JPH0251151B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for simultaneously measuring trace amounts of biological components using a medium element carrier that expands the surface area of a reaction medium. Many methods have been known for quantitative measurement, such as immune reactions based on antigens (including papten) and antibodies, and receptor reactions based on substances such as drugs and hormones and receptors (hereinafter abbreviated as immune reactions). ing. Such methods mainly involve serum, urine,
Quantification of components present in trace amounts in body fluids such as saliva,
It has been applied for qualitative purposes. Trace components include various hormones as in-vivo components, receptors for them, in-vivo enzymes, components specific to pathological conditions, administered drugs and their receptors, and their metabolites, as well as bacteria and viruses. Microorganisms such as
Examples include trace components that exist in various living organisms, such as antibodies against these. Such trace components are mainly measured by applying immune reactions such as antigen-antibody reactions, for example, hormones and antibodies or receptors against them. Methods that utilize the fact that, as a result of immune reactions, these conjugates become insoluble or that large aggregates consisting of a large number of antigen-antibody molecules are formed are already known as classical methods. These are measured by various methods such as directly measuring the amount precipitated in a reaction tube, immunodiffusion method using precipitation in a gel, or method using complement. Furthermore, various methods are well known in which antigens, antibodies, or receptors are labeled by some method, subjected to an immune reaction, and then the labeling activity is measured. Among these, radioimmunoassay (RIA), which uses radioactive substances as a labeling substance, is well known as a widely used highly sensitive measurement method.Although RIA uses radioactive substances, it is safer. As an alternative method, enzyme immunoassay (EIA), which uses an enzyme label instead of a radioactive substance label, has been widely used in recent years. EIA is a method in which an enzyme is bound to an antigen or a receptor such as an antibody or receptor, the degree of immune reaction is measured using the enzyme activity as a label, and the amount of antigen or receptor is measured from the results. It is. In such a case, a receptor or antigen having specific binding ability for the component to be measured is used, and the antigen or receptor as the component to be measured is caused to compete with the enzyme-labeled antigen or receptor, Then, when the bound and non-bound substances generated by the immune reaction are separated or their enzyme activity is measured without separation, or when a receptor or antigen that has specific binding ability for the substance to be measured is used. is used to react with the antigen or receptor to be measured, and then the conjugate is reacted with a compound capable of binding to the enzyme-labeled antigen or a compound capable of binding to the receptor, thereby producing a sandwich. Thereafter, the enzyme activity may be measured. Conventionally, when utilizing these various immune reactions, one of the components to be measured is
The method only carried out one reaction, and there were many drawbacks to measuring a wide variety of components. That is,
Measuring multiple components requires a large amount of samples such as serum equivalent to the number of target components, and a wide variety of reagents are used for each measurement, which also requires a long time. It was hot. In contrast, a solid phase body in which two types of antibodies are immobilized on a carrier is used, and samples containing the two types of antigens to be measured are added and reacted, and a labeled compound of one type of antigen between them is first reacted. A method in which the labeling activity of the antigen is measured, followed by reacting with a labeling compound of another antigen and measuring the labeling activity (JP-A-56-78598, No. 52-15815, No. 56)
-96248) is known. In these measurement methods, beads and test tubes, which have traditionally been used as solid phase substances, are used as antibodies, microparticles with various distinguishable particle sizes are used as carriers, or beads and microparticles are used instead of beads or microparticles. Because a carrier with multiple fins is used and a solid phase is used on which two or more types of antibodies are immobilized, it is difficult to react trace components with extremely small amounts of samples. However, it is extremely difficult to calculate the titer of this immobilized antibody, and the measurement requires a reaction to sequentially measure each component.
This had the disadvantage of requiring a longer measurement time. The present inventors have researched and developed a method that can easily and accurately measure various trace components in a living body simultaneously over multiple components,
The purpose of this is to use a reaction tube for immune reaction as a carrier, and to use it as a carrier to be added to the reaction medium of immune reaction, the carrier has a shape that can be inserted into the immune reaction part of the reaction tube, and is external. It is formed so that the diameter maintains a slight distance from the inner diameter of the reaction tube, and the distance between the reaction tube and the element carrier is the same.It is an element carrier for the medium that expands the surface of the reaction medium. By using this carrier as one or more carriers, when reacting a small amount of sample, it is possible to expand the surface area of the reaction medium containing a small amount of sample liquid using the medium element carrier. This method provides a simple and good multi-component simultaneous measurement method that can be measured using a very small amount of sample liquid. The present invention can also be applied to EIA and RIA, as well as competitive reactions and sandwich immunoreactions. First, a reaction tube as a carrier used in the present invention may be a reaction test tube for immune reactions conventionally used in various EIAs and RIAs; for example, a reaction tube made of glass or plastic may be used. is used in any form. In addition, the element carrier has a shape that can be inserted into the immune reaction part of the reaction tube, and is formed so that its outer diameter maintains a slight distance from the inner diameter of the reaction tube, and the element carrier is formed so that it can be inserted into the immune reaction part of the reaction tube. It may be a medium element carrier that is formed so that the space is the same, expands the surface area of the reaction medium, and has a shape that can be divided into one or more parts as appropriate.
In addition, in the case of an immune reaction, any type of material that can immobilize a receptor or antigen that has specific binding ability with the component to be measured, and which can be added to the reaction tube, is usually made of glass or an insoluble material. Various things such as polysaccharides and insoluble proteins are granular, plate-shaped, bullet-shaped,
It is used in shapes such as a spear. Such carriers used in the present invention include:
An insoluble carrier used in general EIA and RIA is used. especially antibodies or antibody fragments
When insolubilizing Fab' etc., the target to be bound to an insoluble carrier is generally an antibody protein, so a carrier similar to that used in the production of immobilized enzymes to immobilize enzyme proteins is used. is used. Examples of this carrier include insoluble protein carriers such as albumin and gelatin treated with glutaraldehyde, agarose, epichlorohydrin treatment or cyanogen bromide treatment such as cellulose and dextrin, and insoluble insoluble protein carriers treated with an amination reagent. Semi-synthetic polymer carrier (polysaccharide), acrylonitrile, acrylic acid, acrylic ester, methacrylic acid, methyl methacrylic acid, vinyl alcohol, vinyl acetate, styrene, aminostyrene, divinylbenzene, acrylamide, ethylene, maleic anhydride, Polymers or copolymers such as crotonic acid, insoluble synthetic polymer carriers (polymer resins) obtained by treating these with aminating reagents, and other insoluble inorganic carriers such as silane compounds treated with aminating reagents. It may also be used by combining them. In addition, various known means may be used to introduce amino groups into this insoluble carrier, such as amination by reduction of nitrile groups, γ of hydroxyl groups, etc.
