JPS5984161A - Solid-phase carrier for immune measurement - Google Patents

Abstract

PURPOSE:To enable quick immune reaction under a uniform condition without the need for any laborious operation and with the use of an extremely small amt. of an immune reaction medium by using a styrene polymer incorporated therein with a sulfonic acid group in constituting a solid-phase carrier for immune measurement. CONSTITUTION:A solid-phase carrier made of polystyrene molded to a prescribed shape is subjected to a pretreatment for one hour at 5 deg.C and thereafter it is put into a cooled concd. sulfuric acid and is brought into reaction preferably for 6hr. The solid-phase carrier is removed from said acid after the reaction and is thoroughly washed with distilled water. When the carrier is further thoroughly washed repeatedly with 100mM phosphoric acid buffer soln. (8.0pH), a polystyrene solid-phase carrier 1 incorporated therein with the sulfonic acid group is obtd. The resulting carrier 1 has an extremely large amt. of immobilizing antibody per carrier and therefore a satisfactory immune measurement is made possible with an extremely small amt. of the antibody as well. Since the coupled antibody exists in an extremely stable form, the carrier withstands long-time and frequent use and has a good rate of maintaining the activity of the antibody.

Description

[Detailed description of the invention] The present invention relates to a novel solid phase carrier for immunoassay.

Many means are known for carrying out antigen-antibody reactions (conventionally) and similar reactions in a large capacity.

This method is mainly used to detect components that exist in trace amounts in body fluids such as blood and blood. , t, is applied to qualitative, etc. Trace components include physiologically active substances such as various hormones as biological components, isozymes in the body, administered drugs and specific antibodies against drugs, antigen components derived from microorganisms, and specific antibodies against microorganisms and their antigen components. antibody,
Furthermore, micro-M existing in various living organisms such as these metabolites
゛Ingredients can be mentioned.

Such trace components are mainly measured by applying antigen-antibody reactions or similar reactions in immune reactions, such as hormones and antibodies or receptors against them.

Methods that take advantage of the fact that as a result of antigen-antibody reactions, the combination thereof becomes insoluble or a large aggregate consisting of a large number of antigen/antibody molecules are formed are already known as classical methods. Are these straight tubes the amount that settles in the test tube? 1
method for determining l11, immunodiffusion method for precipitation in gel,
Alternatively, it is measured by a method using complement that binds to an antigen-antibody complex. Also, a method is well known in which various Nobutin 1 antigens and antibodies are labeled by some method and the labels are measured. Among these methods, radioimmunoassay (IA) is widely used as a highly sensitive measurement method, but RIA uses radioisotopes and is a safer measurement method in place of radioisotope labels. In recent years, enzyme immunoassay (BIA), a measurement method using enzyme-labeled substances, has been widely used.

EIA includes drugs and antigens (fills) and butenes:
In this method, an enzyme is bound to an antigen (hereinafter simply referred to as an antigen) or an antibody, the degree of antigen-antibody reaction is measured using the enzyme activity as a label, and the degree of antigen-antibody reaction is measured from the results. .

In such a measurement method, when the substance to be measured is an antigen or an antibody, a labeled antigen or antibody is competitively reacted with an unlabeled (non-labeled) antigen or antibody. In addition, there are cases where antigen-antibody bound substances and non-bound substances are separated and measured during a competitive reaction, and cases where they are measured without separation, and the Sand-Deutsch method is also mentioned.

The assay method using the in-phase carrier for immunoassay of the present invention is an assay method used in the competitive reaction method or the Sand-Deutsch method among these methods, and in particular, the assay method using the solid phase carrier for measurement, which is an insoluble carrier, is used as an antibody. And antigen 1! This is a method used for constantization (insolubilization). More specifically, the measurement method using the in-phase carrier for immunoassay of the present invention uses an in-phase carrier in which IgG or its first antibody or second antibody is immobilized on a polystyrene solid phase carrier into which sulfonic acid groups have been introduced, and is carried out by the Kita method. It is.

