JPS60500973A - Insoluble substances treated to prevent nonspecific protein binding - 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] , an insoluble substance treated to block specific protein binding. Technical field The present invention is directed to the use of living organisms on solid carriers for diagnostic tests, enzyme treatments, affinity purification, etc. It relates to chemical or immunological substances.

Background technology Soluble biological substances that land on solid supports can be used in diagnostic tests, enzymatic treatments and affinity treatments. It is often used in sticky products. For example, encapsulating antibodies or antigens onto solid supports. Because of this, its immunological partner can be easily removed from the 4N, compound of many substances. can be done. Similarly, when an enzyme is attached to a solid support, it is added to the reaction mixture. can be easily removed from the flow or used in continuous flow processing. Heterologous radioimmunology assays and enzyme immunoassays attach one or more reactants to a solid phase and release free It depends on its ability to be separated from the reactants. Agglutination assay (presence of antigen or antibody in fluid) (in order to determine the (carrying appropriate immunological material). Cells, Cell Components and Bacteria The separation and fixation of is aided by solid-bound antibodies or antigens. biological grains for example, by specifically attaching to solid bodies coated with appropriate antibodies and antigens. , fractionation from other rice 1″I particles can be performed. Immobilization of biological particles 1″ suitable This can be done by specific encapsulation of small particles coated with specific antibodies or antigens. this The existence of small lees is a substance that can be easily detected. It may also contain a trainer or electron-dense material.

The main difficulty in using the above-mentioned homologous carriers is that soluble biological substances Make sure to check the quality! 1 and prevent undesired substances from sticking to the carrier. It is to be. The small size of these problems is due to excessively high background and low sensitivity during the test. and material loss and low purity in affinity purification and enzymatic treatments. Ru.

A solution to the first of these problems is the incorporation of proteins and peptides into polymeric solids. It is made by conjugate bonding. For example, in U.S. Special Terms No. 3,845,852- used cyanogen halides to activate water-insoluble polymers, and then activated water-soluble proteins. It describes how to combine.

U.S. Patent No. 3,857. ! According to No. 131, water-soluble sulfodiimides are proteins. can be used as a condensing agent to bind the nail coalescing carrier particles. US patent No. 4,001. ．． 583 As described in No. 3, the surface of biological materials is coated with glutaraldehyde. can be covalently bonded to plastic materials. U.S. Patent No. 4,048 , No. 723, proteins are bonded to latex having carboxylic acid amide groups on the surface. Sudame's three-step method is demonstrated. Protein or peptide polystyrene latte A method for producing the compound is described in U.S. Pat. No. 4,118,349. In the case of , the bond is made by an aromatic diazonium compound. bifunctional alde Immunology via reaction with diamines mediated by carbodiimide followed by reaction with hydrogen A two-step process for attaching target materials to latex polymers is described in U.S. Pat. No. 4,140. efi It is featured in issue 2. The above-mentioned methods are known for bonding biological substances to polymeric materials. as well as other methods (for example, "Chemical Modif" by Kiefer) cation Of proteins, Haptens, And5o lid Supports”, Immunological Methods, Academic Press, 1878 , pp. 137-150) is a hydrophobic bond that was usually used before the covalent bond method. There is no doubt that it is more reliable. However, these methods does not eliminate the problem of nonspecific fixation and sometimes makes it worse.

U.S. Pat. No. 4,284,786 describes water-soluble polyhydroxy compounds, preferably The aminopolysaccharide is transferred to a latex carrier, preferably a carboxylated polymer, with a water-soluble carbon. rubodiimide, Woodward reagent K (N-ethyl-5-Tokuhyo GO-50097 :((5) phenyl-inxazolium-3'-sulfonate) or water-soluble chloroformier An invention has been described which solves some of the problems mentioned above by covalently bonding through It is listed. Latin 1, the 7-mino group of the aminopolysaccharide that does not bind to the sugar carrier is hydrated. Converts to roxyl group. The polysaccharide is then activated with periodate to produce glucose. Some of the rings are oxidized to dialdehydes. Afterwards, the immunologically active material is added to this React with activated polysaccharide. Reaction with immunologically active materials is dialdehyde This must be done within a short time after the formation of the species. The reason is that dialdehyde-containing polysaccharides This is because they undergo relatively rapid deterioration. That is, pre-activated latex- Polysaccharide particles cannot be easily stored and may contain any desired immunologically active material. It cannot be shipped to the end user where the material may adhere. Furthermore, immunologically The thick base produced by the reaction of the amine group of the active material with dialdehydes is borax. It is necessary to stabilize it with sodium dichloride. This results in an excess of immunologically active It is necessary to carry out at 0° C. to keep denaturation to a minimum after removal of the material.

The latex particles in U.S. Pat. No. 4,284,788 are It has a surface completely covered with aminopolysaccharides that bind to the carboxyl groups of this is in a solution other than a protein that is a partner for the attached immunologically active material. proteins from adhering to latex particles. The problem with this method is that the dial Formation of dehydes, reactions with dialdehydes and reduction of Schiff bases require skill and care. This is a reaction that requires significantly more money and time.