-Introduction of aminoalkyl groups by reaction with aminopropyltriethoxysilane, or conversion of hydroxymethyl groups to aldehyde groups by periodic acid oxidation, and introduction of amino groups by reacting the aldehyde groups with diamines, polysaccharide hydroxyl groups is converted to an imidocarbonate group by cyanogen bromide reaction, the amide group is converted to an iminohalogenide group by a phosphorus halide compound, and this functional group is further treated with a functional group-introducing reagent to introduce a new functional group as appropriate. It's okay. Among these insoluble carriers, suitable for processing include polymer resins such as nylon, polystyrene, polyacrylamide, and vinyl chloride, insolubilized polysaccharides such as cellulose and dextran, and inorganic materials such as glass. The element carrier in such a carrier may be bullet-shaped, spear-shaped, or similar in shape, but it may be attached to the surface of another solid molded object such as metal or plastic in such a way that the shape of the element of the present invention is maintained. Preferably, the medium element is formed to expand the surface area of the reaction medium and has the same shape as the immunoreaction tube used. Next, the element carrier of the present invention will be explained with reference to the drawings. In FIG. 1, a medium element carrier as an element carrier is generally indicated by reference numeral 1. The element carrier 1 is made of a polymeric resin material such as nylon 6,6, and is composed of an element body 2 and a mounting leg 3 protruding from the center of the upper surface 2a of the body 2. Although another operating rod (not shown) is attached to the mounting leg 3, the mounting leg 3 itself may have an extended shape for use as an operating rod. The element main body 2 is molded into a bullet shape, and its outer diameter is formed to maintain a slight clearance from the inner diameter of the reaction tube 4. A wedge-shaped projection 5 is provided at the top of the lower peripheral surface of the element body 2.
The protrusion 5 maintains a certain gap 6 between the arcuate outer circumferential surface 2b of the element body 2 and the arcuate inner circumferential surface 4a of the reaction tube 4. Further, instead of this wedge-shaped projection 5, a convex projection may be used. In short, any shape may be used as long as the projection 5 can prevent the element body 2 from coming into close contact with the arcuate inner circumferential surface 4a of the reaction tube 4. The height of this protrusion may be such that the width of the element main body 2 and the inner wall surface of the reaction tube 4 are the same. As shown in FIG. 2, the upper surface 2a of the element main body 2 may be provided with a square cut groove 8 centered on the mounting leg 3. The end of the cut groove 8 is opened at the peripheral edge of the element main body 2 in order to weaken the surface tension of the reaction liquid on the extension of the upper surface 2a. Further, the groove shape of the cut groove 8 may have any cross-sectional shape such as a semicircular shape, an elliptical shape, a trapezoidal shape, a triangular shape, or a quadrangular shape. Since the element carrier of the present invention has the above-described structure, when the element body is immersed in the reaction liquid in the reaction tube, the protrusions of the element body come into contact with the inner bottom surface of the reaction tube, and the element body A slight gap is formed between the outer peripheral surface and the corresponding inner peripheral surface of the reaction tube (see FIG. 1). Therefore,
Even if the amount of reaction liquid is small, by immersing the element body, the liquid is guided into the gap and the reaction liquid that rises to the top of the element body flows in the cut groove and reaches the surface of the element itself. Loss of tension action. Therefore, the reaction liquid comes into contact with the outer peripheral surface of the element main body under uniform conditions (in the form of a layer of 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 element main body 2, as shown in FIG. 3A. Moreover, as another modification, as shown in FIG. As another modification, as shown in FIG. 3C, the elements may be arranged symmetrically in the circumferential direction of the upper surface 2a of the element main body 2. In this manner, the cut grooves 8 may have any shape as long as their terminal ends are opened at the peripheral edge of the upper surface portion 2a. FIG. 4 shows another modification of the element 1. The element body 2 in this example is shaped like a spear. Therefore, the reaction tube 4 used has a tapered shape along the element body 2, and the element can be modified and processed according to the shape of various commercially available reaction tubes. The rest of the structure of the element main body 2 is the same as that of the example shown in FIG. 1, so the same numbers are given to the parts having the same structure, and the explanation thereof will be omitted. Furthermore, as mentioned above, in order to perform multiple immune reactions without also increasing the surface area of a small amount of reaction solution, a carrier having multiple elements in the shape of a plate or rod may be used as a solid phase. . Insoluble antibodies, etc. (antigen, receptor ) and prepare a solid phase. The solid phase may be prepared by introducing a functional group into the carrier in advance to bind antibodies, etc., and processing it into a specific shape, or by introducing the functional group after processing. For example, if the reaction tube or element carrier is nylon 6,6,
It is processed in advance into the above-mentioned shape, and then activated by dimethyl sulfate or immicroscopic method, treated with hexamethylene diamine and glutaraldehyde to bind an antibody, such as a second antibody, and then immunized with the first antibody. A solid phase body may be obtained by immobilization by reaction. Furthermore, when the antibody is a dextran gel, such as Sephadex, it is processed in advance into the above-mentioned shape, then activated with BrCN, and the antibody is bound to and immobilized thereon. Furthermore, in the case of polystyrene, etc., after processing, it may be fixed after being chemically treated with sulfuric acid, or it may be fixed by physical adsorption. In the case of glass, etc., it may be aminoalkylsilylated using an organic amino group-introducing silane reagent such as a γ-aminoalkyltriethoxysilane compound, and then immobilized based on this amino group by a known immobilization means. Furthermore, in general, when immobilizing chemically, a functional group in the molecule of the carrier or an introduced functional group and a functional group in the molecule of the antibody, such as an amino group, a hydroxyl group,
A polyfunctional compound capable of bonding the two based on a functional group such as a carboxyl group, a thiol group, or an aldehyde group is used. Examples of various known polyfunctional compounds include diisocyanate compounds such as hexamethylene diisocyanate and 2,4-toluene diisocyanate, diisothiocyanates such as hexamethylene diisothiocyanate, and succinic aldehyde. , dialdehydes such as glutaraldehyde and adipaldehyde, N,N'-ethylene bismaleimide, N,N'-0-phenylene dimaleimide, bisdiazobenzine, N,N'-
Polymethylene biiodoacetamide, diethylenemalonimidate, dimethyladipineimidate, 3-(2'-benzothiazolyldithio)-propionic acid, 3-(2'-pyridyl-N-oxide-dithio)propionic acid, 6 Sulfide carboxylic acids such as -N[3-(2'-benzothiazolyl-dithio)propionyl]caproic acid or its reactive derivatives such as succinimide ester, p-nitrophenyl ester, acid chloride, imidate, etc. -85900), maleimidocarboxylic acids such as maleimidobenzoic acid, maleimidophenyl acetic acid, maleimidophenylpropionic acid, or reactive derivatives thereof. This polyfunctional compound can also be used as a functional group introduction reagent for introducing a functional group into a carrier. Furthermore, in general, when obtaining a solid phase, an inert medium such as a buffer solution of pH 6 to 8, acetone, methanol, ethanol, dioxane, dimethyl sulfoxide, dimethylacetamide, tetrahydrofuran, or a mixed medium thereof is used. The carrier and the polyfunctional compound are reacted using a medium at cooling or room temperature. Thereafter, the insoluble carrier is washed if necessary, and then an antibody or the like is further added and reacted using the same inert medium to cause the insoluble carrier to react with the antibody or the like. Furthermore, in order to obtain an antibody-second antibody-first antibody conjugate obtained by binding a first antibody with a second antibody, for example, using an antibody processed into a desired shape, Based on the means of obtaining the counterpart,
