JPS61252215A - Substance for fixing molecule containing amino group and itsproduction and use - 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 Field of the Invention The present invention relates to new devices and new techniques in the covalent immobilization of molecules containing active amino groups. Specifically, the present invention is applied to the detection and quantification of proteins present at low concentrations in body fluids such as serum. More specifically, the present invention provides an immunoassay method (Is door unoas).
It is applied to the immobilization of antibodies and antigens that can be used for the quantification of antigenic substances using the technique of ``Say''.

BACKGROUND OF THE INVENTION Immunoassay techniques are increasingly used to determine the presence or concentration of antigenic substances that are present in low concentrations in serum or other body fluids. The above technology has the advantage that the antibody specifically binds to the corresponding antigen, and that the reaction [antibody Ab + antigen Ag - (antibody antigen AgAb)] follows the principle of agglutination reaction. .

A variety of immunoassay techniques have recently been utilized, which can be classified according to the nature of their binding. The most commonly used method is Competitive Binding Assay (Competitive Binding Assay).
y) and non-competitive sandwich assays (Non-
competitive Sandwich As5a
y). Whatever technique is used, labeled antibody or antigen conjugates must be used in detection; the most widely used labels include radioisotopes, enzymes, and fluorophores. I can do it. Methods using these labels include radioimmunoassay [hereinafter sometimes abbreviated as RIA], enzyme immunoassay [hereinafter EI
[sometimes abbreviated as A] and fluorescence immunoassay [hereinafter F
It is sometimes abbreviated as IA].

Radioactive isotope 125, 14G or 3 as a label
Radioimmunoassay using H is still the most widely used immunoassay method due to its extremely high sensitivity. Many of the above RIAs can detect as small an amount of antigen or antibody as IQ-16 moles. R.I.
Despite this high sensitivity, A has a number of disadvantages arising from the dangers of using isotopes that emit high-energy gamma and beta rays in the detection method and problems with the disposal of radioactive waste. In recent years, many attempts have been made to develop new enzyme immunoassay systems and fluorescent immunoassay systems that have sensitivity and measurement speed comparable to RIA. These systems utilize enzymes or fluorophores in place of radioactivity as a means for detecting antigen φ antibody binding reactions.

Engwall and Perlman study immunochemistry (I+s+
suno-chemigtry ) 8:8? l (I
Since first described in 971), methods using enzymes as labeling substances have been developed. Examples of descriptions in this regard include U.S. Patent Nos. 3,654,090 and 3,791
No. 932, No. 3,850,752 and No. 387
9.262, description of each specification and journal reference to immunological method (Journal of Immunological Method)
munological methods) 1:24
7 (I972) and Methods of Enzymology
702419 (I980). Each of the methods described above is a heterogeneous E I
It is based on the A (Heterogenous EIA) measurement principle. This measurement principle includes the step of separating the reacted enzyme-labeled component from the unreacted enzyme-labeled conjugate. These assay methods are particularly suitable for detecting and measuring substances such as antigens or antibodies with a molecular weight of 10,000 or more that are present in body fluids.

In addition, biochemical competition biophysical e-research fist D municesi g 7 (Eliachem, Bio
ph2s.

Res, Goms, ) 47:84B (I972)
Also of interest are the homogeneous enzyme immunoassays described in U.S. Pat. No. 3,817,837. These assays use /\butene (Hapt
The relationship between en) and the enzyme is such that when butene binds to an antibody, the enzyme activity changes. Since homogeneous EIA does not require a separation step, these methods can usually be carried out very quickly. These assays are used in a very wide range of applications, including the determination of therapeutic drug levels, the assay of misused drugs, and the determination of other antigenic substances with molecular weights greater than 10,000.

Fluorescent immunoassays have a sensitivity approximately equal to that of RIA. This method is less reliable compared to RIA because high and variable blanks appear very often. Some typical FIA measurement principles are based on Methods of Enzymology.
Enzymology)? 4:3.28゜80.7
8 and 87 (I981) and U.S. Patent No. 3,90
No. 1,654.

Chemiluminescence has also been utilized in immunoassay processing. Chemiluminescence refers to light produced as a result of chemical changes. This involves converting the free energy of a chemical reaction into light energy. The well-known chemiluminescence reaction uses hydrogen peroxide as the oxidant and a catalyst (e.g. Fe3, Cu2
There is an oxidation of luminol (5-amino-1,4-dihydroxyphthalazine) in basic solution in the presence of Co2. Some representative chemiluminescent immunoassay methods are described in U.S. Patent No. 4.104,02
No. 9, No. 4,375,972, No. 4,380,580
It is stated in each specification of the issue.

Electron spin resonance for use as a labeling means in immunoassay procedures is described in US Pat. No. 3,850,578. This method requires relatively expensive equipment and free electrons yielding a label, which is a potential carcinogen.

Enzyme immunoassays are used not only in the field of clinical analysis, but also in microbiology, viral biology, and biochemistry.

Its scope of application is also expanding to the fields of tissue culture and immunology. Among these assay methods, representative ones include:
To make it convenient to process many tests in parallel,
A plastic micro titration dish (formerly known as a crotch plate) with 96 wells (Well; 8
A method carried out in a titer dish (titer dish) can be mentioned. The above wells (capacity, 0.3 mjL) were used to read the absorbance of each content along the vertical axis using a high-speed colorimeter with automatic X-Y movement and display mechanism, similar to a spectrophotometer cuvette. function as expected.

ETAs using commercially available microtitrators are almost always heterogeneous immunoassays, using physical adsorption of the primary antibody or antigen to the solid surface of the well. Although the tertiary structure varies,
Due to weak hydrophobic van der Waalska interactions between protein and polymer, approximately 1 to 200 ng
A relatively weak bond density of /ctn' is obtained. Adsorptive binding between antibodies and antigens has clear limits in the measurement range, and errors arise due to low binding density and various desorptions of antibody φ antigen complexes. It is widely used not only in FIA but also in heterogeneous competitive RIA.

Recently, antibodies or antigens have been synthesized onto solid-phase (e.g., Journal of
Several methods have been disclosed for attachment via covalent bonds to polystyrene beads (reported in Immunological Methods 34:315 (I980)). In the above method, polystyrene beads are treated with nitric acid and reduced with an alkaline solution of sodium dithionite to form an aminopolystyrene surface. The beads are activated with glutaraldehyde and used to covalently attach antibodies. Beads producing these covalently immobilized antibodies were also used in sandwich-type immunoassays. These assays, which use polystyrene beads to which antibodies are covalently attached, have been shown to have higher surface densities of antibodies, shorter incubation times, and improved accuracy and range. A similar method is published in Journal of Immunological Methods 57:87 (I983
)It is described in. In the above method, Schistosoma mangoni antigen is covalently attached to the surface of a 96-well polystyrene microtitration dish. The microtitrator was later used to measure antibodies in patient serum in enzyme immunoassays.

In the above operation, an aminopolystyrene surface is formed in the well by a nitration reaction using a mixture of fuming nitric acid in glacial acetic acid and reduction using alkaline sodium dithionite. Antigenic proteins are N-chain molecules that act as short coupling molecules that bind a single protein.
Ethyl-N'-(3-dimethylaminopropyl)-lupodiimide and superic acid are used to attach to the aminopolystyrene surface. This report also shows that the assay performed using covalently bound antigen has higher sensitivity compared to that using physically adsorbed antigen.

SUMMARY OF THE INVENTION The present invention provides novel methods and means for covalently immobilizing molecules such as antibodies or antigens to various polymer surfaces. The present invention also provides an instrument that can obtain a linear detection relationship over a wide concentration range of a substance to be detected and quantified when applied to an immunoassay. Furthermore, the present invention can be used for the immobilization of all molecules having active amino groups for measurement or production purposes, such as enzyme immobilization, DNA immobilization, cell immobilization, virus immobilization. It can be used for immobilization of bacteria, affinity chromatography, etc.

The device of the present invention has a plastic structure (e.g.