Disclosure of invention According to one example of the present invention, when a protein is accompanied by other proteins, specific binding partner for specific binding proteins. Compositions for use are described. This composition contains specific binding proteins and other proteins. contains a water-insoluble support having a surface capable of entraining molecules. In addition, ``2nd composition The first substance of the support surface mainly consisting of first and second substance surface parts. The first parenchymal portion is sufficiently covered to substantially prevent protein binding to the first parenchymal portion. Contains a polysaccharide coating that does not cover 2 substantial parts.

According to another embodiment of the invention, the aforementioned composition is further attached to a second substantial surface portion. Contains biological material.

Furthermore, according to another example of the present invention, when a protein is accompanied by other proteins, biological It specifically binds to a specific binding protein that is a specific binding partner for a target substance. A method of forming a water-insoluble surface of a solid support is provided. This method is specially A water-insoluble surface capable of entraining foreign binding proteins as well as other proteins; It mainly consists of a surface part and a second substance surface part, and the first substance is coated with polysaccharide. using a solid support having a surface where one portion is covered and a second substantial portion is uncovered; It means something.

According to yet another example of the invention, the specific binding moiety for the first biological substance A specific binding protein having a first binding site that acts as a binder is accompanied by other proteins. A method is described for assaying aqueous sample solutions containing Ru. This method allows the aqueous sample solution to be accompanied by specific binding proteins as well as other proteins. a first water-insoluble surface covered by a polysaccharide coating; and a second substantial surface portion to which a first biological substance is attached. and a first solid support having a . First solid support for sample aqueous solution The amount of specific binding protein bound to the first biological substance separated from the body and landed in the body To detect.

Other examples of the invention include a first substantial surface portion and a second substantial surface portion consisting essentially of a first substantial surface portion and a second substantial surface portion. It binds to specific binding proteins that serve as specific binding partners for biological substances. It is a method of reducing the adhesion of unwanted proteins to water-insoluble surfaces that have the ability to Ru. This method involves shielding a first parenchymal surface portion with a polysaccharide coating and The purpose is to apply a substance to the second parenchymal surface area.

Furthermore, other examples of the invention include a specific binding partner for a first biological substance; a first binding site that is a binding partner for a second biological substance; and a second binding site that is a binding partner for a second biological substance. binding) ′ζ[☆ and potentially accompanying the specific binding protein with other proteins The present invention provides kits for assaying samples with increased specificity and sensitivity. This key The kit can entrain the specific binding protein and other proteins, and the shielded a first parenchymal surface portion and a second parenchymal surface portion to which the first biological substance is attached; a solid support bearing a first water-insoluble surface; This Ki71□hasa Furthermore, specific binding proteins and other proteins can be entrained, and polysaccharide coated The first parenchymal surface part that was shielded by a plurality of solid particles each having a second insoluble surface consisting primarily of a solid surface portion; Contains children.

The present invention provides that the polysaccharide coating is applied to the surface of a solid support where proteins can normally be entrained. If a first portion is covered and a specific biological material is applied to the remainder of the surface, the The only proteins that are not specific binding partners for physical substances are biological substances. It is based on the discovery that even the part of the surface that has been deposited cannot adhere to the surface of the support. It is something. The partially coated composition of the present invention, which is relatively easy to manufacture, The adhesion of biological knowledge is all 1N surface GO-500973 (6) It's very easy. In general, these compositions are based on theory 1. <cheap (this can be manufactured) . Moreover, the m substitute of the present invention is the name.

Form F'U for attachment of desired biological material, 11. i″i′ to store or hold a ship I can do it.

The best form for carrying out the uninvention 03, accompanied by Ito-White 1e↓ or other proteins When a specific binding partner is a specific binding partner for a biological substance, A composition useful for specifically binding to is provided in 1. Here ゛'I 16 Physics A specific substance called a toner is a substance that acts as a specific binding partner for a specific binding protein. It is widely used in j-me without indicating the substance of meaning. Here is an example of this work V1: , enzymes, antibodies, natural receptors such as thyroxine-binding globulin and ahidin , clobins such as hemoglobin, lens proteins, surface antigens, N [↓ There are monopolymer antigens, etc. The specific binding protein is water-insoluble. The desired function can be made with protein. Is there a long list of such substances? It was unveiled on August 4th, 2015 in the United States.

Composition 1-! 'lj! 'I! Target combination A F white τ Rinami Hini and other 1 A1 Contains a water-insoluble support that disrupts the surface that can entrain white matter. This solid support The body may be formed of microscopic or gigantic grains or large expansible surfaces such as walls, flat plates, wells, etc. j, both of which can be used to separate protein 41 formulations. Ru.

According to the invention, if the water-insoluble support is in the form of nine particles, these particles preferably has a specific gravity close to that of water so that it can be stably suspended in an aqueous medium. Gaga The particles usually have a diameter of about 0.2 microns to 1 cm.