First, a second antibody is bound. Next, the first antibody may be added to this and bound based on an immune reaction, and the immunoactivity of the first antibody obtained in this way is hardly deteriorated, so it is a particularly preferable solid phase. When using two or more of the solid phase bodies obtained in this way, for example, a reaction tube solid phase body and one element solid phase body or an element solid phase body that can be separated into two or more. or a combination of multiple element solid phase bodies that can be separated into two or more. In particular, as an element solid phase that can be separated into two or more, for example, a plurality of element carriers shown in Fig. 1 are used, and one element is immobilized with a target antibody, etc., and another element is used with a different purpose. An antibody or the like is immobilized to obtain a solid phase body on which a plurality of different antibodies or the like are immobilized. These solid phase bodies are then cut into respective sizes. For example, when making a solid phase of two types of elements, if the result of combining them constitutes one element, you can cut them to an appropriate size, and simply cut them into symmetrical halves. In addition, if the density of the faces differs, the element may be constructed using each fragment cut according to the ratio according to the density, for example, a fragment of 1/4 size. By using an element fragment and an element fragment of 3/4 size, the low-density part is made into a 3/4-sized fragment, and the high-density part is made into a 1/4-sized fragment, which are then glued together to form an element. Further, when preparing a solid phase body of three or more types of elements, each fragment may be cut according to the number thereof, and then these fragments may be combined and used as an element in the original shape. Furthermore, when combining the fragments of the element solid phase, they may be simply integrated into a shape that can be separated according to the cut surface of the element body, or the element may be combined at the joining site of the cut surface of each element fragment. Each fragment may be integrated with a joint portion for maintaining the gap distance between the main body and the reaction tube. In particular, in the latter case, it is preferable to use a fragment of the element main body as a carrier in advance and immobilize an antibody or the like onto it to form a solid phase. Furthermore, when using a solid phase consisting of a plurality of plate-shaped or rod-shaped elements that are separable as described above, antibodies, etc. are immobilized on the entire surface of each carrier so that the entire surface can be used as a solid phase. It is preferable to form a bonding area in the vicinity of the contact area of each solid phase body in order to maintain the gap distance. Furthermore, the components to which enzymes are bound, such as antibodies, antigens, haptens, etc., and the components to which radioactive isotopes are bound, which are used in measurements, are prepared using known EIA or
The means for labeling used in RIAs are utilized. In addition, various known enzymes such as those belonging to oxidoreductases, hydrolases, transposases, lyases, isomerases, and ligases can be used as enzymes as labeling substances. In order to measure two or more components to be measured, such as antigens and antibodies, which have specific binding properties to the antigen, etc., by using two or more insoluble antibodies etc. immobilized on a carrier in this way, Known EIA or
Measurement method by RIA is applied. For example, the small test tube used is 15 x 105 mm (inner diameter
12.8mm±0.1mm), elements having the respective dimensions shown by symbols A to I in FIG. 5 are used. That is, A: hemispherical part with a radius of 6 mm, B: the height of the cylindrical part is 10 mm, C: the height of the conical protrusion part is 0.4 mm,
D: radius of cone of conical projection 0.5mm, E: radius
0.75mm semicircular groove, F: Center distance between opposing grooves 7.5mm, G: Diameter of convex part for inserting the operating rod 4mm, H: Diameter of operating rod insertion hole 2mm, I: The element body consists of a cylindrical part with a height of 3 mm (height 10 mm, diameter 12 mm) and a hemispherical part with a radius of 6 mm, and can be separated or divided. Under these conditions, the amount of immunoreaction buffer is 100 μl.
The liquid containing the component bound to the labeled substance is
Approximately 100μ and test liquid (serum, urine, etc.) 100μ
It is sufficient to use degree. Further, at this time, the width (thickness) of the reaction liquid was uniformly formed at 0.4 mm.
In the above-mentioned element bodies, an insulin antibody is immobilized on one element body, a glucagon antibody is immobilized on the other element body having the same shape, and then each element has the same shape, that is, the first The top surface shape cut into the cut shape shown in the figure is made into semicircular element fragments, and the obtained semicircular fragments of the insulin antibody solid phase body and semicircular fragments of the glucagon antibody solid phase body are obtained. It is used as a solid phase for the separable element body. Examples of other simultaneous measurements include simultaneous measurement of α-fetoprotein, CEA, and ferritin, simultaneous measurement of T ₃ and T ₄ , and other appropriate combinations of various steroid hormones and peptide hormones. This can be done by using a divisible solid phase that incorporates various specific antibodies. It is also clear from the above description that the wall of the reaction tube may be used as one solid phase that can be divided. The reaction temperature and time vary depending on the object, but for example, in the case of simultaneous measurement of insulin and glucagon, the reaction time is 5°C and 8 to 48 hours. Generally, the time period is appropriate depending on the component to be measured.
The temperature and the concentration of the component to be measured can be selected. Furthermore, the amount of each liquid used may be adjusted based on the small test tube used and the size of the element used therein. Examples of methods for measuring insulin and glucagon content in serum are as follows. Buffer solution in a small reaction tube (15 x 105 mm, inner diameter 12.8 mm ± 0.1 mm), which is a solid phase substance for glucagon antibody.
Add 100μ of enzyme-labeled insulin solution, 50μ of enzyme-labeled glucagon solution, and 100μ of a solution containing insulin and glucagon at a constant concentration.
Next, an element which is a solid phase body of an insulin antibody having the size and shape described above was added to this, and the mixture was incubated at 5°C for 15 minutes.
Perform time incubation. After the reaction is complete, add 5 ml of distilled water or physiological saline to wash this element, and then add 500~500ml of enzyme substrate solution in advance.
Transfer the element to another small test tube in which 1000μ of the sample has been dispensed, and perform the enzyme reaction at 37°C for 30-120 minutes. Add 1.5 to 2.0 ml of reaction stop solution to stop the reaction, remove the elements, and then measure the components consumed and produced based on the enzymatic reaction. After the washing solution is removed from the reaction tube using an aspirator or decantation, about 2 to 3 ml of the enzyme substrate solution is added in the same manner as above to allow the enzyme reaction to occur, and then the absorbance of the solution is measured. The amounts of insulin and glucagon in the subject sample are determined from the labeling curve determined from the absorbance of a labeling solution of known concentration. Although it is measured as described above, a more detailed explanation will be added. 6,6-nylon having the shape of the element of the present invention (see Figure 5) (hereinafter referred to as 6,6 nylon measuring element) was activated by the immicro method using phosphorus pentachloride, and hexamethylene diamine and glutaraldehyde were activated. Processing yields a spacer-introduced 6,6 nylon measuring element. This is reacted with the IgG fraction of rabbit anti-guinea pig IgG serum to immobilize the IgG. Next, this was reacted with guinea pig insulin antibody serum, and 6.