It takes the form of vials, cuvettes, beads, 96 (microtitrator with II wells). In immunoassay applications, antibodies can be covalently immobilized to the above structures using the novel method and used in heterogeneous enzyme immunoassays or fluorescent immunoassays. The above method is simpler than prior art techniques that simply utilize adhesive protein binding, and is more accurate than current techniques that utilize covalent bonding systems.

In the present invention, for example, the antibody (protein) used as an unlabeled antibody in a sandwich-type enzyme immunoassay or fluorescence immunoassay is covalently bonded to a long spacer molecule (the spacer itself is also covalently attached to the polymer surface). By doing so, it can be immobilized on the polymer surface by a covalent bond. The spacer molecule is synthetic poly-. The spacer molecule is
The polymer is covalently attached to the surface by chemically or photochemically initiating graft polymerization using two or more suitable monomers and a free radical generating system. - of the monomer is CH2=CRWZ (
The Z group has a structure that is capable of chemically reacting with reactive amino acid groups of proteins. Therefore, protein is
Covalently attached to a spacer molecule. The second monomer spaces the active groups Z along the spacer molecule. Moreover, the above monomer has a structure of CH2=CRY (the Y group is a group capable of imparting the necessary hydrophilicity to the long spacer molecule). Graft polymerization of the above monomers is carried out in various solvents or mixtures thereof, from 0°C in the photochemically initiated method, or from 40°C in the chemically initiated method, in these solvents. and in an inert gas. After the grafting is completed, the grafted plastic structure is washed to remove unreacted monomer, ungrafted polymer, organic solvent, and residual initiator. and antibodies in an aqueous buffer solution,
It is covalently immobilized on the activated spacer molecule.

The plastic structure containing the immobilized antibody can be dried (after neutralizing the active groups remaining on the spacer molecule) and used in a competitive enzyme immunoassay, a sandwich-type enzyme immunoassay, or a fluorescent immunoassay. .

The present invention therefore provides surface-treated polymeric materials for use in the immobilization of molecules containing effective amide 7 groups. the substance has a plurality of polymers bonded thereto;
The polymer is formed from a mixture of at least two monomers, one of the monomers containing an active group effective for binding to the amine 7 group in the immobilized molecule, and one of the monomers containing a plurality of the active groups. is dispersed on the polymer at a distance between the substance and the substance. The present invention also provides a method for producing a conjugate of the substance and the polymer and a method of use specific to the processing of an immunoassay. .

Therefore, by applying the present invention to immunoassays, long spacers can be used instead of polyclonal antibodies immobilized by adhesive, which are currently used as antigenic substances in heterogeneous enzyme immunoassays or fluorescent immunoassays. Antibodies that are covalently immobilized via a molecule (the spacer itself being covalently attached to a polymeric support) can be used. The present invention is thus useful for detecting the concentration or presence of various types of antigenic substances and other biological substances present in body fluids. The description will be made with reference to the immobilization of antigens and antibodies in methods and the like. Therefore, the term antigen or antigenic substance as used herein generally refers to a substance against which polyclonal or monoclonal antibodies can be produced. Examples of the antigen include human IgG, human IgM, human IgA, mouse IgG, and human fibrinogen.

The present invention is also useful for covalently immobilizing antigens for use in a variety of homogeneous competitive radioimmunoassays, enzyme immunoassays, and fluorescent immunoassays. Examples include protein A.

However, the present invention can also be used to immobilize molecules having an effective amino bonding group for purposes such as analysis and production by bonding them to plastic materials. For example, the present invention can be used to covalently immobilize polypeptides, polysaccharides, or proteins such as enzymes, DNA, viruses, bacteria, or cells.

DETAILED DESCRIPTION OF THE INVENTION These and other features of the invention will be described in detail below with reference to the accompanying drawings.

FIG. 1 is a block diagram illustrating the grafting process in the formation of long spacer molecules covalently attached to a polymer surface and the covalent immobilization of antibodies by activated spacer molecules.

Figure 2 shows mouse IgG and human IgG and IgM.
Figure 2 shows a plot of absorbance versus the natural logarithm (log) of antigen concentration, with standard error line (3σ), showing results obtained using polyclonal antibodies bound by conventional adhesion in three enzyme immunoassays for .

Figure 3 shows mouse IgG and human IgG and IgM.
Plots of absorbance versus natural logarithm (log) of antigen concentration with standard error lines showing results obtained using polyclonal antibodies covalently immobilized on long spacers in three enzyme immunoassays for (3σ).

As noted above, the invention will be described with particular reference to immobilization of antibodies or antigens.

A representative method of covalently attaching an antibody or antigen to a polymer surface via a spacer molecule according to the present invention is 1.
The present invention is illustrated in FIG.

The main feature of this method is the use of covalent bonds (as a result,
In addition to linking the antibody to the polymer surface in an irreversible manner, it also creates a space between the antibody molecule and the polymer surface via a synthetic chain molecule (described herein as a spacer molecule). It's also about putting it down.

As explained in detail below, the spacer molecule is formed by grafting several types of monomers,
Produces chain polymers that covalently attach to the polymer surface of plastic materials.

One of the monomers provides an active site rZJ that splits along the spacer molecule and covalently binds to the antibody. Therefore, the antigen to be measured can be bound to an antibody and a tracer or labeled antibody that tracks the antigen for analysis.

Antibody molecules bound to the polymer surface by long (up to several hundred angstroms in length) spacer molecules have a parallel Although the increased freedom of movement and rotational movement of immobilized antibody molecules, which exhibit movements very similar to dissolved molecules, the high degrees of freedom with respect to translational and rotational movement of immobilized antibody molecules is Improves the kinetics of antigen binding in competitive immunoassays. This effect is even more pronounced in sandwich-type enzyme immunoassays where the presence of a second antibody conjugated to the enzyme further promotes steric aggregation around the sandwich complex.

With this treatment method, the incubation time required for antigen binding and second antibody-enzyme binding can be significantly reduced without any obvious deterioration of the measurement results.

Additionally, it is important to note that the present invention provides superior control over the density of antibodies or antigens covalently immobilized on the polymer surface. The protein surface density of antibodies and antigens obtained by adhesive bonding varies greatly, and depends on the structure of the protein, the protein concentration used in the coating solution, the conditions at the time of adsorption treatment such as temperature and pH, etc. These antibodies or antigens vary depending on the surface structure of the plastic to which they are adsorbed. Adhesive conjugations of the type practiced in the prior art generally suffer from low usable yields, but the density of antibodies on plastic surfaces is acceptable. The sticky bonds of antigens, especially low molecular weight antigens, are more unstable, and the low surface density of antigens limits the sensitivity and analyte range of these assays (Dyna
micRange). As shown in Figure 1, antibodies or antigens with free amino groups from lysine residues react with the active group Z on the spacer molecule to form a covalent bond that permanently immobilizes the protein molecule. In the above procedure, the yield of bound antibody or antigen is therefore greatly increased compared to prior art adhesive binding. Furthermore, the amount described can also be easily controlled by a quenching reaction of the reactive Z group on the spacer molecule.

Furthermore, it is recognized that the present invention is superior in controlling non-specific protein binding compared to the prior art. Nonspecific binding of proteins (eg, antibody-enzyme conjugates) is a well-documented phenomenon and is probably one of the major sources of error in heterogeneous immunoassays.

Non-specific protein binding in standard adhesive enzyme immunoassays and fluorescence immunoassays is determined by adding the appropriate protein (e.g. bovine serum albumin [Bovine Serum AIbu+sin;
BSA], porcine gelatin) at high concentrations saturates the plastic surface and reduces and prevents non-specific binding of the antibody-enzyme conjugate or the antibody-fluorophore conjugate. Saturation of the surface with BSA molecules reduces binding of the antibody-enzyme conjugate by increasing steric aggregation around the antibody-antigen complex, while decreasing the sensitivity of the immunoassay. However, in the present invention.

Since the antibody or antigen molecule is covalently attached to the polymer via a long spacer molecule, the spacer effectively isolates the molecule from the surface saturated with BSA or gelatin, so that steric aggregation is negligible. becomes. Furthermore, by using a suitable monomer in the formation of the spacer molecule, the spacer molecule itself can have hydrophilic properties that prevent non-specific binding.