In conventional immunoassay methods using insoluble carriers, spherical carriers or paper disk-shaped carriers made of nylon, polystyrene, or glass are used as carriers for immobilizing antibodies. I was doing it, but
The number of antigens and antibodies immobilized on these insoluble carriers was small and unsatisfactory.

The present inventors previously attempted to improve solid phase carriers for immunoassays that have these various drawbacks, and invented a homophase carrier molded in a special shape, for example, a bullet shape (Japanese Patent Application No. 101365/1982). However, polystyrene, which is commonly used as a solid phase carrier, has disadvantages such as weak physical adsorption, easy detachment of bound antibodies, and lack of adsorption needles. Zl- could not be said to be practical either.

The present invention has been researched and developed in order to improve the above-mentioned drawbacks, and its purpose is to convert a styrene-based polymer measurement carrier, which is an insoluble carrier, to fuming sulfuric acid or anhydrous. The in-phase carrier introduced with sulfonic acid groups obtained by treatment in sulfuric acid at a low temperature has an extremely high capacity to bind immune components such as antigens and antibodies by 7 to 1 times more than untreated carriers. Knowing that it is good,
More preferably, this carrier is formed into a special structure, the contact area is increased in the reaction tube by the measurement homomorphic substance, and the contact surface of the reaction liquid with the measurement solid phase carrier is made into a uniform layer. It does not require complicated operations such as stirring or shaking, and uses significantly less immunoreaction medium.
An object of the present invention is to provide a solid phase carrier ligature for immunoassay, which allows the immune reaction to be carried out quickly and under uniform conditions.

The present invention can also be applied to EIA and RIA. Furthermore, the present invention can be applied to competitive reaction methods such as the in-phase method and measurement methods using the sandwich method.

As the insoluble carrier for the styrenic polymer used in the present invention, general 1fllA-? An insoluble carrier of styrenic polymer used in RIA is used.

The styrenic polymer may be a monopolymer made from styrene, such as styrene and aminostyrene, divinylbenzene, acrylamide, ethylene, methacrylic acid, methacrylic acid, methyl methacrylate, acrinitrile vinyl alcohol, acetic acid. vinyl,
It may also be a copolymer with butadiene and maleic anhydride. This styrenic polymer as an insoluble carrier for measurement 111'' carrier may be used in spherical form,! It is preferable for Samurai to have a bullet shape, 4A shape, or similar shape, and is formed by adhering it to the surface of another solid molded object such as gold H or plastic so that the shape of the in-phase support for measurement of the present invention is maintained. Even if it's something.

Next, preferred structural examples of the solid support for measurement of the present invention will be explained with reference to the drawings.

In FIG. 1, the solid phase carrier for measurement is generally indicated by the reference numeral 1. The solid phase carrier for measurement is made of a polystyrene polymer resin material, for example, and is provided with a carrier body 2 and an upper surface 2a of the body 2 protruding from the center.
It'll consist of 3. A music operating rod (not shown) is fitted into the mounting leg 3 and attached, but the handle τ1 leg 3 itself may be shaped like a long rope to be used as an operating rod. The carrier body 2 is molded into a bullet shape, and its outer diameter is reactive. ! It is formed to a diameter that maintains a small gap with the inner diameter of r4. A wedge-shaped projection 5 is integrally provided on the top of the lower peripheral surface of the carrier body 2, and the projection 5 connects the arcuate outer peripheral surface 2b of the carrier main body 2 and the arcuate inner peripheral surface 4a of the reaction tube 4. K so that a constant gap 6 is maintained between the two. Further, instead of the wedge-shaped projection 50, a convex projection may be used. In short, any shape may be used as long as the projections 5 can prevent the carrier main body 2 from coming into close contact with the arcuate inner circumferential surface 4a of the reaction tube 4. The height of the protrusion ξ is 1 between the carrier body 2 and the inner wall surface of the reaction tube 4.
6 (only need to be the same. As shown in FIG. 2, the top surface 2a of the carrier main body 2 is provided with a square cut groove 8 with one mounting leg 3 in the center. The terminal end of the groove 8 is opened at the peripheral edge of the carrier main body 2 and is opened at the upper surface portion 2a.
In order to reduce the surface tension of the reaction solution on the critical line of 6, the shape of this cut m8 may be semicircular, elliptical, trapezoidal, triangular, square, etc. The shape may be shifted by h.