6-Nylon element-anti-guinea pig IgG-
Prepare insulin antibody. Similarly, the IgG fraction of rabbit anti-guinea pig IgG serum was reacted with
A 6,6-nylon element-anti-guinea pig IgG-glucagon antibody is prepared by reacting the guinea pig glucagon antibody serum with the antibody immobilized with IgG. Then this 6,6-nylon element-anti-guinea pig IgG-insulin antibody, and 6,
6-Nylon element-anti-guinea pig IgG-
Each solid phase body of glucagon antibody is cut in half using a stainless steel cutter to form a semicircular shape. Each element fragment is then combined to form an element body. Next, this element body is reacted with serum containing a β-galactosidase-insulin conjugate, a β-galactosidase-glucagon conjugate, and various concentrations of labeled insulin and glucagon, or both,
After washing, the element body is removed, each fragment is separated, and β-galactosidase activity is measured to prepare a standard curve. The insulin and glucagon contents in the sample are simultaneously measured using a standard curve using standard insulin and glucagon. As described above, by using the distinguishable labeled compound of the present invention, two or more immune reactions can be performed in a short time with an extremely small amount of sample reaction solution, and as a result, trace amounts of components can be accurately detected with a small amount of sample. And it can be measured in a short time. Next, examples of the measurement method will be described, but it goes without saying that the present invention is not limited thereto. Example 1 Simultaneous measurement of insulin and glucagon by EIA [Preparation method of solid phase [test tube]-A] Polystyrene test tube (15 x 105 mm, inner diameter 12.8 ±
2 ml of sulfuric acid was added to 2 ml of sulfuric acid (pH 8.0) and reacted at 5°C for 6 hours to introduce SO ₃ C ^- groups. ) and reacted overnight at 5°C to obtain test tubes in which 8 μg was immobilized per tube.
Next, add a 30,000-fold dilution of anti ^-16-29 glucagon fragment serum (guinea pig) to this test tube (0.25
%BSA, 5mM EDTA, 3mM _MgCl2 , 0.15
Add 2 ml of diluted solution consisting of 0.01 M phosphate buffer (PH7.4) containing mM NaOl, 0.1% _NaN3 ,
After reacting overnight at °C,
^16-29 glucagon fragment antibody] conjugate was obtained. [Preparation method of solid phase body [element]-B] A polystyrene element having the size and shape shown in Fig. 5 is activated with sulfuric acid, SO ₃ ^- groups are introduced, and this is injected with anti-guinea pig IgG rabbit serum.
0.1M phosphate buffer (PH) containing 20μg of IgG fraction
8.0) Polystyrene element solid support was immersed in 1 ml and reacted overnight at 5°C to obtain a polystyrene element solid support on which 10 μg of IgG was immobilized each. Next, this element solid phase was immersed in 1 ml of a 23,200-fold diluted serum containing guinea pig insulin antibody (described in the previous section) and allowed to react overnight at 5°C to form a [element-anti-guinea pig IgG-insulin antibody] conjugate. A solid phase was obtained. [Preparation method of β-galactosidase-insulin] 60 mg of insulin was added to 0.1 M phosphate buffer (PH
8.0) Dissolve in 17 ml, add 1.7 ml of dimethylformamide containing 6.5 mg of 3-(2'-benzothiazolyl-dithio)propionic acid succinimide ester, react for 1 hour under cooling, and after the reaction, the pH becomes 5.0 and precipitates. This precipitate was dissolved in 4.0ml of 0.1M phosphate buffer (PH7.0) (8.9mg/ml as insulin), 10.0μ of this was collected, and 6mg of β-galactosidase was added to it. The mixture was added and reacted for 1 hour, and then the reaction solution was transferred to Cephadex G.
In a column (1.5 x 120 cm) packed with −100
Elute with 0.01M phosphate buffer (PH7.4) containing 0.15M NaCl, collect 65-73ml fractions [(insulin)-CO-( _CH2 ) ₂ -S-(β-galactosidase)] Insulin-β-galactosidase conjugate (β-galactosidase) consisting of the amino group of insulin and the thiol group of β-galactosidase is
-Gal-labeled insulin) (insulin 2.4 μg/ml and a conjugate of 1 molecule of insulin per molecule of β-galactosidase, and has 95% of the activity of the insulin conjugate against insulin antibodies) obtained). [Method for preparing β-galactosidase- ^19-29 glucagon fragment] 0.4 mg of glucagon fragment (19-29) at 50
Dissolve in 0.4 ml of mM phosphate buffer (PH8.0), add 10μ of 100 mM EDTA aqueous solution and 0.1% 3-
625μ of a dimethylformamide solution containing (2'-benzothiazolyl-dithio)propionic acid succinimide ester was added and reacted at 5°C for 1 hour. After the reaction, this was transferred to Cephadex G-15 (1.5 x 40
The pass-through fraction was obtained by gel filtration with a 3-cm column (containing a derivative in which a 3-(2'-benzothiazolyl-dithio)propionyl group was introduced at the N-terminus of glucagon fragment (19-29)). Then,
50 containing 20 μg of this 3-(2′-benzothiazolyl-dithio)propionyl group-introduced derivative.
Add 50mM phosphate buffer (PH7.0) containing 6.19mg of β-galactosidase to 2ml of mM phosphate buffer (PH7.0).
7.0) was added and reacted at room temperature for 1 hour. The reaction solution was gel-filtered through a Cephadex G-150 (1.5 x 90 cm) column, and the fraction that passed through was collected and bonded to the N-terminus of the glucagon fragment (19-29) and the thiol group of β-galactosidase. A β-galactosidase-glucagon fragment (19-29) conjugate (purity 95%) (β-Gal labeled glucagon) was obtained. Simultaneous measurement method of insulin and glucagon by EIA Immune reaction buffer (0.25% BSA, 5mM EDTA, 3mM MgCl ₂ ,
100μ of 0.15M NaCl, 0.01M phosphate buffer (PH7.4) containing 0.1% _NaN3 , 50μ of β-Gal labeled insulin (20.0pg as insulin), 50μ of β-Gal labeled glucagon (as glucagon fragment)
2.0pg) 50μ, and standard solution (5-640μ/ml as insulin and 25-640μ/ml as glucagon)
Add 100 μ of a liquid containing 3200 pg/ml, add 1 piece of solid phase-B (element), and incubate at 5°C.
The reaction was carried out at night. After completion, 5 ml of distilled water was added and solid phase B was first extracted while being washed.
0.1% BSA, 0.15M NaCl, 1mM
0.01M phosphate buffer containing _MgCl2 , 0.1% _NaN3 (PH
6.7) Solution consisting of 85 parts and 15 parts of methanol solution containing 200mM mercaptoethanol] Solid phase substance-B was added to a small test tube containing 500μ, and reacted at 37°C for 1 hour. After the reaction is complete, remove the reaction stop solution [100 m
After adding 2 ml of M glycine-NaOH buffer (PH 11.0) to stop the reaction and removing solid phase-B, the absorbance of the solution at 420 nm was measured. Next, after removing the washing solution by decantation, the solid phase body A was prepared using the β-galactosidase activity measurement solution (described above).
2 ml was added and reacted at 37℃ for 2.5 hours. After the reaction was completed, 500μ of reaction stop solution [400mM glycine-NaOH buffer (PH11.0)] was added to stop the reaction, and the absorbance at 420 nm of the reaction solution was measured. A quantitative curve was created. The results are shown in Figure 7, and in Figure 7, 〇-〇 indicates the quantitative curve of insulin, and △
−△ indicates the quantitative curve of glucagon;
The simultaneous measurement system for insulin and glucagon of the present invention showed an extremely good quantitative curve.