In the present invention, the method for covalently bonding antibodies or antigens to polymeric (plastic) structures consists of a two-step process. As shown in FIG. 1, spacer molecules are first formed on the surface of a plastic structure using a treatment method that chemically or photochemically initiates the grafting of two or more monomers. An antibody or antigen is then covalently bound to the spacer molecule. Preferably, one of the monomers (which are graft polymerized and thus form spacer molecules on the polymer surface) is a vinyl monomer having a 5-shell structure.

CH2=CR+ -W-Z R1 group is a hydrogen atom, a methyl group, or a carbon atom number of 16
Hereinafter, an aliphatic or aromatic group having 1 to 6 carbon atoms is preferred. The X group is an active ester group and must have the following membrane structural formula.

R3 and R4 groups are hydrogen atoms or hydrocarbon groups having 16 or less carbon atoms, preferably 1 to 6 carbon atoms, and the structure of the hydrocarbon group is aliphatic, aromatic, or aliphatic. It can have oxygen, nitrogen, sulfur, fluorine, bromine and chlorine atoms as heteroatoms. The R3 and R4 groups may or may not be bonded to each other, and when R3 and R4 are bonded, they may both form an aromatic group. One preferred structure includes an active ester group of the membrane structural formula below.

In the above-membrane structural formula, n is l, 2 or 3.

The W group that connects the X group to the vinyl group has 16 or less carbon atoms, preferably 1 to 6 carbon atoms, and its structure is aliphatic,
Consists of aromatic or a combination of aliphatic and aromatic. The W group can also contain oxygen, nitrogen, and sulfur atoms as heteroatoms. The above active ester is a derivative of N-diacylhydroxylamino, and is known to easily react with an amino group on a ricin residue in a protein to form an amide derivative. The protein molecule is covalently bonded to the active ester-bearing group via the amide derivative. The above amide forming reaction has an optimum pH in the range of pH 7.5 to 8.5. The amide-forming reaction is also known to have a very high aminolysis/hydrolysis ratio of about 106, thus ensuring high yields of the amide-forming reaction even at very low protein concentrations. Other preferred features of the active ester group include:
Since the above ester group can be easily suppressed by ammonium ions or primary amino groups containing compounds,
One of the points that can be mentioned is that it is possible to appropriately and preferably control protein binding.

The second monomer that is graft-polymerized to form the spacer molecule has the following general formula.

CH2=CR2-Y In the above general formula, R] is a hydrogen atom, a methyl group, or has 16 or less carbon atoms, preferably 1 to 6 carbon atoms.
It is an aliphatic or aromatic group. The Y group is an aliphatic and/or aromatic group having 16 or less carbon atoms, and can contain an oxygen atom, a nitrogen atom, and a sulfur atom as heteroatoms. The groups provide spacing between the active X group on the spacer molecule and the polymeric material and also provide the spacer molecule with favorable hydrophilic properties. The above Y group is preferably an aliphatic group having 1 to 6 carbon atoms. In particular, when a relatively hydrophilic spacer is required, the Y group is a hydroxyl group, an amide group, a carboxyl group, or It is preferable to contain a wood-philic group similar to these.

Furthermore, a third monomer can also be graft polymerized to form a spacer molecule. The third monomer has the following - membrane structural formula.

CH2=CR5-X-Rs C=CH2 In the above membrane structural formula, R5 is a hydrogen atom, a methyl group, or an aliphatic or aromatic group having 16 or less carbon atoms, preferably 1 to 6 carbon atoms. It is. The X group has 16 or less carbon atoms, and its structure is aliphatic, aromatic, or a combination of aliphatic and aromatic. The X group can also contain oxygen, nitrogen, and sulfur atoms as heteroatoms. Further, the X group preferably has 1 to 6 carbon atoms. The above divinyl compound is added as a crosslinking agent or a branching agent to act on the structure of the spacer molecule.

The molar ratio of the monomers CH2= CR+ -W -Z, CH2=CR2-Y and CH2= CRs -X -Rs C= CH2 used in the grafting operation depends on the number of active groups (binding sites) on the spacer molecule. and determine the spacing, hydrophilicity of the spacer molecule and crosslinked structure of the spacer molecule. It will be clear that the above method creates more widely spaced binding sites, which is necessary to enable accurate measurements of high concentration substances.

Chemically initiated grafting processes are initiated by free radical generating compounds such as azo compounds, organic peroxides and peresters. All of the above compounds decompose at certain temperatures, producing free radical species that initiate polymerization and grafting reactions. The chemically initiated grafting process is carried out at a temperature ranging from 40°C to the boiling point of the solvent used in the process. The most suitable solvents are those that have good solubility, are inert towards the monomers, do not dissolve or swell the grafted plastic structures, and have a boiling point above 50 °C. It is. Examples of types of solvents that can be used include alcohols, nityls, esters, ketones, and the like. All grafting operations must be carried out under an inert gas, e.g., at atmospheric or higher pressure, in nitrogen or argon. Reaction times vary, but are usually between 2 and 10 hours. within the time range.

Photochemically initiated grafting processes use UV radiation and photoactive compounds that generate free radicals, such as aromatic ketones. The above compound absorbs ultraviolet rays of a specific wavelength having a high quantum yield and generates free radical species that initiate grafting and polymerization reactions. The preferable ultraviolet rays used in the present invention include 320 to 400 nm, preferably 360 nm. The next example is near-ultraviolet rays. The UV spectrum in the above region is a black light fluorescent discharge lamp,
In the normal case, the energy density of the UV irradiation used in the present invention ranges from 25Wh/rn' to 1oOW.
It is within the range of h/rn'. The photografting treatment operation is carried out at a temperature from 0° C. to the boiling point of the solvent used in the treatment.

The same types of solvents used for chemically initiated grafting can also be used for photografting processing operations. The most typical of the above solvents are alcohols, ethers, esters and ketones. Preferred photoinitiating compounds have triplet energies (Triplet
Energ7; E T) is 67kg Φ force Lolly 1
It is known to be a mole of benzophenone. In addition, the inert gas is generally nitrogen or argon under atmospheric pressure.The reaction time obtained varies, but usually 10
The duration is from 1 minute to 2 hours.

The grafted plastic structure is washed with an organic solvent and deionized water to remove precipitated copolymer, organic solvent, unreacted monomer, and decomposition products of the starting compound. The grafted plastic construct is
Generally, it is used immediately for antibody or antigen binding, or it can simply be vacuum dried, sealed in a polyethylene bag, and refrigerated. It has been found that when stored under anhydrous conditions, the grafted plastic remains active for covalently immobilizing antibodies or antigens for at least six months.

Typical plastic materials that may be used include polystyrene, polyvinyl chloride, polyethylene, polypropylene, polycarbonate and polyacrylate.

Covalently binding the antibody or antigen to the spacer molecule is carried out in an aqueous, non-nucleophilic buffer solution at pH 7.5 to 8.
．． 5 and at temperatures ranging from 0°C to 37°C.

It is a short period of time, from 1 hour to 18 hours. The protein concentration in the binding solution can be as low as 1100 n/mJL, but when using antibodies it can be as low as about sgg/mJL.
It is common to set it as a degree.

Surface stabilization of plastic surfaces coated with antibodies or antigens can be carried out using solutions containing bovine serum albumin, egg albumin, nonionic surfactants such as Tween-20 (registered trademark), or mixtures thereof. In some cases, it is advantageous to include a certain amount of ammonium ions in the stabilizing solution in order to suppress the active ester groups remaining on the spacer molecules.

Plastic surfaces coated with antibodies or antigens can be lyophilized and stored under anhydrous conditions at subzero temperatures for at least 12 months.

By using the bonded structures of the present invention, not only can higher concentrations of substances be measured, but the optical clarity of the substances is maintained even in high concentration applications, thus ensuring accurate identification of the bound substances to be analyzed. Measurement becomes possible.