Since the structure is similar to that of the n++j constant-loss, double-phase carrier of the present invention, when the carrier body 2 is immersed in the reaction liquid placed in the reaction tube 4, the protrusions 5 of the carrier body 2 are connected to the reaction tube 4. A slight gap is formed between the outer circumferential surface of the carrier body 2 and the inner circumferential surface of the reaction tube 4 corresponding to the button (see Fig. 1).
. Therefore, even if the amount of reaction liquid is small, the carrier body 2
By immersion, the liquid is guided into the gap and raised to the upper surface of the carrier body 2, and the reaction liquid flows through the notch O8 and loses the surface tension effect on the upper surface. Therefore, the reaction liquid contacts the entire outer circumferential surface of the carrier body 2 under uniform conditions (in the form of debris with a constant thickness).

As a modification of the cut grooves 8 as described above, they may be arranged radially in the radial direction of the upper surface portion 2a of the carrier main body 2, as shown in FIG. 3(A)K. Also, as another modification,
As shown in FIG. 3(B), the carrier body 2 is arranged linearly in the diametrical direction of the upper surface 2a of the carrier main body 2. Alternatively, they may be arranged symmetrically in the circumferential direction of the upper surface portion 2a of the carrier body 2.

In this way, the cut /i\8 may have any shape as long as its terminal end is open 11 at the peripheral edge of the upper surface portion 2a, and it can be formed in any shape.

FIG. 4 shows another modification of the solid phase support 1 for measurement. The carrier body 2 in this structural example is formed into a spear shape. Therefore, the reaction tube 4 is also tapered along the carrier body 2. Therefore, as will be understood from this, the carrier body may be modified and processed according to the shape of various commercially available reaction tubes. The other structure of the carrier body 2 in the embodiment shown in FIG. 4 is the same as that in the embodiment shown in FIG. , the explanation is omitted.

As mentioned above, the solid phase carrier can be formed into various shapes, and an actual measurement example for forming the backing of a bullet-shaped solid phase carrier, for example, will be explained with reference to FIG.

For example, the small test tube used is 15X1 (15ynWL)
In the case of an inner diameter of 12.8 m, m±n, 1 my+L), the carrier main body 2 has the dimensions of each part KIA indicated by the reference numeral or number in Fig. 5.
made. That is, A: hemispherical portion with a radius of 6 mm, B: height of the cylindrical portion: 10 mm, C: height of the conical protrusion)
, 4m'WL % D: Radius of cone of conical protrusion 0
．． 5tntn% E: Semicircular groove with a radius of 0.75 mm, F: Center distance between opposing grooves 7.5 mm, G
: Diameter of the convex part for inserting the operating rod 4 m, m%H:
The diameter of the operation rod insertion hole is 2 mm.I: The height of the protrusion for insertion of the insertion rod is 3 mm. Measure the buffer solution on the carrier body 1(] 0 /i
About β, the liquid that binds the labeled substance is 10
0/l, e culm degree and test liquid (serum, urine, etc.) are 1()θ
A value on the order of μm may be used.

In addition, at this time, the width (thickness) of the liquid layer of the reaction solution was (1.4 mm
It is formed uniformly with L.

The carrier body formed as in Example 2-1 is added to fuming sulfuric acid or anhydrous sulfuric acid solution, usually under cooling, for example,
The sample is immersed for 2 hours or more at a temperature of about 0.degree. C. to react and introduce sulfonic acid groups.