In addition, when measuring insulin and glucagon in the test sample (serum, plasma, etc.), use a standard solution of 100μ
Instead, 100μ of the sample was added, the same operation as above was performed, and the amounts of insulin and glucagon in the sample were determined by comparing with the quantitative curve. Example 2 Simultaneous measurement of insulin and glucagon by RIA [Preparation method of solid phase [test tube]-A] Prepared in the same manner as above. [Preparation method of solid phase [element]-B] 50 6,6 nylon elements having the size and shape shown in Figure 5 were stirred for 2 days in 60 ml of benzene containing 4 g of phosphorus pentachloride and 4 g of pyridine. Afterwards, it was washed four times with benzene to obtain an immicrolided 6,6 nylon element.
Add 50 ml of an aqueous solution (PH 11.5) containing 100 mM hexamethylene diamine, react at room temperature for 1 hour, wash thoroughly with 0.1 M phosphate buffer (PH 8.0), and add 4% glutaraldehyde to this solution.
Add 50ml of 0.1M phosphate buffer (PH8.0) and react at room temperature for 1 hour.
8.0), and spacers were added to 6,6 nylon elements (50 pieces) in 50 ml of 0.1 M phosphate buffer (PH 8.0) containing 5 mg of the IgG fraction of anti-guinea pig IgG serum (rabbit). The mixture was reacted overnight at 5° C. to obtain a nylon element solid phase on which 50 μg of IgG was immobilized per piece. Next, a 23200-fold dilution of serum containing guinea pig insulin antibodies [0.25% BSA, 5mM EDTA, 3mM
50 _ml ( ₂₅₀
mg of insulin antibody) and reacted overnight at 5°C to form a solid phase of [nylon element-anti-guinea pig IgG-insulin antibody] conjugate.
I got a B. [Method for preparing ¹²⁵ I-insulin] ¹²⁵ I-insulin can be prepared using WMHunter,
FC Greenwood et al.'s method [Nature (London)]
194, 495 (1962)] and gel filtration was performed using a Sephadex G-25 column (0.9 x 20 cm) to obtain ¹²⁵ I-insulin. [ ¹²⁵ I-[Tyr ¹⁸ ]-Glucagon (18-29) preparation method] (A) Production of [Tyr ¹⁸ ]-Glucagon (18-29) H-Tyr-Ala-Gln-Asp-Phe-Val-
Production of Gln-Trp-Leu-Met-Asn-Thr-OH Boc-Tyr-(Bzl- _Cl2 )-Ala-Gln-Asp
(OBzl) −Phe−Val−Gln−Trp−Leu−Met−
Asn-Thr(Bzl)-OBzl 2.04g (1mM), anisole 6ml, ethanedithiol 0.94mm (10m
M), 131 mg (1 mM) of skatole, and 0.62 ml (10 mM) of dimethyl sulfide were added. to this,
Introduce 70 ml of anhydrous hydrogen fluoride (HF) and heat at 0°C.
After stirring for an hour, HF was quickly distilled off under reduced pressure. 100 ml of Et ₂ O was added to the residue and the resulting precipitate was collected. This is dissolved in 100 ml of 50% acetic acid, centrifuged, and the supernatant liquid is lyophilized. Dissolve this in 20 ml of 8M urea solution and 20 ml of acetic acid, add 2 ml of thioglycolic acid, reduce at 45°C for 20 hours, and then use a Sephadex LH-20 column (4.2 x 137 cm).
and eluted with 50% acetic acid. The eluate was fractionated into 7 ml portions, and the 74th to 80th fractions were collected and lyophilized to obtain [Tyr ¹⁸ ]-glucagon (18-29).
Yield 120mg, Rf ² 0.73 (cellulose), amino acid analysis values: Asp1.97(2), Thr0.95(1), Gln2.07(2),
Ala0.98(1), Val0.94(1), Met0.98(1), Leu1(1),
Phe0.97(1), Tyr0.94(1), Trp1.05(1), the above raw materials were produced as follows. (1) P(19−29)Boc−Ala−Gln−Asp(OBzl)
−Phe−Val−Gln−Trp−Leu−Met−Asn
-Thr(Bzl)-OBzl [1] P(20-29) (Synthesis reported in Example 1 of JP-A-56-81540) 12.4 g (7.5 mM)
TFA 40ml, dimethyl sulfide 20ml, ethanedithiol 20ml, and skatole 983
After stirring at room temperature for 50 minutes, TFA was distilled off under reduced pressure. Ether was added to the residue, and the resulting precipitate was dissolved in a mixture of 150 ml of dry DMF and 50 ml of N-methylpyrrolidone. to this
Boc-Ala-OH2.0g (1.4 times equivalent) HOBt1.4
Add g (1.4 times equivalent) and WSCD1.92ml (1.4 times equivalent), adjust the pH to 7 with NMM, stir overnight, then add DMF50ml, HOBt0.51g,
0.71 g of Boc-Ala-OH and 0.69 ml of WSCD were added and stirred overnight. After distilling off DMF under reduced pressure, it was poured into ice water, and the resulting precipitate was washed twice with hot ethanol to obtain 11.35 g of the target product (yield: 87.8%).
I got it. TLC; Rf ¹ 0.75 (silica gel) Amino acid analysis; Asp1.92(2), Thr0.88(1),
Glu1.94(2), Ala0.88(1), Val0.91(1), Met0.95
(1), Leu1, Phe0.93(1), Trp0.71(1), (2) [Tyr ¹⁸ ]P(18−29)Boc−Tyr(Bzl−
Cl ₂ )−Ala−Gln−Asp(OBzl)−Phe−Val−
Gln−Trp−Leu−Met−Asn−Thr(Bzl)−
OBzl[2] P(19-29)[1] 8.61g (5mM)
40ml TFA, 20ml dimethyl sulfide, 20ml ethanedithiol, and 983mg skatole
After stirring at room temperature for 50 minutes, under reduced pressure.
TFA was distilled off. Ether was added to the residue, and the resulting precipitate was dissolved in a mixed solution of 150 ml of DMF and 50 ml of N-methylpyrrolidone. To this, Boc−Tyr
(Bzl- _Cl2 )-OH3.08g (1.4 times equivalent)
HOBt0.95g (1.4 times equivalent), WSCD1.28ml
(1.4 times equivalent) was added, the pH was adjusted to 7 with NMM, and the mixture was stirred at room temperature for 48 hours. After distilling off DMF under reduced pressure, ice water was added, and the resulting precipitate was washed three times with hot ethanol to obtain 9.00 g of the target product (yield:
88.0%). TLC; Rf ¹ 0.76 (silica gel) Amino acid analysis value; Asp1.90(2), Thr0.91(1),
Glu1.96(2), Ala0.89(1), Val0.90(1), Met0.93
(1), Leu(1), Phe0.95(1), Tyr0.92(1),
Tvp0.68.(1), In addition, the abbreviations described in this specification have the following meanings. Ala; L-alanine Gln; L-glutamine Asp; L-aspartic acid Asn; L-asparagine Phe; L-phenylalanine Val; L-valine Trp; L-tryptophan Leu; L-leucine Met; L-methionine Thr; L-threonine Boc; t-butoxycarbonine Bzl; benzyl OBzl; benzyl ester TFA; trifluoroacetic acid NMM; N-methylmorpholine DMF; dimethylformamide HOBt; 1-hydroxybenzotriazole WSCD; N-ethyl, N'-3- Dimethylaminopropyl-carbodiimide Bzl-Cl ₂ ; 2,6-dichlorobenzyl The following carriers and developing solvents were used for thin layer chromatography (TLC). 1; Chloroform-methanol-acetic acid (80:25:2) Silica gel manufactured by Merck & Co.