The solid support itself must be inert to immunological diagnostic tests. many materials can be used as a water-insoluble support. Of particular interest is the U.S. patent no. 4.0413,723, 4,118,349, 4,140,682 and and latexes such as those described in US Pat. No. 4,264,786. can be used Glass surfaces are described in US Pat. No. 4,1139,138. other useful Polymers are described in U.S. Pat. Nos. 3,619,371, 3,700,809, 3. No. 853,987, No. 4,108. ! No. 1172 and No. 4,201,763 It has been shown in history. Each of these patent specifications describes various biological substances, particularly proteins. A wide range of binding groups for binding to white matter have been described. solid support is latex It is preferable to check for active groups that can form covalent bonds with polyhydroxy compounds. It is. Therefore, the latex support has carboxyl groups, amino groups, or It is possible to have an active +1 group such as a group that can be converted into a group. Illustrated on latex support The active groups for 1 are -coOu, -Cf3Nl (active hydrogen-containing groups such as 2, primary and and a secondary amine group or nitrile group. US VF Fraud No. 4,284.7H describes a number of latex materials that are particularly suitable for use in the present invention. Book The preferred latex material for practicing the invention is polystyrene. Especially acrylic polystyrene combined with carboxyl-containing J compounds such as acid, methacrylic acid, etc. Also included.

The protein is coated on the surface of the water-soluble support for 1' (coflu'). iゑ The first substance table of the water-insoluble support [substantially prevents binding to the 111 portion] For many aI! Apply the intellectual coating. In addition, this amount of 11th class cortein This is the second real table of supports! Fl! It is required that the 41f portion not be covered. Ru. The polysaccharide coating should be 5,000 daltons or more, especially 10 It is formed from polysaccharides characterized by relatively high molecular weight, 1'-silicon equals 1,000,000 tardors or more. Tawa class 11+-k 'II') Records and flags 1--The weight of the Combined or co-combined, like neuraminic acids, uronic acids, glycosamines It can be a +D+j compound of carbohydrate compounds. Furthermore, the dye +3”n is A combination of λ゛1 or cross-carrying copolymers, or their 411-type eta and condensation It can be combined with monomers, especially epoxides. Especially interesting Sugars include dextran, ficoll, sucrose and epichlorohydrin. Synthetic copolymers of loose or hyaluronic acid'.1.

Approximately 2 to 6) ↓ (alkylene groups with 4 atoms r) bonded to the functional groups of polysaccharides I am interested in the amine groups normally attached to the chain. Potassium present in diamines and polysaccharides Particularly convenient are reaction products with ruboxyl functions. For example, J, of Imm unolog! 1,114.704-709 (1975).

According to the invention, from 1,000 to 1,000,000 or more as mentioned above. It is possible to use a relatively high molecular weight amine polysaccharide such as Ficoll, which has a normal molecular weight. Particularly suitable. By using aminophycols having a molecular weight of about 4oo, ooo Excellent results were obtained.

coating only a first substantial surface portion of the water-insoluble surface; and a second substantial surface portion of the water-insoluble surface; In order to ensure that the polysaccharides remain without being covered with polysaccharides, the amount of attached polysaccharides is calculated using the water-insoluble table. Approximately 0.5 x 10-' to 3 x 10-' per cm' of total surface area, g It is necessary to control within the range of . This means that the molecular weight of the polysaccharide is approximately 400,000 nm. In case of about 700-. This corresponds to 4000 molecules/pL2.

According to one example of the invention, the biological material is attached to the second substantial surface portion of the water-insoluble surface. do. This attachment can be done by hydrophobic adsorption, electrostatic bonding, covalent bonding, or a combination thereof. I can do it. Preferably, the biological material is covalently attached to the second substantial surface portion.

When a biological substance is covalently bonded to the second substantial surface portion, such covalent bonding is optional. This can be achieved in a number of ways. using activators such as water-soluble carbodiimides. This is due to the formation of adducts with two carbonyl groups on the latex surface. Next Amine bonds are created by reacting carbodiimide adducts with amino groups on biological materials. remains on the latex support. Other useful activators are Woodward's reagent K (N -ethyl-5-phenyl-isoxazolium-3'-sulfonate) or water-soluble It is chloroformate.

Using insoluble materials to specifically bind to proteins present in a mixture The composition can be used in cases where: This relationship is confirmed by antibacterial protein binding assay. It is encountered in cell identification, cell segmentation, cytology, histology, etc. vary widely in treatment This invention generally applies to insoluble materials as particles or larger structures. and a protein mixture, the biological substance and the specific protein on the surface are combined. This includes allowing sufficient time for binding between binding partners. Next, Wash the body surface to free it from non-specific binding proteins, leaving only specific proteins. .

Of particular interest is the use of labeled or unlabeled particles. La as a sign These include geoisotopes, light materials, magnetic materials, enzymes, enzyme substrates, color dyes, etc.

Such labels can be attached to water-insoluble surfaces, polysaccharides or biological materials, and Preferably, it binds to water-insoluble surfaces or polysaccharides. Label throughout the particle if desired Can be uniformly dispersed.

and a first binding site that is a specific binding partner for the first biological substance. and a second binding site that is a binding partner for a second biological substance. For assaying samples potentially containing specific binding proteins in association with other proteins. I am particularly interested in the position that provides the kit. Such kits include slides, wells and a giant expansible surface wall defined on a support such as. Support is specific binding It has a first water-insoluble surface that can entrain proteins and other proteins. This first The water-insoluble surface includes a first substantial surface portion shielded with a polysaccharide coating and a first and a second substantial surface portion to which biological substances have adhered. Also, the kit each have a second insoluble surface that can entrain specific binding proteins and other proteins. The solid particles include a plurality of solid particles. This second insoluble surface is a polysaccharide coating. A first parenchymal surface portion that is shielded and a second parenchymal surface that has a second biological substance attached thereto. It consists of the main part and I- from the surface part. These particles are labeled with a label that can generate a detection signal. can be done. For example, particles may be colored with a coloring agent such as a dye. , and the signal is simply the color itself. Alternatively, use other signs such as those described above. can do.

