce active monomers. This active monomer molecule is vapor-deposited and polymerized on the surface of the target carrier substrate, thereby obtaining a degree of polymerization.
The substrate surface can be coated with more than 500 polymers. In this case, a substrate having durability under the above-described reduced pressure conditions is used.

The thickness of the film of polyparaxylylene or the like covering the surface of the carrier substrate may be appropriately determined according to the material, properties, form, etc. of the substrate. Is sufficient as long as it does not break due to friction and impact between the substrates, and is generally 0.1 μm to 50 μm, preferably 1 μm.
It is desirable to set the range to about m to 20 μm. When the carrier base material is a chromatographic paper or a glass fiber paper, it is necessary to be careful not to close the voids, and the film thickness of the polyparaxylylene or the like is in a range of about 0.1 μm to 1 μm. In many cases, it is appropriate to use it. On the other hand, in the case of a metal carrier substrate used in a usage form in which a strong impact is applied, a film thickness having a strength corresponding thereto is necessary, for example, in a range of about 4 μm to 20 μm. Is often preferred.

In order to suppress non-specific adsorption, BSA (bovine serum albumin),
It is preferable to use it for immunoassay after treating with bovine serum, casein, skim milk and the like.

As the thermoplastic resin constituting the skeleton of the carrier, for example, polystyrene having an average particle size of 0.1 to 20 mm, polypropylene, ethylene-vinyl acetate copolymer, nylon,
At least one of polyethylene, styrene-butadiene copolymer, polyvinyl chloride, a copolymer containing these as a main component, and a mixture thereof can be preferably used.

As the magnetically responsive powder carried on the thermoplastic resin, a so-called soft ferrite powder of, for example, 0.01 to 10 μm can be preferably used.

The magnetically susceptible powder may be mixed at the time of producing beads made of the above thermoplastic resin and contained inside or supported on the surface, and the method of supporting on the surface may be a method by adsorption, a method by heat fusion. Etc. can be used.

The degree of inclusion of the magnetically responsive powder in the beads varies depending on the strength of the magnetic field acting for carrier flow, the degree of fluidity of the carrier, and the like, and is not necessarily determined uniformly. % To 80%, preferably about 5% to 40%.

A simple iron or stainless steel ball can be used as a core.

(Function and Effect) The carrier of the present invention has the above-mentioned structure of having a surface coating layer of polyparaxylylene or the like having a very high ability to adsorb and immobilize an antibody or the like, so that polyparaxylylene or the like can be obtained. The excellent property of adsorbing and immobilizing proteins can be used, and antigens or antibodies can be easily immobilized without performing other chemical treatments. (Hereinafter referred to as "blocking") can sufficiently suppress non-specific adsorption to the carrier.

The object to be coated with the polyparaxylylene or the like of the present invention is not particularly limited in its shape and the like as long as the pressure resistance at the time of the coating treatment is satisfied. Has the effect of providing a carrier having a non-specific adsorption ability easily and widely, and furthermore, by coating the polyparaxylylene or the like by a treatment without heating the substrate itself, the carrier substrate Because the mechanical strength of is also improved, the mechanical strength is low, fragile,
Alternatively, there is also an effect that the use of various materials whose use as the carrier base material is limited at present is opened due to properties such as being unstable to heat.

Furthermore, there is an effect that a carrier for immunoassay such as magnetically responsive beads can be easily and suitably prepared by the present invention. In other words, the carrier whose structural part is a thermoplastic resin containing a magnetically responsive powder is not particularly limited in its shape, and can be coated with a surface such as polyparaxylylene, and the antibody can be applied thereto. A carrier for immobilizing an antigen, an antibody, or a physiologically active substance as designed can be produced by adsorbing such a substance.

Such magnetically responsive beads and the like are prepared by preparing beads made of a thermoplastic resin or the like in which a magnetically responsive powder is supported, and covering the surface with polyparaxylylene or the like, and furthermore, covering the surface with an antibody or the like. The above carrier can be produced by adsorbing.

By immobilizing an antigen, an antibody, or a physiologically active substance on the magnetically responsive beads or the like, the sensitivity of the measurement system can be enhanced by promoting the antigen-antibody reaction while stirring with a magnetic stirrer or the like, and also efficiently. B / F separation can be realized, which is useful for rapid and quantitative operation of immunoassay.

(Examples) Hereinafter, the present invention will be described based on examples shown in the drawings, but the present invention is not limited to only these examples.

Example 1 Production of Carrier for Immobilizing Antibody Ethylene-vinyl acetate copolymer particles 1 having an average particle size of 1.5 mm
And 200 g, average particle mixing the soft ferrite 20g of diameter 0.3 [mu] m MnF ₂ 0 ₄ of the ZnFe ₂ 0 ₄ and components, for 30 minutes at 110 ° C., stirred for to magnetosensitive powder containing heated Beads Was manufactured. The particle size of the beads was 1.5 mm.

Next, 50 g of the magnetically responsive powder-containing beads were vapor-deposited and coated with 10 g of dimonochloroparaxylylene (manufactured by Union Carbide Co., Ltd.) using a vacuum vapor deposition device to prepare the antibody-immobilizing carrier of this example. The operation of vapor deposition coating is performed under the conditions of evaporation (sublimation) temperature of 175 ° C, thermal decomposition temperature of 680 ° C, and vacuum degree of 0.1 torr.
This was performed using a para-xylylene vapor deposition apparatus manufactured according to the method of P. Kramer et al. Described in J. Polum Sci., Vol. 22, pages 475-491 described above. The thickness of polyparaxylylene is 2μ
m.