Avt5721, 2; 1-butanol-pyridine-acetic acid-
Water (15:10:3:12) Merck cellulose
Avt5716 (B) Preparation of ¹²⁵ I-[Tyr ¹⁸ ]-glucagon (18-29) Using [Tyr ¹⁸ ]-glucagon (18-29) obtained in (A) above, WMHunter, FC
The method of Greenwood et al. [Nature (London) 194 ,
495 (1962)] and gel-filtered with a Sephadex G-25 column (0.9 x 20 cm).
¹²⁵ I-[Tyr ¹⁸ ]-glucagon (18-29) was obtained. [Simultaneous measurement of insulin and glucagon by RIA] Solid phase body [test tube]-A contains 100μ of immune reaction buffer (above), ^125I -insulin (10.0pg as insulin) 50μ ^125I- [Tyr ¹⁸ ]-glucagon ( 18-29) (4.5pg as glucagon) 50μ,
and standard solution (5-640μU/as insulin)
ml), contains 25-3200 pg/ml as glucagon)
Add 100μ and add solid phase (element)-B to this.
One piece of was added and the reaction was carried out at 5°C overnight. After completion of the test, 5 ml of physiological saline was added, and solid phase material B was first extracted while being washed, transferred to a new test tube, and its radioactivity was measured. Separately, the radioactivity of solid phase material A was measured after the washing liquid was removed by decantation. The results are shown in Figure 8.
Further, in FIG. 8, ○-○ indicates the quantitative curve of insulin, and Δ-△ indicates the quantitative curve of glucagon.The simultaneous insulin and glucagon measurement system of the present invention showed an extremely good quantitative curve. In FIG. 8, the horizontal axis shows insulin and glucagon concentrations, and the vertical axis shows B/Bo (%). Example 3 CEA and ferritin by Sanderuch method
Simultaneous measurement with EIA [Preparation method of solid phase substance [test tube]-A] Polystyrene test tube (15 x 105 mm, inner diameter 12.8±
Add 2 ml of concentrated sulfuric acid to 0.1 mm), react at 5°C for 6 hours to introduce -SO ₃ ^- group, and add 0.1 M phosphate buffer (PH8. 0) Add 2 ml and react at 5℃ overnight.
[Test tube - EIA antibody] Solid phase body A of the conjugate was obtained. [Preparation method of solid phase body [element]-B] 50 elements made of 6,6 nylon at 30% (V/
V) Add to 50 ml of a methanol solution containing dimethyl sulfate, react at 37°C for 50 minutes, wash thoroughly with methanol, add 50 ml of an aqueous solution (PH 11.5) containing 100 mM hexamethylene diamine, and react for 1 hour at room temperature. Wash thoroughly with M phosphate buffer (PH8.0), add 50 ml of 0.1M phosphate buffer (PH8.0) containing 4% (V/V) glutaraldehyde, and react at room temperature for 1 hour. After washing with 0.1M phosphate buffer (PH8.0), a spacer was introduced into a 6,6 nylon element. These elements (50 pieces) were added to 50 ml of 0.1 M phosphate buffer (PH8.0) containing 5 mg of the IgG fraction of anti-ferritin rabbit serum.
[Element-
Ferritin antibody] A solid phase body B of the conjugate was obtained. [Preparation method of β-Gal-labeled ferritin antibody] An IgG fraction was obtained from ferritin antiserum by ammonium sulfate fractionation, and the antibody was further purified by affinity chromatography using Sepharose 4B antibody on which ferritin was immobilized. 4 mg of purified antibody
Dissolve in 1.8ml of 0.1M phosphate buffer (PH8.0) and add 3-
200 μg of a dimethylformamide solution containing 14.88 μg of (2'-benzothiazolyl-dithio)propionic acid succinimide ester and 20 μg of a 100 mM EDTA aqueous solution were added, and the mixture was reacted at 5° C. for 1 hour. After the reaction, this was mixed with Cephadex G-15 (1.5×
The flow-through fraction was obtained by gel filtration with a 40cm column (3-(2'-
[Including derivatives with benzothiazolyl-dithio)propionyl groups introduced]. Next, 3.33 mg of β-galactosidase was added to 2 ml of 50 mM phosphate buffer (PH7.0) containing 1.5 mg of this 3-(2′-benzothiazolyl-dithio)propionyl group-introduced derivative.
2 ml of 50 mM phosphate buffer (PH7.0) was added, and the mixture was reacted at room temperature for 2 hours. Transfer the reaction solution to Sephadex G-
The gel was filtered through a 150 (1.5 x 90 cm) column and the flow-through fraction was collected to obtain a β-galactosidase labeled ferritin antibody conjugate (purity 98%) (β-Gal labeled ferritin antibody). [Preparation method of β-Gal-labeled CEA antibody] An IgG fraction was obtained from CEA antiserum by ammonium sulfate fractionation, and the antibody was further purified by affinity chromatography using Sepharose 4B antibody on which CEA was immobilized. 4 mg of purified antibody in 0.1M phosphate buffer (PH8.0) 1.8
3-(2′-benzothiazolyl-dithio)pyropionic acid succinimide ester dissolved in
200μ of dimethylformamide containing 14.88μg, and
20μ of 100mM EDTA aqueous solution was added and reacted at 5°C for 1 hour. After the reaction, this was gel-filtered through a Cephadex G-15 (1.5 x 40 cm) column to obtain the flow-through fraction [3-(2'-benzothiazolyl-dithio)propionyl group was introduced into the amino group of the CEA antibody. [including derivatives]. Then,
50 containing 1.5 mg of this 3-(2′-benzothiazolyl-dithio)propionyl group-introduced derivative.
50mM phosphate buffer (PH7.0) containing 3.33mg of β-galactosidase in 2ml of mM phosphate buffer (PH7.0)
2 ml was added and reacted at room temperature for 1 hour. The reaction solution was gel-filtered through a Cephadex G-150 (1.5 x 90 cm) column, the flow-through fraction was collected, and β-galactosidase labeled CEA antibody conjugate (purity 98%) (β
-Gal-labeled CEA antibody) was obtained. [Simultaneous measurement of CEA and ferritin by EIA] In solid phase body [test tube]-A, add 200 μl of immune reaction buffer (above) and standard solution (2.5 to 2.5 μl as CEA).
160ng/ml, and as ferritin, 5~
320ng/ml) was added thereto, one solid phase [element]-B was added thereto, and the reaction was carried out at 37°C for 5 hours. After completion, the internal solution was removed by suction, and 5 ml of physiological saline was added to wash the solid phase bodies-A and B. This was combined with β-Gal-labeled ferritin antibody and β-Gal-labeled ferritin antibody.
Immunoreaction buffer containing Gal-labeled CEA antibody
The reaction was carried out for 18 hours at room temperature.
After finishing, add 5 ml of physiological saline and first add solid phase-B.
The sample was taken out while being washed, and added to a small test tube to which 500μ of the β-galactosidase activity measurement solution (above) had been added in advance, and allowed to react at 37°C for 3 hours.