The enzyme immunoassays for mouse IgG, human IgA, human IgG, human IgM, and human fibrinogen exemplified below were performed using goat or rabbit polyclonal antibodies. All of the above testing methods are
It is a sandwich type in which a second antibody-enzyme conjugate is used for photometric or fluorescent measurements of products formed by enzyme catalysis. Antibody-enzyme conjugates include alkaline phosphatase using p-nitrophenyl phosphate as a substrate or 2,2°-azodi(3-ethylbenzthiazoline-6-sulfonate) (ABTS) as a chromogen.
One of the horseradish peroxidases used was radish peroxidase. The alkaline phosphatase conjugate contains the fluorescent substance 4-methylumbelliferyl 7,
liferyl phos-phate) was also used. Photometric measurements of enzyme products were carried out using a 96-well plate reader (Guinatec MR6).
00) was used. For the fluorescence measurement method, a plate reader (Microfluoro manufactured by Dynatic Co., Ltd., pre-registration Ie') equipped with 96 wells was used.

As shown in FIGS. 2 and 3, a plate with 96 wells coated with antibodies by adsorption and a covalently immobilized plate of the present invention were used to detect mouse IgG and human IgG. By sequentially comparing enzyme immunoassays for IgM and human IgM, it is readily seen that the antibody-adsorbed assay decreases in the region of high antigen concentration. The available detection range, indicated by the overlapping error line in the plot of FIG. 2, is from 0.8 ng to 20 ng of antigen for adsorption coated plates.

With the covalently coated plate shown in Figure 3, the available detection range is extended from 0.8 ng to 200 ng of antigen. Furthermore, when using the covalently immobilized antibodies of the present invention, the error line (3σ) is clearly reduced.

This may be due to the high antibody concentration, the permanent immobilization of the antibody to the polymer surface, or the reduction of steric aggregation by long spacer molecules that immobilize the antibody and provide greater freedom of movement for the antibody. This is due to

The grafted polymeric materials of the present invention can be used in immunoassay processing methods as well as for the identification and measurement of all substances that have an active amino group attached to an active group (i.e., an active ester group) provided on the spacer molecule. It will be clear that the method can be used for all types of detection procedures, as well as for the type of label used and whether an enzyme immunoassay, a fluorescent immunoassay, a radioimmunoassay or a cold-light immunoassay is used. There are no particular limitations; in addition, the present invention is applicable to other utilization methods that require the immobilization of the above-mentioned molecules (e.g., D
It can also be used for denaturation of NA).

[Example 1] Acrylamide (purity is at the level used for electrophoresis)1.
50 g and N-acryloxysuccinimide 1. O
Og 2-propatool (moisture content 0.2% or less) 7
0. Dissolved in OmjL at 25°C using a magnetic stirrer.

2.2″-7zobis(2-methylpropionitrile)
(AZOBIS) 50 mg was dissolved in 0.50 ml of A7Tone, and this solution was added to the above 2-propertool solution, stirred for another 2 minutes using a magnetic stirrer, and then filtered with a sintered glass filter. .

Two 96-well polystyrene microtitration plates (LimbroJ) were filled with the above-mentioned filtered solution containing AZOBIS and monomers that generate free radicals. -Propertool solution was filled to 0.280 ml per well. The plate was placed in a vacuum oven heated to 157°C. The oven was rapidly evacuated until the pressure dropped to 100 mbar and filled with pure (99.9%) argon until the pressure reached 1000 mbar. In order to further reduce the oxygen concentration in the oven, the above operation was repeated two or more times. The plate was placed in the oven at 57°C for 4.5 hours. A white precipitate appeared in the wells in about 1 hour.

After 4.5 hours, remove the plate from the oven and heat it for 30 minutes.
Leave for a minute to cool.

The plate was washed five times with deionized water in a 96-well microtitration plate washer. The time required for washing the two plates was less than 3 minutes.

A solution of goat anti-mouse IgG antibody (manufactured by Tago, affinity purification) (5 g/ml of antibody in 0.05 M sodium phosphate buffer, pH 8.5) was added to the wells of the washed plate. Filled with 15 mu.

The plate was left at 20°C for 1 hour and then stored at 4°C for 18 hours. The plate was further washed three times with deionized water, and each well was treated with 1% bovine serum albumin and 0.0% bovine serum albumin.
300 mfL of pH 8.5 sodium phosphate buffer containing 0.02% sodium azide was charged. Plates were kept at 20°C for 4 hours and at 4°C for an additional 18 hours.

The plate was then washed three times with deionized water again.

Preparation of antigen or sample with 1% bovine serum albumin, 0
002% sodium azide, and calcium chloride 2
Diluted in pH 8, 2(7)O, 1M Tris-HCl buffer containing mM and added in a 0.150 m volume to the wells. Each sample or standard sample was analyzed in at least triplicate.

Standard samples were used in the range of 0.8 ng to 200 ng per well. Plates were incubated for 18 hours at 4°C. The plates were again washed three times with deionized water before adding the enzyme-antibody conjugate. Goat anti-mouse Ig
G antibody/alkaline phosphatase conjugate (manufactured by Tavo, affinity purified) was prepared in 0.1M hydrochloric acid buffer at pH 8.2 containing 1% bovine serum albumin, 0.02% sodium azide and 2mM calcium chloride. It was diluted so that the abundance ratio in the liquid was 1:1000. 0.150 mM of the conjugate solution was distributed to each well except for the blank well. The plates were incubated for 2 hours at 37°C and 1 hour at 20°C. After washing three times with deionized water, the wells were injected with 0.5mM p-nitrophenyl φ phosphate solution (2mM magnesium chloride).
1.0 at pH 8.2 containing M and 0.5 mM zinc chloride.
0.150 m (in Tris/HCl buffer) was filled.

The rate of increase in the absorbance of the wells at 410 nm was measured using digital equipment.
The plates were read on a Guinatec MR600 automatic plate reader connected to a VANII/750:1 monitor (Ment). The computer accepts the kinetic data as is, and performs statistical and graphical analysis such as plotting absorbance against the natural logarithm (log) of antigen concentration and plotting variation values against the natural logarithm (log) antigen concentration. to be processed.

[Example 2] Acrylamide (purity is at the level used for electrophoresis)1.
50g, N-acryloxysuccinimide 0.70g
and N,N"-methylin-bis-acrylamide 0.
030g to 2-propertool (moisture content 0.2% or less) 70. Dissolved in OmfL at 25°C using a magnetic stirrer. 2. Dissolve 50 mg of 2°-7 zobisu(2-methylpropionitrile) (AZOBIS) in 0.50 ml of acetone, add this solution to the above 2-propanol solution, and stir magnetically for an additional 2 minutes.
After stirring using a stirrer, the mixture was filtered through a sintered glass filter.

The filtered 2-propanol solution containing free radical-generating AZOBIS and monomer was added to two 96-well polystyrene microtitration plates (Limbro) at 0.0. It was charged to 280mA. The plate was placed in a vacuum oven heated to 57°C. The oven was rapidly evacuated until the pressure dropped to 100 mbar and filled with pure (99.9%) argon until the pressure reached 1000 mbar. In order to further reduce the oxygen concentration in the oven, the above operation was repeated two or more times. The plate was placed in the oven at 57°C for 4.5 hours. A white precipitate appeared in the wells in about 1 hour. 4
After 5 hours, the plate was removed from the oven, allowed to cool for 30 minutes, and washed five times with deionized water in a 96-well microtitration plate washer. The time required to wash the two plates was 3.
It took less than a minute.

Goat anti-mouse IgG antibody (manufactured by Tavo, affinity purification) solution (pH 8.5) was added to the wells of the washed plate.
Antibody 5Pg in 0.05M sodium phosphate buffer
/mfL) was loaded with 0.150 mu. The plate was left at 20°C for 1 hour and then stored at 4°C for 18 hours. The plate was further washed three times with deionized water, and each well was filled with 0.300 mJl of sodium phosphate buffer, pH 8.5, containing 1% bovine serum albumin and 0.02% sodium azide. Plate is 4 hours 2
It was kept at 0°C and then at 4°C for 18 hours. The plate was then washed three times with deionized water again.