 Note that the beads were rotated and stirred through the vapor deposition process.

The following experiment was performed using the carrier having the polymonochloroparaxylylene coating layer as described above.

Immobilization of antibody on carrier The above 2000 carriers containing magnetically sensitive powder were mixed with 4 ml of 0.1 M
The cells were suspended in a sodium carbonate buffer (pH 9.5), 250 μg of anti-TSH (thyroid stimulating hormone) monoclonal antibody was added, and the mixture was shaken at room temperature for 16 hours. After washing 5 times with phosphoric acid / physiological saline, the suspension was suspended in 0.5% bovine serum albumin, 0.1 phosphate buffer, pH 7.5 for 3 hours at room temperature, and the adsorption site was blocked on the carrier surface.

TSH Enzyme Immunoassay Twelve carriers each having immobilized anti-TSH monoclonal antibody were placed in a polypropylene cup having a diameter of 10 mm and a height of 20 mm. A solution obtained by diluting an anti-TSH monoclonal antibody-alkaline phosphatase conjugate recognizing an epitope different from that of the antibody immobilized on the carrier with bovine serum.
50 μ and 100 μ of standard human serum containing a known concentration of TSH were dispensed, and reacted while stirring the carrier at 37 ° C. for 40 minutes using a magnetic stirrer.

After washing 5 times with 0.1% Tween 20 / phosphate buffer, 100 μM of 1 mM 4-methylumbelliferyl phosphate pH 10.0 was added, and the mixture was shaken at 37 ° C. for 10 minutes. Then, 0.14 EDTA (ethylenediaminetetraacetic acid) containing 0.1M
The reaction was stopped by adding 500 μM of a phosphate buffer.

The solution after the reaction was stopped was changed to an excitation wavelength of 360 nm and a fluorescence wavelength of 450 nm.
The fluorescence intensity was measured in nm, and the results are shown in FIG.

These results indicate that highly sensitive immunoassay can be performed using the polyparaxylylene-treated magnetic beads. Incidentally, the minimum detection sensitivity of TSH calculated from the y-section and its 2SD in Example 2 was 0.003 μIU.

Also, during the immobilization of the antibody on the beads and the measurement on the immune side in the examples, no detachment of the ferrite from the beads was observed.

Example 2 (Immunoassay with Various Papers) Cellulose paper (Whatman ET31) and glass fiber paper (Whatman GF-B) were coated with dimonochloroparaxylylene to a thickness of 0.5 μm. The conditions for coating and the like are the same as in the first embodiment.

In the above operation, a coating of dimonochloroparaxylylene can be formed without heating the cellulose paper.

The above-mentioned polyparaxylylene-coated paper was punched out to a diameter of 6 mm and suspended in a 0.1 M phosphate buffer pH 8.0 containing an anti-progesterone antibody for 5 hours. The amount of antibody per punched piece (hereinafter, referred to as “disk”) was 10 μg. Then, the antibody solution was discarded, and the antibody solution was suspended in 0.1 M phosphate buffer (pH 8.0) containing 0.5% BSA and left overnight.

Paper not coated with polyparaxylylene was similarly treated for comparison.

Each of the above disks was placed in a test tube, and 0.5% BSA / 0.5% containing progesterone-alkaline phosphatase was added.
A 1 M phosphate buffer (pH 8.0, 500 µ) was added, and the mixture was reacted at room temperature for 1 hour. After washing three times with 0.1% Tween 20 phosphate buffer, 10 mM paranitrophenyl phosphate solution pH
0.5 ml of 10.0 was added and left at 37 ° C. for 30 minutes. Then 1M
The reaction was stopped by adding 2 ml of a phosphate buffer solution containing EDTA, and the absorbance at 405 nm of the solution was measured.

 The results are shown in Table 1 below.

A higher absorbance in Group 1 compared to Groups 1 and 2 indicates that the polyparaxylylene-coated paper has a higher antibody immobilization ability. Group 3 was measured in the same manner as in Groups 1 and 2 except that the antibody was not bound to the paper treated with polyparaxylylene, and the non-specific adsorption of the paper coated with polyparaxylylene was extremely low. ing.

This result indicates that a product obtained by adsorbing an antibody on polyparaxylylene-coated paper can be suitably used as a carrier for immunoassay.

[Brief description of the drawings]

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram showing the relationship between TSH and fluorescence intensity when performing an enzyme immunoassay of TSH using an anti-TSH monoclonal antibody-immobilized carrier constituted by applying the present invention.

Claims (4)

(57) [Claims] 1. A carrier for immobilizing an antigen, an antibody or a physiologically active substance, comprising a coating layer made of polyparaxylylene or a derivative thereof provided on the surface for immobilizing the antigen, the antibody or the physiologically active substance. 2. The carrier for immobilizing an antigen, an antibody or a physiologically active substance according to claim 1, wherein the polyparaxylylene is represented by the following general formula: (However, in the above formula, n is 500 or more on average, and R ₁ and R ₂ each represent hydrogen or halogen, and may be the same or different.) 3. The method according to claim 1, wherein the base material constituting the carrier is formed into a bead-shaped or plate-shaped predetermined shape using a thermoplastic resin. Carrier for immobilizing antigens, antibodies or biologically active substances. 4. The carrier for immobilizing an antigen, an antibody or a physiologically active substance according to claim 1 or 2, wherein the substrate constituting the carrier contains a magnetic substance.