2 ml of reaction stop solution (100mM glycine-NaOH buffer (PH11.0)) was added to stop the reaction, and after removing solid phase B, the absorbance of the solution at 420 nm was measured. In A, after the washing solution was removed by decantation, 2 ml of the β-galactosidase activity measurement solution (above) was added, and the reaction was allowed to proceed at 37°C for 5 hours.
0.5 ml of NaOH buffer (PH 11.0) was added to stop the reaction, and the absorbance of the reaction solution at 420 nm was measured.
As shown in Figure 9, and in Figure 9, 〇-〇 are
A quantitative curve of CEA is shown, and Δ-Δ is a quantitative curve of ferritin. The CEA and ferritin simultaneous measurement system of the present invention showed an extremely good quantitative curve. Example 4 Thyroxine [T ₄ ] and thyronine [T ₃ ]
Simultaneous measurement by EIA [Preparation method of solid phase body [element]-A] Four polystyrene element fragments shown in Fig. 6 (element fragments obtained by dividing the element into four equal parts in Fig. 5) are combined (dividable) into four pieces. The element was activated with sulfuric acid and SO ₃ ^-
IgG of anti-rabbit IgG goat serum
100mM phosphate buffer (PH) containing 20μg of fractions
8.0) Polystyrene element solid phase was immersed in 1 ml and reacted overnight at 5°C to obtain 8.4 μg of IgG immobilized on each polystyrene element solid phase. Next, this element solid phase was immersed in 1 ml of an 8000-fold dilution of anti-T ₄ -OMe serum (rabbit) (described in the previous section) and allowed to react at 5°C overnight to determine the [element-anti-rabbit IgG-T ₄ antibody] conjugate. Solid phase body-A was obtained. [Preparation method of solid phase body [element]-B] A polystyrene element having exactly the same shape as shown in Fig. 6 (phthalocyanine blue was mixed in during molding and colored pale blue) was treated in the same manner as above to obtain anti-rabbit IgG- An immobilized element solid phase body was obtained. Next, the solid phase body was immersed in 1 ml of a 20,000-fold dilution of T3 _- OEt serum (rabbit) and reacted overnight at 5°C to form a solid phase body of the [element-anti-rabbit IgG- _T3 antibody] conjugate.
I got a B. [Preparation of solid phase element [element]-C] One fragment of the solid phase element-A that was divided again and three fragments of the solid phase element-B were separated into two pieces with sharp edges and a stopper in the center. They were combined in a coupling machine having a T ₄ , T ₃ -solid phase for simultaneous measurement [element]-C was prepared. [Preparation method of T ₄ -OMe] 600 mg of T ₄ (manufactured by Sigma) was suspended in 15 ml of methanol, dried hydrogen chloride gas was blown into the suspension at -10°C until saturation, and the suspension was stirred at room temperature for 20 hours. 0 more
After blowing in dry hydrogen chloride gas at °C, the precipitate precipitated was collected by filtration, washed with methanol containing hydrogen chloride gas, dried under reduced pressure in a desiccator containing solid sodium hydroxide, and then recrystallized with methanol-ether. , the desired T ₄ -OMe 582.5 mg (yield 91.2%) was obtained. [Preparation method of β-Gal-labeled T ₄ -OMe] 2 mg of T ₄ -OMe was dissolved in 90% dimethylformamide, 10
%100mM phosphate buffer (PH8.0) 2ml,
1.78 ml of dimethylformamide containing 1.78 mg of 3-(2'-benzothiazolyldithio)propionic acid succinimide ester was added, and the mixture was allowed to react for 1 hour under cooling. The reaction solution was added to a LH-20 (1.5 x 40 cm) column, and 90% dimethylformamide, 10%
Gel filtration was performed using a 100 mM phosphate buffer (PH5.0) as a solvent. The eluate was fractionated into 2 ml portions, and 3-(2'-benzothiazolyl-dithio) was added to the amino group of _T4 .
Fraction containing a propionyl group-introduced derivative (12
~15th item) was collected. Next, β-galactosidase 2 was added to 2 ml of a 10% dimethylformamide solution in a 90% phosphate buffer (PH8.0) containing 8 μg of this 3-(2′-benzothiazolyl-dithio)propionyl group-introduced derivative.
mg was added thereto, and the mixture was allowed to react at room temperature for 2 hours. The reaction solution was added to a Sephadex G-150 (1.5 x 90 cm) column, gel filtration was performed with 10 mM phosphate buffer (PH7.4) containing 0.15 M NaCl, and the flow-through fraction was collected. ₄ and the thiol group of β-galactosidase.
-Galactosidase- _T4 -OMe conjugate (purity 93
%) (β-Gal labeled _T4 -OMe) was obtained. [Preparation method of T ₃ -OEt] 500 mg of T ₃ (manufactured by Sigma) was suspended in 15 ml of ethanol, dried hydrogen chloride gas was blown into the suspension at -10°C until saturation, and the suspension was stirred at room temperature for 20 hours. The reaction solution was filtered, the filtrate was concentrated under reduced pressure, benzene was added, and the deposited precipitate was collected and dried. Furthermore, the target T ₃ -OEt was 515 mg (yield 93.7%) by suspending and washing in benzene.
I got it. [Preparation method of β-Gal-labeled T ₃ -OEt] 2 mg of T ₃ -OEt was dissolved in 90% dimethylformamide, 10
Dissolve in 2 ml of 3-% phosphate buffer (PH8.0) and
2.06 ml of dimethylformamide containing 2.06 mg of (2'-benzothiazolyldithio)propionic acid succinimide ester was added, and the mixture was allowed to react for 1 hour under cooling. Add the reaction solution to a LH-20 (1.5 x 40cm) column, add 90% dimethylformamide, 10% 100m
Gel filtration was performed using M acetate buffer (PH5.0) as a solvent. The eluate was fractionated into 2 ml portions, and fractions (12th to 15th fractions) containing a derivative in which a 3-(2'-benzothiazolyl-dithio)propionyl group was introduced into the amino group of _T3 were collected. Next, 2 mg of β-galactosidase was added to 2 ml of a 10% dimethylformamide solution in 90% phosphate buffer (PH 8.0) containing 7.2 μg of this 3-(2'-benzothiazolyl-dithio)propionyl group-introduced derivative, and the mixture was heated at room temperature. The mixture was allowed to react for 2 hours. The reaction solution was added to a Sephadex G-150 (1.5 x 90 cm) column, gel filtration was performed with 10 mM phosphate buffer (PH7.4) containing 0.15 M NaCl, and the flow-through fraction was collected. β-galactosidase-T _{3 -OEt} conjugate (purity:
95%) (β-Gal labeled T ₃ -OEt) was obtained. [Preparation method of T ₄ -BSA conjugate] Add 25 mg of T ₄ -OMe to 17.5 ml of dimethylformamide.
Contains 6 mg of glutaraldehyde, dissolved in
Add 16ml of 10mM phosphate buffer (PH8.0) and stir at room temperature.
The reaction was allowed to proceed for 30 minutes. Next, 20 ml of 10 mM phosphate buffer (PH8.0) containing 113.6 mg of BSA was added to this.