Antigen or serum sample preparations were diluted in 0.1 M Tris Φ-HCl buffer, pH 8.0, containing 1% bovine serum albumin, 0.02% sodium azide, and 2 mM calcium chloride and added to the wells. .150mM volume was added. Each sample or standard sample was analyzed in at least triplicate. Standard samples range from 0.8 ng to 2 per well.
It was used in a range of 00 ng. Plates were incubated for 18 hours at 4°C. The plates were again washed three times with deionized water before adding the enzyme-antibody conjugate.

Goat anti-mouse IgG antibody/wasabi radish peroxidase conjugate (manufactured by Tabo, affinity purified),
1% bovine serum albumin, 0.01% methiolate and 1% sodium chloride in a 0.05M sodium phosphate buffer with pI (a, 5) in a 1:1 ratio.
It was diluted to 000. Conjugate solution 0.150m
jL was distributed to each well except for the blank well. The plates were incubated for 1 hour at 20°C. After washing three times with deionized water, the wells were treated with 0.1% 2.2°-Azo-di(3-ethylbenzthiazoline-
0.150 ml of 6-sulfone) (ABTS) solution (in citric acid/phosphate buffer pH 5.0 containing 0.003% hydrogen peroxide) was charged. The absorbance of the well is 410
In nm, Digital Equipment Co., Ltd. VAN
The plates were read on a Dynatic MRf;OO automated plate reader connected to a II/750 computer. The computer performs statistical and graphical functions as described in Example 1.

[Example 3] Acrylamide (purity is at the level used for electrophoresis) 21
．． 5g and N-acryloxysuccinimide 10.
0 g of solution was added to 2-propatool (moisture content 0.2%).
(below) prepared using a magnetic stirrer at 25° C. during 1000 mi. Another solution containing 72 g of AZOBISO was added to 5. This solution was added to the above 2-propertool solution under stirring. After the above solution was filtered through a sintered glass filter, a volume of 0.280 m3 per well was distributed to 36 wells of a 96-well microtiter plate (manufactured by Linpro).

The plate was placed in a vacuum oven heated to 57°C. The oven was rapidly evacuated until the pressure dropped to 100 mbar and filled with pure (99.9%) argon until the pressure reached 1000 mbar. In order to further reduce the oxygen concentration in the oven, the above operation was repeated two or more times. The plates were placed in the oven at 57°C for 4.5 hours. A white precipitate appeared in the wells in about 1 hour. After 4.5 hours, the plates were removed from the oven, allowed to cool for 30 minutes, and washed five times with deionized water in a 96-well microtitration plate washer.

Goat anti-human IgM antibody (manufactured by Tavo, affinity purification) solution (antibody 5 #g/mf in 0.05 M sodium phosphate buffer, pH 8.5) was added to the wells of the washed plate.
(containing L) was loaded at 0.150 mM. 20 plates
After being left at 4°C for 1 hour, it was stored at 4°C for 18 hours. The plate was further washed three times with deionized water and each well was filled with 0.0% sodium phosphate buffer, pH 8.5, containing 1% bovine serum albumin and 0.02% sodium azide.
．． Filled with 300 mfL. Plates were kept at 20°C for 4 hours and at 4°C for an additional 18 hours. The plates were washed three times with deionized water, the remaining water was shaken out of the wells, and finally sealed in a polyethylene bag and stored at -18°C.

The plate described above with goat anti-human IgM antibody covalently immobilized did not show any decrease in enzyme immunoassay for human IgM for 12 months after being stored at -18°C. 4] Acrylamide (purity is at the level used for electrophoresis) 19
．． 3g and N-acryloxysuccinimide 9.0
A solution of g was prepared in a 900 m 2-propanol (moisture content below 0.2%) solution at 25°C using a magnetic stirrer. Another solution containing 84 g of AZOBISIo was prepared in 15% of acetone, Omi, and this solution was added to the above 2-propertool solution under stirring. After filtering the above solution with a sintered glass filter, 0.280 mJl per well was added to 36 wells of a 96-well microtiter plate (manufactured by Linpro).
The amount was distributed.

The plate was placed in a vacuum oven heated to 57°C. The oven was rapidly evacuated until the pressure dropped to 100 mbar and filled with pure (99.9%) argon until the pressure reached 100 mbar. In order to further reduce the oxygen concentration in the oven, the above operation was repeated two or more times. The plate was placed in the oven at 57°C for 4.5 hours. A white precipitate appeared in the wells in about 1 hour. After 4.5 hours, the plate was removed from the oven and left to cool for 30 minutes. The plate was then washed five times with deionized water in a 96-well microtiter plate washer. The plates were dried for 24 hours at 25° C. in a vacuum of o, i mbar.

Immediately, it was sealed in a polyethylene bag (DRIERITE (registered trademark) was placed inside the bag to absorb remaining moisture). Note that the above-mentioned dry elite is intended to absorb moisture that may cause hydrolysis of the active ester group on the spacer molecule. When stored at 20°C, the above plate can be stored for 3 months and at 4°C.
It did not show any loss in antibody or antigen binding activity for 6 months when stored for 6 months.

[Example 5] In the same manner as in the method described in Example 2, two plates each having 96 wells (manufactured by Guinatec, Microfluor B [formerly crofluor"B"; registered trademark) were It was grafted using N-acryloxysuccinimide and acrylamide and coated with goat anti-mouse IgG antibody (manufactured by Tabo).

Preparation of antigen or sample with 1% bovine serum albumin, 0
．． 02% sodium azide and calcium chloride 2m
p) Ia containing M was diluted in 0.1 M) Lis-HCl buffer and added in a volume of 0.150 ml per well.

Each sample or standard sample was analyzed in at least triplicate. Standard samples were used ranging from 33 picograms to 9.0 nanograms (9000 picograms). Plates were incubated for 18 hours at 4°C, then
Washed three times with deionized water.

Goat anti-mouse IgG antibody O alkaline phosphatase conjugate (manufactured by Tavo, affinity purification) was added to 0.5 μM of pi (8,2) containing 1% bovine serum albumin, 0.02% sodium azide, and 2 mM calcium chloride. 1
M) Diluted in Lis/HCl buffer to an abundance ratio of 1:1000.

0.150 mM of the conjugate solution was distributed to each well except for the blank well. The plates were incubated for 2 hours at 37°C and 1 hour at 20°C. After washing three times with deionized water, the wells were treated with a 0.5 mM 4-methylumbelliferyl phosphate solution (pH 8, containing 2 mM magnesium chloride and 0.5 mM zinc chloride).
2 l.

0.200 mjL of OM) in Lis-HCl buffer was filled. The rate of increase in fluorescence was measured using Microfluoro (formerly croFLOUR; trademark) reader MR600 manufactured by Guinatec.
Measurements were made using an automated plate reader.

The excitation wavelength was 350 nm and the emission was measured at 480 nm. Statistical and graphical processing functions are added to the kinetic data, such as plotting fluorescence (arbitrary units) against the natural logarithm (log) of antigen concentration and variation values against the natural logarithm (log) antigen concentration. [Example 6] Similar to the method described in Example 2, a microtiter polystyrene plate with 96 wells (Linpro) was used. ) were grafted using N-acryloxysuccinimide and acrylamide. Similar to the method described in Example 1, the plates were coated with antibody (goat anti-mouse IgG antibody) and then inactivated using 1% BSA. However, Il
A second antibody labeled with buffer solution 75 terminal phosphatase was added to each well, and the same procedure was immediately repeated with the sample or standard sample in the same volume of buffer.

The plate was then incubated at 37°C for 2 hours. Other procedures, such as adding substrate and reading absorbance at 410 nm, were similar to the standard analytical method described in Example 1.

[Example 7] Similar to the method described in Example 2, two microtiter polystyrene plates (manufactured by Linpro) each having 96 wells were treated with N-acryloxysuccinimide and acrylamide. Grafted.

Plates were incubated at -4°C using human fibrinogen in 0.05M sodium phosphate buffer at pH 8,5.
Cover at. Ten wells are used for testing each concentration of fibrinogen. The amount of fibrinogen used per well was O'. 2 for 15m1 capacity
000, 500.