Production method The compositions of the invention can be manufactured in a number of ways, some of which are described below. Ru.

nine popes The compositions of the invention are water-insoluble proteins that can entrain specific binding proteins and other proteins. It can be manufactured by preparing a solid support having a surface. the surface a first substantial surface portion of said surface is coated with a polysaccharide coating, while a second substantial surface portion of said surface is coated with a polysaccharide coating; The real surface area is not covered with a polysaccharide coating. This can be done by coating the surface with, for example, glass. Achieved by adding nitro groups by nitrification using a mixture of acid and sulfuric acid Ru. After washing the acid mixture off the surface, a readily available reducing agent such as stannous chloride is added to hydrochloric acid. Used in conjunction with nitro to reduce the nitro group to an amino group. Wash the solid support again and remove the halogen. A cyanide component is attached to the 7-mino group. Cyanogen halides usually have low water solubility. It is added as an ethanol solution to the aqueous solution that comes into contact with the solid surface. halogenation It is believed that cyanide is actually in the form of a monoethoxy derivative when added. Voluntary excess Wash away excess cyanogen chloride. Next, the residual halogen in the cyanogen halide fil Hydroxyl group (pka: approx. 10-') where the compound dissociates into hydronium ion It is thought that the surface is hydrolyzed to form a net negatively charged surface. This is Karboki Helps promote electrostatic binding of aminopolysaccharides to surfaces that do not necessarily have sil groups. Two. Aminopolysaccharide molecules are then attached to the surface. formed in this way The intermediate product is a water-insoluble surface having an amine polysaccharide electrostatically attached to at least a first surface portion. It is a solid support having. This intermediate product is then reacted with cyanogen halide. can cross-link adjacent amine polysaccharide molecules to form useful compositions. Wear.

This reaction with cyanogen halides is at least as difficult as possible due to its acidity and ionic strength. It serves to free the second surface portion from the amine polysaccharide coating.

Biological substances such as those described above may be attached to the second substantial surface portion of the water-insoluble surface. can. This can be achieved through hydrophobic adsorption, electrostatic binding or through the use of activators as described above. can be achieved in particular by covalent bonding.

Tomohiro” Another method for producing the composition of the present invention is to prepare a water-insoluble composition having carboxyl groups on its surface. The purpose is to prepare a support. sufficient to cover the surface with a monolayer of amine polysaccharide. The surface is contacted with an amine polysaccharide in a nominal amount. For example, excessive amounts of amyl Noficol can be contacted with the surface in an aqueous solution. Electrostatic amine polysaccharide Retained on the surface by bonding. This is because acids and highly concentrated salt solutions result in the removal of polysaccharides. It is also known for this reason.

Excess aminopolysaccharide is removed from the solid support by washing with water. After that, a halo like the one mentioned above Adding an ethanolic solution of cyanogenide to a solid surface coated with amine polysaccharide . At least a considerable portion of the amine group converts cyanogen halide into a cyanogen halide adduct. Sufficient amounts are used to add and cross-link the various amino groups to each other.

At the end of this reaction, a first substantial surface portion of the water-insoluble surface is coated with the aminopolysaccharide. a second substantial surface portion of the water-insoluble surface is not covered with the amine polysaccharide; . Although it is believed that the surface is originally coated with electrostatically bound amine polysaccharides, After reaction with the cyanogen halide component, the surface is no longer completely covered with aminopolysaccharides. This was experimentally confirmed. Instead, such surface areas are exposed to biological materials. Available for binding. Furthermore, after reaction with the cyanogen halide component, or highly concentrated salt solutions no longer remove the aminopolysaccharide. Generally on water-insoluble surfaces The amount of aminopolysaccharide remaining in the water is approximately 0.5XI per cm' of water-insoluble surface area. O-'~3X10-'g.

Biological substances can be attached to a second substantial surface via hydrophobic adsorption or covalent bonding as described above. Another method of making the compositions of the present invention is by depositing the carboxylic acid on the carboxylic acid. The water-insoluble surface containing active groups such as sil groups is insufficient to cover the entire surface. For reaction with amine polysaccharides and water-soluble carbodiimides in a single reaction step. Ru. The product obtained in this way can be converted into a carbodiimide by the carboxyl group, for example. containing an amine polysaccharide covalently bonded to the first substantial surface portion of the water-insoluble surface via do. A second substantial surface portion of the surface is not covered by amine polysaccharide molecules. remain.

Again, the biological material may be attached to the second stromal surface portion by any desired method. I can do it.