The mixture was further reacted at room temperature for 2 hours. The reaction solution was sufficiently dialyzed against distilled water, the dialyzed solution was freeze-dried, and the BSA-T ₄ -OMe conjugate (binding molar ratio 1:
8.6) was obtained. [Preparation method of T ₃ -BSA conjugate] 25 mg of T ₃ -OEt was dissolved in 17.5 ml of dimethylformamide, and 10 molar solution containing 7 mg of glutaraldehyde was added under stirring.
Add 16ml of M phosphate buffer (PH8.0) and incubate for 30 minutes at room temperature.
Allowed to react for minutes. This then contains 118mg of BSA.
20 ml of 10 mM phosphate buffer (PH8.0) was added, and the mixture was further reacted at room temperature for 2 hours. The reaction solution was dialyzed with distilled water, and the dialyzed solution was freeze-dried to obtain a BSA-T ₃ -OEt conjugate (bond molar ratio 1:12.6). [Preparation of antibodies] 1 mg of each BSA-T ₄ -OMe and BSA-T ₃ -OEt conjugate (95 μg as T ₄ -OMe, 116 μg as T ₃ -OEt) was added to a 10 mM phosphate buffer containing 0.15 M NaCl (PH7. 4) Dissolve in 1 ml and add Freund's Complete Adjuvant (Freund's
Complete adjuvant) 1.0 ml was added and thoroughly mixed to obtain an emulsion. Next, each rabbit was injected with 2 ml of the emulsion into the skin of the fingers and toes and subcutaneously at several locations on the back. The same amount of emulsion was subcutaneously injected 8 times every 2 weeks,
Whole blood was collected 10 days after the final immunization, left at room temperature for 60 minutes to coagulate, and then incubated at 3000 rpm for 10 minutes.
Anti- _T4 -OMe serum (rabbit), respectively, by centrifugation for min.
Anti- _T3 -OEt serum (rabbit) was obtained. [Simultaneous measurement of T ₄ and T ₃ by EIA] In a test tube (15 x 105 mm, inner diameter 12.8 ± 0.1 mm), 100 μl of immunoreaction buffer (described above) and β-Gal labeled T ₄ −
OMe (approximately 2.0ng as _T4 ) 50μ, β-Gal labeled _T3
−OEt (approximately 0.1 mg as T ₃ ) 50μ, and standard solution (10-640 mg/ml as T ₄ , 0.5-32 ng/ml as T ₃ )
ml of solution) was added, one solid phase [Element]-C was added thereto, and the reaction was carried out at 37°C for 3 hours while rotating the solid phase at 30 minute intervals.
After completion of the reaction, 5 ml of distilled water was added and the solid phase -C was removed while being washed. Divide the washed solid phase into one solid phase fragment for T ₄ and three solid phase fragments for T ₃ , and immerse each in a test tube to which 2 ml of β-galactosidase activity measurement solution has been added in advance. _Solid phase fragments for 30 min at 37 °C, T ₃
The solid phase fragments were allowed to react for 2 hours. After the reaction is complete, add 0.5ml of reaction stop solution [400mM glycine-NaOH buffer (PH11.0)] to stop the reaction.
After removing the solid phase, 420nm of each liquid
The absorbance at was measured. The results are as shown in Figure 10, in which 〇-〇 indicates the quantitative curve of T ₄ , △-△ indicates the quantitative curve of T ₃ ,
The simultaneous measurement system for T ₄ and T ₃ of the present invention showed an extremely good quantitative curve. In addition, when measuring T ₄ and T ₃ in a test sample (serum, plasma, etc.),
The same operation as above was carried out by adding 100μ of the sample instead of 100μ of the standard solution, and the amounts of T ₄ and T ₃ in the sample were determined by comparing with the quantitative curve.

[Brief explanation of the drawing]

The drawings show several preferred embodiments of the present invention; FIG. 1 is an enlarged partial sectional view of the element, FIG. 2 is a plan view thereof, and FIGS. FIG. 4 is an enlarged sectional view of the element of the second embodiment; FIG. 5 is an actual measurement diagram of one example for manufacturing the element; FIG. The figure shows an element fragment divided into four equal parts, Figure 7 is a quantitative curve for simultaneous measurement of insulin and glucagon by EIA, Figure 8 is a quantitative curve for simultaneous measurement of insulin and glucagon by RIA, and Figure 9 is by EIA.
Quantitation curve for simultaneous measurement of CEA and ferritin, 1st
Figure 0 shows the quantitative curve of simultaneous measurement of T ₄ and T ₃ by EIA. Explanation of the symbols: 1 is the element carrier, 2 is the element body, 4 is the reaction tube, 5 is the protrusion, 6 is the gap,
8 is a cut groove.

Claims (1)

[Scope of Claims] 1. The reaction medium has a shape that expands the surface area and can be inserted into the immune reaction part of the reaction tube, and is formed so that the outer diameter maintains a slight distance from the inner diameter of the reaction tube. Reaction with one or more media element carriers that can be used as a separable carrier, which are formed with protrusions on the bottom that form the same gap as the reaction tube and the element carrier. The surface area of the reaction medium is expanded in the reaction by using a tubular carrier and using two or more solid phase bodies each having a substance that has specific binding ability for the component to be measured immobilized on each of the carriers. A multi-component simultaneous measurement method characterized by reaction. 2. The multi-component simultaneous measurement method according to claim 1, wherein the media element carrier that can be used as one or more separable carriers is a media element carrier that can be separable into two or more. 3 The reaction medium has a shape that expands the surface area of the reaction medium and can be inserted into the immune reaction part of the reaction tube, and is formed so that the outer diameter maintains a slight distance from the inner diameter of the reaction tube, and during this time A media element carrier and a reaction tube carrier that can be used as one or more separable carriers that are formed with protrusions forming the same gap on the bottom so that the gap between the reaction tube and the element carrier is the same. A liquid containing the component to be measured and a solid phase body having the binding ability to immobilize a substance having specific binding ability for the component to be measured on two or more of the carriers. Simultaneous multi-component measurement according to claim 1, characterized in that the labeled compounds are reacted, their solid phase bodies are then collected, and the labeling activity of the labeled compounds bound to the solid phase bodies is measured. Law. 4. The substance that has specific binding ability for the component to be measured is a receptor, the component to be measured is an antigen, and the labeled compound that has binding ability is a labeled antigen, or The substance that has specific binding ability to the antigen is the antigen, the component to be measured is the receptor, and the labeled compound that has the binding ability is the labeled receptor, or the substance that has specific binding ability to the component to be measured is the labeled receptor. The substance that has the ability is the receptor,
The component to be measured is an antigen, and the labeled compound having binding ability is a labeled receptor having binding ability to the antigen to be reacted after the receptor and antigen have reacted, or the component to be measured is The substance that has specific binding ability is the antigen, the component to be measured is the receptor, and the labeled compound that has the binding ability binds to the receptor for reaction after the antigen and receptor have reacted. 4. The method for simultaneous multi-component measurement according to claim 3, wherein the multi-component simultaneous measurement method is performed using the same or different measurements. 5. The method for simultaneous measurement of multiple components according to claim 3 or 4, wherein the label is an enzyme or a radioactive substance. 6. The method for simultaneous multi-component measurement according to claim 1, 3 or 4, wherein the solid phase body is a carrier-second antibody-first antibody.