100, 50, 1O and 1ng. After washing, the plate was washed with 1 ml of 0.05 M phosphate buffer at pH 8, 5.
%BSA solution for 4 hours. Inactivate at room temperature.

After removing excess BSA by washing, 1% Na
cjL, 1:5 abundance ratio in 0.05M phosphate buffer containing 1% (7) BSA and 0.01% methiolate
Add horseradish peroxidase-labeled goat anti-human fibrinogen antibody diluted to 0.00 to the plate.

Each well contains 0-15 mJL of antibody. Incubate the plate with the enzyme for 1.5 hours at room temperature. After washing, add 0.15 mJl of 1% ABTS in pH 5, 0 citrate/phosphate buffer containing 0.03% hydrogen peroxide to each well. Add substrate and read plate 5 minutes later.

[Example B] Acrylamide (purity is at the level used for electrophoresis)1.
50g and N-acryloxysuccinimide 0.7
00g to 2-propatool (moisture content 0.2% or less)
70. Dissolved in OmJL at 25°C using a magnetic stirrer.

2,2'-7zobis(2-methylpropionitrile)
(AZOBIS) 50mg 0.

50m! This solution was added to the above 2-propertool solution, stirred for another 2 minutes using a magnetic stirrer, and then filtered through a sintered glass filter.

The above filtered 2-propatool solution containing free radical-generating AZOBIS S and monomer was added to two 96-well polyvinyl microtiter plates (manufactured by Dynatic) at a concentration of 0.20 per well.
It was filled to a level of mA. The plate was placed in a vacuum oven heated to 57°C. The pressure of the above oven is 100
It was rapidly evacuated until it dropped to mbar and filled with pure (99,9%) argon until the pressure reached 1000 mbar. In order to further reduce the oxygen concentration in the oven, the above operation was repeated two or more times. The plate was placed in the oven at 57°C for 4.5 hours. A white precipitate appeared in the wells in about 1 hour. After 4.5 hours, remove the plate from the oven and let it cool for 30 minutes.
The wells were washed five times with deionized water in a microtiter plate washer with 3 wells. The time required to wash the two plates was less than 3 minutes. A goat anti-mouse IgG antibody (manufactured by Tavo, affinity purification) solution (pH 8.5) was added to the wells of the washed plates.
Antibody 5g/m in 0.05M sodium phosphate buffer
0.150m sentences (including standing) were filled. 20 plates
After being left at 4°C for 1 hour, it was stored at 4°C for 18 hours. The plates were further washed three times with deionized water and each well was filled with 0.0% sodium phosphate buffer, pH 8-5, containing 1% bovine serum albumin and 0.02% sodium azide.
゜20 m i was filled. Plates were kept at 20°C for 4 hours and at 4°C for an additional 18 hours. The plate was then washed three times with deionized water again.

Preparation of antigen or sample with 1% bovine serum albumin, 0
．． 02% sodium azide and calcium chloride 2m
M.paa, diluted in 2 O, 1M Tris-HCl buffer, was added to the wells in a volume of 0.150 nJL. Each sample or standard sample was analyzed in at least triplicate.

Standard samples were used in the range of 0.8 mg to 200 ng per well. Plates were incubated for 18 hours at 4°C. The plates were again washed three times with deionized water before adding the enzyme-antibody conjugate.

Goat anti-mouse IgG antibody/alkaline phosphatase conjugate (manufactured by Tavo, affinity purified) was added to 0.1 M solution at pH 8.2 containing 1% bovine serum albumin, 0.02% sodium azide, and 2 mM calcium chloride.
) It was diluted in a lithium-hydrochloric acid buffer at an abundance ratio of 1:1000.

0.150 mJL of conjugate solution was distributed to each well except for the blank well. The above plate was heated at 37℃ for 2 hours.
and incubated for 1 hour at 20°C.

After 3 washes with deionized water, add 0.5 mM to the wells.
(7) P-nitrophenyl-phosphate solution (pH 8,217 containing 2 nM magnesium chloride and 0.5 mM zinc chloride) 1.0 M in Lis-HCl buffer) 0.
150 mjL was filled. The rate of increase in the absorbance of the wells at 410 nm was read on a Dynatic MR600 automatic plate reader connected to a Digitator 2 Φ Equipment VAN 1/750 computer.

The computer accepts the kinetic data as is, and performs statistical and graphical analysis such as plotting absorbance against the natural logarithm (log) of antigen concentration and plotting variation values against the natural logarithm (log) antigen concentration. to be processed.

[Example 9] Acrylamide (purity is at the level used for electrophoresis)1.
50g and N-acryloxysuccinimide 0.7
0g to 2-propatool (moisture content 0.2% or less) 7
0. Dissolved in OmJl at 25°C using a magnetic stirrer. a-cumyl-peroxyneodecanoate (a-cus+y peroxyneodeca
noate) 50, 0 p+1 to acetone 0=50
This solution was added to the above 2-propertool solution, stirred for an additional 2 minutes using a magnetic stirrer, and then filtered through a sintered glass filter.

96 ([1]) The above filtered 2-propanol containing a-cumyl-peroxyneodecanoate and the monomer that generates free radicals was added to two polystyrene microtiter plates (manufactured by Linpro) equipped with wells of 1. The tool solution was filled to 0.280 mjL per well. The plate was placed in a vacuum oven heated to 57°C. The oven was rapidly vacuumed until the pressure dropped to 100 mbar, and The plate was filled with pure (99,9%) argon until the mbar was reached. To further reduce the oxygen concentration in the oven, the above operation was repeated two more times. The plate was placed in the oven for 5
It was kept at 7°C for 4.5 hours.

A white precipitate appeared in the wells in about 1 hour. After 4.5 hours, the plate was removed from the oven, allowed to cool for 30 minutes, and washed five times with deionized water in a 96-well microtitration plate washer. The time required for the two plate washing operations was less than 3 minutes.

Goat anti-mouse IgG antibody (manufactured by Tavo, affinity purification) solution (pH 8.5) was added to the wells of the washed plate.
5 μg of antibody in 0.05 M sodium phosphate buffer
/ m fL) was filled with 0.150 mjL. The plate was left at 20°C for 1 hour and then stored at 4°C for 18 hours. Further wash the plate three times with deionized water and
1% bovine serum albumin and 0.0
It was filled with 0.300 mJL of a pH 8.5 sodium phosphate buffer containing 2% sodium azide. There are 4 plates
It was kept at 20°C for an additional 18 hours at 4°C. The plate was then washed three times with deionized water again.

Preparation of antigen or sample with 1% bovine serum albumin, 0
．． 02% sodium azide and calcium chloride 2m
p containing M! (0.1M of 8, 2) was diluted in Lis-HCl buffer and added to the wells in a volume of 0.150mM. Each sample or standard sample was analyzed in at least triplicate.

Standard samples were used in the range of 0.8 ng to 200 ng per well. Plates were incubated for 18 hours at 4°C. The plates were again washed three times with deionized water before adding the enzyme-antibody conjugate.

Goat anti-mouse IgG antibody/alkaline phosphatase conjugate (manufactured by Tavo, affinity purified) was added to 0.1 M solution at pH 8.2 containing 1% bovine serum albumin, 0.02% sodium azide, and 2 mM calcium chloride.
) It was diluted in Lisu-Ro-HCl buffer to an abundance ratio of i:1000.

0.150 ml of conjugate solution was distributed to each well except for the blank well. The above plate.

Incubate for 2 hours at 37°C and 1 hour at 20°C. After 3 washes with deionized water, 0.5 m
M p-nitrophenyl phosphate solution (pH containing 2mM magnesium chloride and 0.5mM summer lead chloride)
8,2 1.0M) in Lis-HCl buffer) 0.150m
1 was filled.

The rate of increase in the absorbance of the wells at 410 nm is:

Dynatic MR600 connected to Digital Equipment's VANII/700 computer
automatic play) - read with the reader. The computer accepts the kinetic data as is and performs statistical and graphical analysis such as plotting the absorbance against the natural logarithm (Iag) of the antigen concentration and plotting the variation value against the natural logarithm (log) of the antigen concentration. to be processed.