This method involves the use of an excess of carbodiimide to form a desired compound on a second substantial surface portion. Biological substances and covalently bonded carbodiimide-activated groups (e.g. Others forming the compositions of the invention have the advantage that carboxyl groups) may still be present. In the method, a water-insoluble surface containing live groups (e.g. carboxyl groups) The solid support to be treated is reacted with an excess of an activator compound such as a water-soluble carbodiimide. Carbodiimide (such as a carbodiimide) forms an additive. Any excess activator is washed away. leave Adding insufficient heavy engineering biological material to react with all activation sites . After the reaction is complete, the biological material remains immersed in the second substantial surface portion. next , the surface is washed to remove any reaction products. Water is brought into contact with the surface. , aminopolysaccharide is added to 71111~, which binds to the active group that remains activated by the activator. Match.

The product obtained in this way contains aminopolysaccharide, activity), and the use of an activating agent. It has a biological substance covalently bound to a water-insoluble surface by I7.

The various methods described above involve the use of carboxyl activating groups and carposiimine I activators. However, other activating groups and activating agents, such as the A solid one can be used as appropriate.

Test method The assay method also constitutes an important part of the invention. According to the invention, the first biology a) a first binding part that is a specific binding partner for the target substance; A method is described for assaying samples containing heterogeneous binding proteins together with other proteins. It is. This method does not involve entrainment of specific binding proteins and other proteins. a surface portion of the first fruit and a first biological object shielded with a polyaxenic coating; A first support with a water-insoluble surface having a second jersey surface portion with a texture attached thereto is tested. by contacting with an aqueous solution of the material. Place the sample aqueous solution on the first solid support. from the specific binding protein bound to the carrier and the first biological substance . The support consists of a plurality of cats, which are usually labeled with a detectable label (=1 being kicked. An example of a support and 1. For example, large expansible surfaces such as plates or wells or There is a board that has at least one well. Aminopolysaccharide, biological substance %t’: 'i f' A is as described above.

In a particularly preferred embodiment of the invention, the solid support is a giant expansible surface and T4゛1 converts the specific binding protein and other proteins r' in the form of a divisor of particles. an additional first substantial surface portion shielded with a polysaccharide coating and a second organism; a second water-insoluble surface having an additional second substantial surface portion having a chemical substance attached thereto; 2 a solid support in contact with a first macro-expandable surface, i.e. a plate or well; be. Specific binding proteins are molecules that act as specific partners for a second biological substance. It is chosen to have two binding sites. Then the particles onto the giant expansible surface Observe the degree of sealing.

Moreover, it mainly consists of a first parenchymal surface part and a second parenchymal surface part, and has a biological The ability to bind to specific binding proteins that serve as specific binding hardeners for substances. A method for reducing the force of adhesion of unwanted proteins to water-insoluble surfaces is described. There is. $1 The implementation surface area is shielded with a polysaccharide coating and the biological material is It is attached to the substantial surface area. The shielding and attachment steps can be performed simultaneously or sequentially. I can do it. The surface in this example is typically a giant expansible surface. In this way, Stick-reducing method paves the way for the use of bovine serum albumin on giant expandable surfaces. give This is due to the high cost of bovine serum albumin, and also because of the high cost of bovine serum albumin. Due to the lack of complete uniformity of bovine serum albumin in the This non-uniformity is inherent in the biological production of these reactants.

Inspection jL first fu” According to the invention, the first biological substance is a specific binding partner for the first biological substance. and a second binding site that is a specific binding partner for the second biological substance. Potentially contains a specific binding protein with a binding site along with other proteins A kit for assaying a sample is provided. This kit includes a first water-insoluble surface. a solid support having a large expandable wall, the first water-insoluble surface being a wall surface; It is. This first water-insoluble surface entrains the specific binding protein and other proteins. be able to. the first surface is a first substantial surface shielded with a polysaccharide coating; and a second substantial surface portion to which the first biological substance is attached. . A plurality of solid particles also form part of the kit. Each particle contains a specific binding protein and other and a second insoluble surface that can entrain proteins. This second surface is a first substantial surface portion shielded with a polysaccharide coating and a second biological material; and an attached second substantial surface portion. Generally, these particles It is marked with a sign that can be given a number. Often this sign is simply colored agent, the signal is its color.

Kits according to other examples of the invention include a plurality of particles as in the kits as described above. , wherein the wall support having a first water-insoluble surface has a first biological substance attached thereto. but does not have a polysaccharide coating on its first substantial portion. That cost Instead, bovine albumin or other shielding agents are used.

Next, the invention will be explained with reference to examples.

Example■ Polystyrene microspheres with a diameter of 1 micron (Polysciences Inc.) 50M water-cooled 75% H2SO4 The mixture was suspended in 100 m of ice-cooled 1:1 HNO,:H2so.

The resulting mixture was stirred in a water bath for 30 minutes and then poured into a glass of ice water. and quenched. The microspheres nitrified in this way were washed with water three times using centrifugation. .

Spoon-” Nitrified microspheres were suspended in 100 m of concentrated HC with 110 g of Sn0 and 2H20. The mixture was made cloudy and stirred at room temperature for 10 hours. Microspheres in which nitrate bases have been reduced to amine groups are S It was separated from the n0bun2 solution and washed twice with water by centrifugation. Next, the microspheres are soaked in water, Washing was performed in the following order: O, IN HCI, H2O, O, IN NaOH, and H2O.

I activated microspheres with cyanogen chloride and released amine microspheres for 900 m! :L cold water and dispersed by ultrasonication in a stainless steel ultrasonic bath cleaner.