The sulfonic acid group obtained in this manner is chemically or physically bound to the receptor for antibodies, antigens, and other drugs by applying conventional means to the carrier for measurement to form insoluble antibodies. (antigen, receptor) and hydrates the same phase.

In the in-phase preparation method, C is prepared by introducing a functional group for binding an antibody, etc. to the pre-8C-I4 injection carrier in advance.
It may be processed into a specific shape, or a functional group may be introduced after processing. To obtain this same phase, the styrene-based polymer treated with sulfuric acid and the immune component are brought into contact with each other in an aqueous medium, and most of the components are immobilized by ionic bonds. Furthermore, for K to be immobilized in a general chemical manner, a functional group in the molecule of the styrene-based polymano-insoluble carrier or a functional group introduced therein, a functional group in the molecule of the antibody, etc., such as an amine group,
A polyfunctional compound capable of bonding the two based on a functional group such as a hydroxyl group, a carboxyl group, a thiol group, or an aldehyde group is used. This known polyfunctional compound C is
Diisocyanate compounds such as hegizamethylene diisocyanate, 2.4-)luene diisonanate,
Diisodiocyanates such as hexamethylene diisothiocyanate, dialdehydes such as succinic aldehyde, glutaraldehyde, and azibaldehyde, N1N-ethylene bismaleimide, N,N'-0-phenylene dimaleimide, bis Diazobenzidine, N.

N1-Polymethylenebiiodoacetamide, diethylmalonimidate), dimethyladipineimidate, 3
-(2-benzothiazolyl-dithio)-propionic acid,
Sulfide carboxylic acids such as 3-(2-pyridyl-N-oxide-dithio)propionic acid, 6-N[3-(2'-benzothiazolyl-dithio)globionyl]caproic acid, or their succinimide esters, P-nitrophenyl esters, Reactions of acid chlorides, imidates, etc.\stretchable conductors (see Japanese Patent Application No. 53-85900), maleimidocarboxylic acids such as maleimidobenzoic acid, maleimidophenyl acetic acid, maleimidophenylpropionic acid, and their reactive derivatives.ノLru.

This polyfunctional compound can also be used as a functional group introduction reagent for introducing a functional group into an insoluble carrier.

In addition, to immobilize the immune components and obtain the same phase, for example, a buffer with a pH of 6 to 8, an alcoholic medium such as acetone, methanol, or ethanol, an etheric medium such as diethyl ether, or a mixed medium thereof can be used. The insoluble carrier and the polyfunctional compound are allowed to react in an inert medium such as, at cooling or room temperature. Thereafter, it is washed as necessary, and further added to the same inert medium, and then an antibody or the like is further added and reacted to cause the insoluble body to react with the antibody or the like.

Below is the key of the in-phase carrier? ! As an example of the adsorption of an antibody on a support in the same phase as the method, an example of adsorption of CIgG will be explained in detail.

A polystyrene homophase body molded into a predetermined shape was pretreated at 5°C for 1 hour, then placed in cooled concentrated sulfuric acid and allowed to react at 5°C for 1 to 16 hours, aI or ii:f'i. . After the reaction, the in-phase support was taken out and thoroughly washed with distilled water, and then added with 1 tl OmM phosphate buffer (pH 8,
Washing is repeated sufficiently in step (n) to obtain one polystin solid 10'' into which sulfonic acid groups have been introduced.

This in-phase carrier is inserted into a test tube having a diameter slightly larger than the diameter of the in-phase carrier, which has previously been filled with a small amount of IgG solution, and incubated at 5°C for an appropriate period of time, e.g., 2-16 hours. IgG can be adsorbed.

Currently, the inner diameter is 12.8mm±0. A solid phase carrier with sulfonic acid groups introduced into a bullet shape with a diameter of 12.0 mm as shown in Fig. 5 was used in a test tube of 9 mm in diameter.
Talk about the adsorption of pressure.