[Example 101 Acrylamide (purity is at the level used for electrophoresis) 0.
38g and N-acryloxysuccinimide 0.2
5g of 1-propatool (moisture content 0.2% or less) 7
Dissolved in 0.0mM at 25°C using a magnetic fist stirrer. 1.25 g of benzophenone was added to the above l-
It was added to the propatool solution and stirred for an additional 10 minutes before being filtered through a sintered glass funnel. Two 96-well polystyrene microtitration plates (Linpro) were loaded with the above-mentioned filtered solution containing albenzophenone and mono-, a photoinitiating compound that generates free radical species upon activation of ultraviolet light. The 1-propertool solution was filled to a volume of 0.280 ml per well. The plates were placed in a glass flat bottom container (12 inches long x 12 inches wide x 2 inches high) and sparged with a stream of pure argon for 15 minutes to remove oxygen. The container was placed vertically one inch above nine black light fluorescent lamps that were vertically spaced one inch apart from each other. The above light source (manufactured by Sylvania 1, Fl 5T
36
The discharge lamp and container were cooled with compressed air. The temperature inside the container was measured to be 25° C.

A white precipitate appeared in the wells 10 minutes after turning on the UV discharge lamp. After 1 hour, the UV discharge lamp was turned off and the plate was washed twice with 2-propatool in a 96-well microtitration plate washer and treated with benzophenone, a water-insoluble photoinitiator. was removed. The plate was then washed 5 times with deionized water in the washing device described above. The time required for washing the two plates was less than 5 minutes. Coating the plate with an antibody (goat anti-mouse IgG antibody manufactured by Tabo), inactivating the plate with BSA, adding the standard sample and sample (mouse IgG), wasabi radish peroxidase The operations of adding the second antibody labeled with , adding the substrate, and measuring the absorbance at 410 nm are the same as those of the analytical method described in Example 2.

[Example 11] Acrylamide (purity is at the level used for electrophoresis) 0.
38g, N-acryloxysuccinimide 0.25g
and 1.25 g of benzophenone to 1-propanol (
The mixture was dissolved in 70.0 ml (water content: 0.2% or less) at 25° C. using a magnetic stirrer. After filtering the above solution with a sintered glass filter, the above 1-propertool solution (
0.20 m1/well) was filled. The plates were placed in a glass flat bottom container (12 inches long x 12 inches wide x 2 inches high) and sparged with a stream of pure argon for 15 minutes to remove oxygen. The container was placed vertically one inch above nine black light fluorescent lamps that were vertically spaced one inch apart from each other. The above light source (manufactured by Sylvania,
F 15T8/BLB fluorescent discharge lamp, 18 inches x 778
inch) is 360 nm at the glass bottom of the grafting vessel.
7) Emit UV radiation energy.

After 30 minutes of photoinitiated grafting, the plate was washed twice with 2-propertools in a 96-well microtitration plate washer to remove benzophenone, and further washed in the same manner as described above. Washed five times with deionized water. Coating the plate with an antibody (goat anti-mouse IgG antibody manufactured by Tavo), and sequentially inactivating the plate with BSA.
Adding a standard sample and a sample (mouse IgG);
Adding a second antibody labeled with horseradish peroxidase, adding a substrate, and 41
The measurement of absorbance at 0 nm is similar to the operation of the analytical method described in Example 2.

[Example 12] Acrylamide (purity is at the level used for electrophoresis)1.
15g, N-acryloxysuccinimide 0.75g
and benzophenone 1, 250 g 1-propatur (moisture content 0.2% or less) 210. During OmfL,
The mixture was melted at 25°C using a magnetic φ stirrer.

After filtering the above solution with a sintered glass filter,
A large polystyrene plate (manufactured by Linpro) with 6 wells was filled with the above solution containing the photoinitiating compound and monomer (G, 28 mJL/well). The plate was placed into a photografting device. The grafting and washing operations of the plate are similar to those described in Example 11. The plates were dried for 24 hours at 25° C. in a vacuum of 0.1 mbar and immediately sealed in a polyethylene bag (with DryElite in the bag to adsorb residual moisture). Such moisture may hydrolyze the active ester groups on the spacer molecules. The activated plates showed no loss in antibody or antigen binding activity for 3 months when stored at 20°C and 6 months at 4°C.

[Example 13] Acrylamide (purity is at the level used for electrophoresis) 0.
75g, N-acryloxysuccinimide 0.50g
and 1.25 g of benzophenone in 50 m of acetone.
Water-cooled deionized water containing 0.1 mA of glacial acetic acid was dissolved in fL using a magnetic stirrer at 0° C. and added with slow stirring. The resulting homogeneous solution was quickly filtered through a pre-chilled sintered glass funnel to minimize hydrolysis of the active ester of N-acryloxysuccinimide.

The filling operation into two polystyrene plates (manufactured by Linpro) each having 96 wells and the photografting operation were performed for 30 minutes in a cold room at 4°C. The plate was then washed twice using a 2-proper tool in a 96-well microtiter plate washer.
Washed five times with ice-cold deionized water. Coating the plate with an antibody (goat anti-mouse IgG antibody manufactured by Tavo), sequentially inactivating the plate with BSA,
Adding a standard sample and a sample (mouse IgG);
Adding a second antibody labeled with horseradish peroxidase, adding a substrate, and 410
The measurement of absorbance at nm is similar to the operation of the analytical method described in Example 2.

[Brief explanation of drawings]

FIG. 1 is a block diagram illustrating the grafting process in the formation of long spacer molecules covalently attached to a polymer surface and the covalent immobilization of antibodies by activated spacer molecules. Figure 2 (I), (2) and (3) are obtained using polyclonal antibodies bound by conventional adhesives in three enzyme immunoassays for mouse IgG and human IgG and IgM, respectively. The plot of the absorbance against the natural logarithm (log) of the antigen concentration is shown along with the standard error line (3σ). Figure 3 (I), (2), and (3) show three types of enzyme immunoassays for mouse IgG and human IgG and IgM, respectively. Natural logarithm (log) of antigen concentration showing results obtained using clonal antibodies
A plot of absorbance versus standard error line (3σ) is shown. Engraving of the drawing (no changes to the content) ←-V? '2 -y FIQjlE l

Claims (1)