0.5g cyanogen chloride in 2:l water and alcohol (150ml) ) was added, and the resulting solution was incubated for 30 minutes with intermittent sonication. It was left warm. This converted the amide 7 group into a cyanogen chloride adduct. Centrifuge the suspension Separate the residual salt of cyanogen chloride adduct 2 Microspheres containing hydroxyl groups formed by hydrolysis of compounds are removed, This was washed twice with cold water by centrifugation.

Small spheres of N-2-aminoethyl carboxymethylhyphenol AECM-F Microspheres were suspended in one volume of cold water by sonication, and 1.2g of A solution of AECM-Finyl in 50 nl of water was added. the resulting mixture was left at a constant temperature for 12 hours with intermittent ultrasonication. AECM-finyl bond The microspheres were washed twice with water by centrifugation. These via Whatman #1 paper Vacuum filtration removed any aggregated microspheres. AECM-Finyl used had a molecular weight of about 400,000 and about 80 amine groups per molecule.

'ffLM' in i-Hisian in order to include part of Col 1 ■- AEC: 750 nl of M-finyl electrostatically coated microspheres cooled to O'C. Suspended in water. Add cyanogen chloride (1g) to 200m of l=1 alcohol:water AECM-Finyl was cross-linked by adding the cold axillary solution to the suspension.

Only part of the surface is due to the acidity produced by the formation of 80 when cyanogen chloride reacts. It remained coated with AECM-Finyl. The resulting suspension was diluted with OoC for 3 After incubation for 5 minutes, the microspheres were centrifuged from the suspension. Centrifuge these microspheres Washed three times with cold water. These microspheres were bound to proteins by culturing them at room temperature. . It can be stored for up to a year at approximately 4°C without significant deterioration. I confirmed that.

Disgraceful” polystyrene giant 0℃ (7) 120mM (7) 2: 1 (7) H2S%: Covered with HNO3 After gentle mixing, the mixture was incubated at 0° C. for 15 minutes. Pour out the mixed acid and o 'C with 50% sulfuric acid and then with ice-cold water until the wash water was neutral.

1-Σ Nitrification bulb +10gc7) 5nC1q・2HJ 100++1 (7) Concentrated hydrochloric acid Cover with a solution dissolved in nitrate and heat it at room temperature for 15 hours with occasional stirring to remove nitrofluorocarbons. group was converted to an amino group. Decant the Sn02 solution and add the spheres to water, 0, INHC sentence, water, and 0. Washed with 1N NaOH and then water.

′-hi in autopsy Hisian Amine spheres, 0.1g of cyanogen chloride, 10ml of alcohol and 200mM The tube was covered with a solution of water and incubated at room temperature for 40 minutes. Sphere cyanogen chloride solution The bulbs were then decanted and washed with water in several portions.

18M#GO-500973 (10) AECM-Fukol Sphere, 0.14g (7) AECM-7 equol was dissolved in 120mM (7) water. The mixture was covered with the solution and left to incubate for 12 hours with occasional stirring. AECM-Ficoll The solution was decanted from the bulbs and the bulbs were then washed twice with cold water. in this way A coating in which AECM-Finyl was electrostatically bonded onto the spheres was obtained.

AECM-7 Col -ゞ-31　Murtame'-f'(1)　at jb'y7n Narukori The spheres electrostatically coated with AECM-Finyl were cooled to O'C and 0.18 g of silicon chloride was added. It was covered with 150ml of ice-cold water solution containing Anne and 18nl of alcohol.

Incubation continued for 30 minutes at O'C. Decanting the cyanogen chloride solution from the bulb The spheres were then washed three times with ice-cold water. Only part of the surface reacts with cyanogen chloride Due to the acidity generated by the formation of HC structures during the process, AECM-finyl remains covered. there were. The AECM-finyl used in the reaction has a molecular profile of approximately It had 80 amino groups. The spheres obtained in this way are used for proteins such as RGC, enzymes, etc. react with macromolecules. It can be stored for several months at approximately 0°C without significant deterioration. be able to.

Illusion 1” Egg ni'ha "1 To the AECM-Ficoll microspheres 0.12 milk ram prepared as described above, 1 ．． Form a final protein concentration of 3 micrograms/m in the total volume of 1+n standard saline. Sufficient MOP, C-21 myeloma protein was added to make the protein. After leaving at room temperature for 1 hour , unbound protein was removed by centrifugation and washing three times with standard saline. Then, M OP(, -21 combined microsphere with 0.642 milgram 7m sentence 1'1' in 1 ml of saline containing a labeled mesocoulombic antibody, anti-48. The mixture was incubated at room temperature for 1 hour. 0.1% of unbound labeled protein from microspheres Wash and remove 3 times by centrifugation with saline solution containing bovine serum albumin (BSA). After that, the bound label was counted. As a result, approximately 2001 gG molecules/microsphere The anti-4a content was 0.013% by weight.

Branch cliff ■ Attachment of AECM-Finyl to Carl-Scylated Polystyrene Mimic Globules To determine the extent of AECM-finyl binding to carboxylated polystyrene microspheres. We conducted several experiments to find out.