In the above test tube, add rabbit IgG (10mM phosphate buffer (pH 8,0) solution o, 4m0 (IgGi
25ttg/me as t.

Add 50 μg/me and 100 μg/me into separate test tubes, insert the polystyrene solid phase carrier into which the sulfonic acid group has been introduced, and incubate for an appropriate time (0, 2,
Incubate at 5°C for 4, 6, 8, 16 hours).

After the reaction, pull out the solid phase carrier and add 100mM phosphate buffer (
The protein J of the remaining IgG solution in the test tube was measured by the Cu-Folin method, and the absorption *I
Check out the GG scenery. As shown in Figure 6, the results showed that 10μgA was obtained using an untreated polystyrene solid support.
Approximately 7.5 times for llr phase support, 20 μg and 40
About 10 times more IgG immobilization if using tig/solid phase carrier
t has increased. From this fact, it can be seen that the solid phase carrier of the present invention has a much larger antibody-bound solution than the untreated carrier, and that a small amount of carrier is sufficient for the antibody-bound antibody to have sufficient reactivity.

In addition, the results of an investigation on the stability of the bond between gG and the homologous Ju form are described below.

The binding stability was examined by subjecting the in-phase carrier on which IgG was immobilized to the following two treatments. As a method for dissociating immobilized substances by ionic bonding, treatment with IN hydrochloric acid at room temperature for 3 hours,
10 as the jQTfl method for immobilized substances by physical adsorption.
%SDS at room temperature for 30 minutes, the released Ig
By measuring G, the bonding equation of D and IgG was investigated.

As shown in the table above, it is clear that in the polystyrene peripheral phase carrier treated with sulfuric acid, about 85- of the immobilized IgG binds to the same phase in an extremely stable ionic form. It can be seen that the solid phase carrier of the present invention has extremely good binding stability.

Next, an example will be described in which the activity retention rate of an antibody in the same-phase carrier of the present invention is compared with that of the same carrier.

Anti-guinea pig Ig (Deutsche spp.) I on a 3-piece solid phase carrier
20 μg of gG fraction in 100 full M phosphate buffer (pif
8.0) The adsorbed IgG let was determined by measuring the remaining IgG-If after reacting the solution (approximately 2 ml of antibody-containing 11) at room temperature for 3 hours and at 5° C. overnight. next,
Guinea pig IgG hJ bound to the same phase was determined by an immunoreaction in which guinea pig IgU Ipq was applied to the antibody 1i'11 fixed solid phase carrier overnight at 5°C. Therefore, as shown in the table above, the polystyrene solid phase support exhibits an activity retention rate of 4''A degree, which is almost the same as the untreated same phase support, and is far superior to the nylon solid phase support. The retention rate was good.

In addition, the above-mentioned nylon homogeneous body was used after introducing an amino group by the immicro method and then activating it by the glutaraldehyde method.

As mentioned above, the sulfonic acid group-introduced polystyrene solid phase carrier of the present invention has an extremely large capacity for immobilizing antibodies per carrier, and therefore, it is possible to immobilize antibodies with a width of 4 sq. Not only is this possible, but the bound antibody also exists in an extremely stable form, so it can withstand repeated use over a long period of time, and the activity retention rate of the antibody is also good.
This is an extremely good carrier for the foot test.

In the in-phase carrier of the present invention, the first
In order to obtain a carrier surface-second antibody-first antibody conjugate formed by binding an antibody, it is necessary to use a measurement carrier t/m- which has been processed into a conventional shape, and to obtain the same phase as described above. Based on this, g32 antibody is first bound. Next, the first antibody is added to this and allowed to bind through the immune reaction, and the immune activity of the first antibody obtained in this way is in the same phase, which is preferable for the tube because it hardly deteriorates. be.

In addition, enzyme-conjugated components used in the measurement, such as antibodies, antigens, 21-butene, etc.
``The component to which the L element is bonded is the well-known EIAt-RIA.
The standard rounding method used in In addition, as enzymes as labeling substances, known enzymes,
For example, oxidoreductase, hydrolase, trans1j″L enzyme,
Enzyme power that develops into lyase, isomerase, and ligase!
Available.