[Claims] 1. A surface-treated polymeric material for use in the immobilization of molecules containing effective amino groups, said material having at least one polymer attached thereto, said polymer comprising: formed from a mixture of at least two monomers, one of said monomers containing an active group effective for binding to an amino group in said molecule to be immobilized, and a number of said active groups being associated with said substance. A polymeric material, characterized in that it is dispersed on the polymer at intervals between. 2. The polymeric material further contains a plurality of molecules containing active amino groups, and each of the plurality of molecules is covalently bonded by the amino group to one of the active groups dispersed on the polymer. A surface-treated polymeric material according to claim 1, characterized in that the surface-treated polymer material is characterized in that: 3. A surface-treated polymeric material used in the immobilization of molecules containing effective amino groups, said material having at least one polymer bonded thereto, said polymer having the following general formula (I): A surface-treated polymeric material produced from a mixture of a monomer represented by the following general formula (III) and a monomer represented by the following general formula (III). CH_2=CR_1-W-Z(I) [In the general formula (I), R_1 is a hydrogen atom, a methyl group, or a carbon atom number of 16
Z is a group represented by the following general formula (II); and W is an aliphatic, aromatic or aliphatic group having 16 or less carbon atoms. A group consisting of an aromatic group, or an aliphatic group having 16 atoms or less, an aromatic group, or a group consisting of an aliphatic group and an aromatic group] ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (II) [In the general formula (II) , R_3 and R_4 may or may not be bonded to each other, and may be a hydrogen atom, an aliphatic group having 16 or fewer carbon atoms, an aromatic group, or a group consisting of an aliphatic group and an aromatic group, or an oxygen atom as a hetero atom. , an aliphatic group, an aromatic group, or a group consisting of an aliphatic group and an aromatic group having a nitrogen atom, a sulfur atom, a fluorine atom, a bromine atom, and/or a chlorine atom and having 16 or less carbon atoms, in which R_3 and R_4 are bonded. [In the general formula (III), R_2 is a hydrogen atom, a methyl group, or Number of carbon atoms: 16
Y is an aliphatic or aromatic group consisting of the following aliphatic or aromatic group; and is an aliphatic group, an aromatic group, or a group consisting of an aliphatic group and an aromatic group having 16 or less carbon atoms] 4. In the above general formulas (I) to (III), Z is a group represented by the following general formula. and Y has 1 carbon atom
is an aliphatic group of 6 to 6, and R_1,
4. The surface-treated polymeric material according to claim 3, wherein R_2, X and W contain 1 to 6 carbon atoms. ▲There are mathematical formulas, chemical formulas, tables, etc.▼ [In the above general formula, n is 1, 2 or 3] 5. A patent characterized in that in the above general formula (III), Y contains a hydrophilic group. Claim 3 or 4
A polymeric substance that has been subjected to the surface treatment described in Section 1. 6. Claims 3 or 3, characterized in that in the general formula (III), Y contains a hydrophilic group containing one type of group selected from the group consisting of hydroxyl group, amide, and carboxyl group. A polymeric substance subjected to the surface treatment described in item 4. 7. The surface-treated polymer material according to claim 3, wherein the polymer further contains a monomer represented by the following general formula (IV). CH_2=CR-X-RC=CH_2(IV) [General formula (
In IV), R is a hydrogen atom, a methyl group, or an aliphatic or aromatic group having up to 16 carbon atoms; and X is an aliphatic, aromatic, or aliphatic group having up to 16 carbon atoms. and an aromatic group, or a group consisting of an aliphatic group and an aromatic group having an oxygen atom, a nitrogen atom, or a sulfur atom and having 16 or less carbon atoms as a hetero atom] 8. The above polymer further has the following general formula ( The surface-treated polymeric material according to claim 4, characterized in that it contains a monomer represented by V). CH_2=CR_5-X-R_5C=CH_2(V) [
In general formula (V), R_5 is a hydrogen atom, a methyl group, or a carbon atom number of 16
The following aliphatic or aromatic group; 9. The polymeric substance further contains a plurality of molecules containing an active amino group, and the plurality of molecules are each covalently bonded to one of the Z groups dispersed on the polymer through the amino group. A polymeric substance treated with 10. The polymeric material further contains a plurality of antibody or antigen molecules, and each of the plurality of molecules is covalently bonded by a respective amino group to one of the Z groups dispersed on the polymer. A surface-treated polymeric material according to claim 3, 4, or 7, characterized in that: 11. The polymeric substance further contains a plurality of molecules of one type selected from the group consisting of polypeptides and polysaccharides,
and each of the plurality of molecules is covalently bonded to one of the Z groups dispersed on the polymer through a respective amino group.
A polymeric material subjected to the surface treatment according to item 1, 4 or 7. 12. Claims 3, 4, or 12, characterized in that the polymeric substance includes one type of substance selected from the group consisting of polystyrene, polyvinyl chloride, polyethylene, polypropylene, polycarbonate, and polyacrylate. A polymeric material subjected to the surface treatment described in item 7. 13. The above monomer is decomposed by a compound (either chemical or photochemical) that generates free radicals in an inert gas and in the presence of a solvent inert to the monomer, and grafted. A method for producing a surface-treated polymeric material according to claim 1, characterized in that the polymeric material is obtained by graft polymerization by reacting with a free radical compound (forming a free radical compound that initiates polymerization). . 14. A monomer represented by the following general formula (I) and a monomer represented by the following general formula (III), CH_2=CR_1-W-Z(I) [In the general formula (I), R_1, W and Z is as defined in claim 3] CH_2=CR_2-Y(III) [In general formula (III), R_2 and Y are as defined in claim 3] (i) free radicals dissolved in a solution consisting of a solvent inert to the monomer, in an inert gas, in the presence of the polymeric substance and at a temperature within the range of 40°C to the boiling point of the solvent; (ii) forming the plurality of polymers by washing the polymeric material with deionized water; and (ii) washing the polymeric material with deionized water. 4. The method for producing a surface-treated polymeric material according to claim 3, wherein the surface-treated polymeric material is bonded to the polymeric material using 15. A monomer represented by the following general formula (I) and a monomer represented by the following general formula (III), CH_2=CR_1-W-Z(I) [In the general formula (I), R_1, W and Z is as defined in claim 3] CH_2=CR_2-Y(III) [In general formula (III), R_2 and Y are as defined in claim 3] ( i) Dissolved in a solution consisting of a solvent inert to the monomer (containing a compound that generates photoreactive free radicals), in an inert gas, in the presence of the polymeric substance and from 0°C to the above Radiation treatment at a temperature within the boiling point of the solvent (free radical removal from the free radical-producing compounds mentioned above)
(ii) forming and bonding the plurality of polymers to the polymeric material by: (ii) washing the polymeric material with deionized water; A method for producing a polymeric substance subjected to the surface treatment according to scope 3. 16. In addition to the steps (i) and (ii) above, the manufacturing method according to claim 14 or 15, which comprises the step of (iii) drying the polymeric substance. . 17. The solvent contains one type of substance selected from the group consisting of alcohol, ether, ester and ketone,
The free radical-generating compound contains one or more substances selected from the group consisting of azo compounds, organic peroxides, and peracid esters, and the inert gas is selected from the group consisting of nitrogen and argon. 15. The manufacturing method according to claim 14, wherein the manufacturing method includes one selected type of gas. 18. The solvent contains one type of substance selected from the group consisting of alcohol, ether, ester and ketone,
Claim 15, wherein the free radical-generating compound includes an aromatic ketone, and the inert gas includes one gas selected from the group consisting of nitrogen and argon. Production method. 19. Claim 15, wherein the free radical-generating compound is benzophenone.
Manufacturing method described in section. 20. The reaction time in step (i) above is 2 to 10
18. The manufacturing method according to claim 14 or 17, wherein the manufacturing method is time. 21. The manufacturing method according to claim 15, 18 or 19, wherein the reaction time in step (i) is 10 minutes to 2 hours. 22. In addition to steps (i) and (ii) above, (iii) the surface-treated polymeric material is placed in an aqueous non-nucleophilic buffer solution at a pH within the range of 7.5 to 8.5. ,
Each of said molecules is dispersed on said polymer by treatment with a plurality of molecules each having an active amino group at a temperature in the range of 0°C to 37°C and in the presence of a hydrolyzing reagent. 15. The manufacturing method according to claim 14, comprising the step of covalently bonding one of the Z groups with the amino group. 23. In addition to steps (i) and (ii) above, (iii) the surface-treated polymeric material is placed in an aqueous non-nucleophilic buffer solution at a pH within the range of 7.5 to 8.5. , at a temperature in the range of 0°C to 37°C, and in the presence of a hydrolyzing reagent, each of said molecules is dispersed on said polymer by treatment with a plurality of molecules, each having an available amino group. 16. The method according to claim 15, comprising the step of covalently bonding the amino group to one of the active Z groups. 24. Claim 22 or 23, wherein the hydrolysis reagent contains a substance selected from the group consisting of bovine serum albumin, egg albumin, nonionic surfactants, and mixtures thereof. Manufacturing method described in section. 25. The production method according to claim 22 or 23, wherein the molecule having an effective amino group contains an antibody or an antigen. 26. In a processing method for immunoassay, a polymeric substance subjected to the surface treatment according to claim 1, 3, or 4 is used, and the polymeric substance has an amino group of an antigen or an antibody. A method of immobilizing the antigen or antibody in a spaced relationship with the polymeric substance by binding to a polymer. 27. In a treatment method for immobilizing molecules having an effective amino group, claim 1, 3 or 4
By bonding the amino group of the molecule to the polymer of the polymeric substance using a polymeric substance that has been subjected to the surface treatment described in Section 2, the molecule is placed in a spaced relationship with the polymeric substance. How to fix it.