The microspheres used were prepared using potassium persulfate as a catalyst to prepare 95.2 parts of fresh microspheres. Air-distilled styrene and 4.8 parts of vacuum-distilled methacrylic acid were combined into 60 It was before suspension obtained by polymerization at ℃. After polymerization is complete, wash the micro-31 spheres with diluted NaOH. filtered through glass wool, brought to pH = 7 with dilute II and evaporated by centrifugation. It was washed three times with distilled water and the solid bamboo IW was adjusted to 0.745%. scanning electron microscope Using a microscope, the microspheres are 0.5 microns with a standard deviation of less than 3%6. It was confirmed that the diameter of the tube was broken. 100 microliters obtained in this way The sample had a surface area of 8.5 x 10'-'1' microns.

50mg of I! Ficoll (80 carboxylic acids per 40,000 molecular weight by titration) 66.5 mg of ethylenediamine hydrochlora in 5 nl of H2O Ido-c'9 (containing 25 microcuries (H/4) and 75 mg (3-dimethylaminopropyl)ethyl carbodiimide hydrochloride’ C”-AECM phyco by reacting with (EDAC) at pH = 4.7. Adjusted the file. This C”-AECM finyl is removed from the low molecular weight reactant by gel filtration. and separated by extensive dialysis against distilled water at 4°C. This C"-A ECM7 Iko Jl/ is 112o medium Te 0.91 double drop, 212°cpm/10 Cold (non-G") AEC prepared in the same manner as 9 with microliters M7　ID　Jl/Trial* increases the average molecular weight of 443,000, so C"-AECM Ficoll ζ 1.7 x 10 cpm/molecule under constant conditions.

C”-AECM Ficoll into 100 microliters of carboxylated microspheres. The bond is simply left in contact for the required time in a total volume of 500 microliters of ice or diluted liquid. This is carried out by touching the microspheres with water or a buffer solution etc. using a centrifugal PL. After washing three times, the accumulated washing waste oil and washing microspheres were counted with respect to C''. 1st The table shows the amount of C remaining on the microspheres after being left at a constant temperature in water for a predetermined period of time following washing with water. -Indicates the amount of AECM Ficoll.

Kamejodama HO AEC) l Fukol 1) C he fixed 1 circle IL friend "L Chinako takashi J" Deception "Continuous speed" "Ku" 11 minutes 2120 89B 15 minutes 2120 917 13 hours 2120 834 15 minutes 4140 1028 15 minutes 8280 1094 1) Use 2120cpm The data in Table 1 shows that saturation of the microspheres in the AECM Buifle occurred quickly and 1109 It is shown that microspheres exhibiting 4 cp are obtained.

The effect of pH on the amount of AECM Ficoll bound to the microspheres was then determined.

The microspheres were treated as in Table 1, but the p)l of the binding and washing solution was It was used as specified in the table.

No. L table AECM Fukol, H Yu0 - and others Hl to Ha- L sleep and I L blood 11'/-0111 pH4,70,H,OHio 43B pH5,80, H, OH, Lo 8B+p) IB, 75. H*OHsu0834 PH7,10,Hi10 HqO131? pH7,80, HaOH2O949 PH8,85, kOH, :L08!1B1) 2120cpm used 2) Adjustment of p)l of distilled HsLO with NaOH or Hi AECM-Ficoll binding is significantly less at lower pH values, eg, below about 6, than at higher pH values. In other words, slight A method is provided for controlling the amount of AECM-finyl deposited on the globules.

The data in Table 2 is based on AE (1; 7 amide groups on M-Ficoll and carboxylic acid on microspheres. that salt formation (ionic bonding) between the sil groups is probably responsible for the observed binding. means. This ionic bond has pH = 1/2 Jog for weak acids and weak bases. Kw -1/2. JogKa + 1/2Kb gives maximum equivalent points . Ka and Kb values are unknown for the substances used, but similar compounds, i.e. Evaluate from Ka of acetic acid = 4.73 and KII+ of ethylamine = 3.25. be able to. The equivalence point calculated in this way is pl = 7.74, and the maximum connection is in good agreement with the observed p)I=7.9.

Next, we measured the effect of salts on the amount of AECM-finyl bound to microspheres. Ta. The microspheres were treated as in Table 1, but the binding and washing solutions were as in Table 3. It was as if it were specified by

Husband AECM Fukol? ), ) and 1-7 in 1.

1LLfi!　u　1 Satdanyu 1 animal u H-bun OPBS 155 NS Hi20 403 NS NS 1’94 NS PBS 138 PBS Hyu 0 338 PBS PBS 58 1) Use 2+20cp 2) NS-Standard salt water 3) PBS-phosphate buffered saline, p) l = 7.3 The data in Table 3 is based on the AECM Washing with a diluted ionic solution not only prevents binding but also minimizes Shows that it is easily removed from the sphere. The microspheres in Table 3 were heated using non-radioactive AEG:M fi When left at constant temperature with Ficoll, only about 50 cpm of AECM Ficoll cannot be exchanged. Ta. In the 6th sample in Table 3, a part of AECM ficoll was formed into microspheres with polar O. Indicates that there is a strong ionic bond or a covalent bond.

Table 4 shows the covalent bonding of AECM-Finyl using a water-soluble carbodiimide activator. Shows data regarding.