Using the insolubilized antibody immobilized on the measurement carrier in this way, the specific binding property for this antibody etc. is determined by [)'J-
Measure all components to be determined, such as antigens and antibodies.
For this purpose, a known measurement method using glA spanning RIA is applied.

EXAMPLES Hereinafter, the present invention will be specifically explained with reference to Examples, but the present invention is not limited thereto.

Preparation method of solid phase carrier: As shown in FIG. 5, a polystyrene carrier molded into a bullet shape (diameter 12, OtrLm) was pre-cooled at 5° C. for 1 hour. Next, the above phase was added to cooled concentrated sulfuric acid and reacted at 5° C. for 6 hours. After the reaction, the carrier was taken out, thoroughly washed with distilled water, and then thoroughly washed with 100 mM phosphate buffer (pHLO), and the polystyrene solid phase carrier into which sulfonic acid groups had been introduced was immersed.

Example 2 Preparation method of antibody adsorption in-phase carrier: Rabbit Ig was placed in a test tube (inner diameter 12.8 mm ± 0.1 mm).
1 ()0mM phosphate buffer (pH 8,1)) solution of G (
IgG Jt is husband * 25.50.100μg/ag)
0.4 m and e each, and the sulfone obtained in Example 1 was inserted into the polystyrene solid phase carrier into which a hard group was introduced, and the mixture was heated at 5°C for 0.2, 4, 6.8 and 16 hours. At each time, the solid phase carrier was removed, and the proteins in the remaining IgG solution in the test tube were measured using the Cu-Folin method to determine the non-adsorbed Ig.
GJJN was calculated to determine the amount of IgG adsorbed on the solid phase carrier.

The results are shown in FIG. 6, and it was found that the IgG immobilization rate was about 7.5 to 10 times higher than that of Boristine itself (untreated polystyrene). It was also found that the reaction time reached the maximum adsorption capacity in 2 hours, and then reached an equilibrium state.

[Brief explanation of drawings]

The drawings show preferred embodiments of the present invention, and FIG. 1 is a partial sectional view of the solid phase carrier for immunoassay according to the first embodiment inserted into a test tube, and FIG. Solid phase for immunoassay,
A plan view of the carrier, FIG. 3B)(B), and FIG. A vertical cross-sectional view of the phase carrier, FIG. 5 is an actual measurement diagram of an example for making an in-phase carrier for immunoassay, and FIG. 6 is a graph of IgGJl adsorbed to the 1^1 phase carrier. Explanation ■ is the carrier for immunoassay, 2 is the carrier body, and 4 is the test tube. 9, ρ Figure 3 (a) (b) (c) Between the opposite tip and end 015°C Processing t IgG -O- 0-10μ9/Anonymous old 4th day tree - 0-0-20pg/Koki nki Dankyu (50, Ltg IgG 7m)'?0.4m1)-Moo 6-40.Ll (1/Koho B Odan body (100PcJ
IgG/m〕6)0.4m17)382

Claims (1)

[Scope of Claims] (1) A solid phase carrier for immunoassay consisting of (1) a styrene-based polymer into which a sulfonic acid group has been introduced. (2; Styrenic polymer is polystyrene or a monopolymer consisting of styrene, styrene and aminostyrene, divinylbenzene, acrylamide, ethylene, acrylic acid, methacrylic acid, methyl methacrylate,
The solid phase carrier for immunoassay according to claim 1, which is a copolymer of acrinitrile vinyl alcohol, vinyl acetate, phtadiene, and maleic anhydride. (3) According to claim 1, the carrier is formed into a substantially bullet-shaped, spear-shaped, or similar shape while maintaining a certain gap with the reaction tube, and is made of a styrene polymer having sulfonic acid groups introduced therein. solid phase carrier for immunoassay.