Milk industry lid AECM fucol) 120 people with carbodiimide Rule M tannoji fixed j L male 1” 1 rule flow imitation J bean bed Yu J Ku” 0 1 hour PBS 155 0.008 1 hour PBS fi9? 0.012 1 hour PBS 582 0.025 1 poem PBS 499 0.05 1 hour PBS 488 0.5 38 hours PBS 1? 9 The data in Table 4 is C”-AECM74 Reacting Col (2120 cpm) with microspheres in the presence of various amounts of carbodiimide It was obtained by washing three times with PBS at pH=7.3. Table 4 minute Incubation of spheres and non-radioactive AECM Ficoll is not possible unless EDAC is used. , which shows that the AECM ficols are not commutative. Probably AECM Ficole's a The mino group is covalently bonded to the carboxyl group on the surface of the microsphere via an amide bond. It seems likely that The binding amount of AECM Ficoll was reduced by a large amount of EDAC. The reason for this is the above-mentioned suppressing effect of higher ionic strength (AECM-Finyl microspheres). This can be explained by the suppression of initial ionic binding to However, the AECM Once EDAC is attached to a surface in the presence of EDAC, it becomes a covalent bond. the The reason is that the PBS wash removes this AECM ficoll and the iozo-bound AEC. Because it will remove JI Ficoll! Yes (see Table 3 and comparison).

The assumption of covalent binding was tested and the microspheres were co-identified after washing away excess EDAC with HO. ] summarized in Table 5 to ensure that they remain activated to perform binding. The actual CM "Positive Emi" 1 caused the disaster M1, and the last one was -1. 10.5mgEDACC” 7AECM7 IDL microsphere = fufi’t’ Excess EDAG before addition of CP! ! Removal of I with Hku0 l　820　$539 2 Hri 0 I1 378 3 PBS None 226 4 PBS available 523 The protocol combines microspheres with EDAC (excess amount) and CI+-AECM Ficoll. Incubate for 1 hour and then wash samples 1 and 3 with PBS. It became.

Samples 1 and 3 were then counted. Samples 2 and 4 were incubated with EDAC for one cycle. Leave to stand, wash twice with H,0, add C-AECM Ficoll and dissolve in H2O. The cells were resuspended, incubated for an additional hour, washed with PBS, and counted. tiny The sphere sample was negative for exchange with non-radioactive AECM Ficoll, indicating covalent binding. Ta. These results suggest that EDAC co-binds at the site where PBS exists. Activates ruboxylated microspheres and shows that they remain active even after removal of excess EDAC. vinegar. PBS improves the microspheres of the AECM ficoll by limiting the phase gradient jH of the microspheres. It is clear that binding to the globules is suppressed. Reason 1: PBS or microspheres EDAC: This is because it does not interfere with activation.

We also determined the usefulness of cyanogen chloride for binding AECM Ficoll microspheres, A summary is shown in Table 6.

□　□　-andP,,! −1−−−−5”−A □−KoP−−−−−−−None Hot 967 0.85 471 Cold 55 24] None Cold 7 0. 65 0 Hot 1393 444 No Hot 1098 0.65 359 Heat Yes 1364 8690.05 Hot 1247 0.85 49 Hot 70 88 673 nothing hot 1079 nothing 31 405 4571) A /F= AEI: M7 I=l, hot 2120 cpm (radioactive AECM7 Icol), Cold E0.0Ei9+ng (71+Non-Radioactive AECM Fico le 2) CTC = cyanogen chloride dissolved in 50 microliters of absolute alcohol mg The protocol used was to inject 100 microliters of microspheres into non-C" React with ECM Ficoll in H and without EDAC, and wash 3 times with H and O (1 2nd wash) - reacted with cyanogen chloride for 30 minutes, washed 3 times with H,O (2nd wash) (washing) and reacted with C/<2 or non-〇’ AECM Ficoll for 1 hour. The procedure consisted of washing three times at 0 (third wash) and then counting the microspheres.

The data (Sample 1) shows that non-exchangeable AECM ficoll of approximately 250 CI) AECM ficolls are bonded to microspheres that already have ionic bonds through the use of Show what is possible. However, AEC has a finyl E[]AC; it already has a bonding layer. Treatment of the microspheres with cyanogen chloride facilitates absorption of additional AEC:M finyls. I can't. For example, the third cleaning of the sample No. 4 in Table 6 is treated with cyanogen chloride. Contains 88% of the counts added to. Minor details regarding the second and third trials The excess count of 250 or more is clearly due to the ionic binding of AECM Ficoll. This is due to The mechanism by which cyanogen chloride binds AECM Ficoll non-exchangeably is Possibly cross-linking amine groups with several adjacent AECM ficoll molecules-1- It depends. The AEf1M ficoll molecules cross-linked in this way were prepared in PBS. Molecules that bind tightly so that they cannot be washed away (for example, sample 6 in Table 3), held in microspheres. The remainder is washed away.

Briefly, AECM Ficoll was added to 100 microliter microspheres in H4O. The maximum value of 11094cp achieved for ionic bonding is 7570 molecules/surface area Equivalent to 1 square micron. This is I094cpm/(1,7X 10-” cpm/molecule) equal to ). This means that the AECM Ficoll fraction is 13.210-P on the surface 1-. If we assume that the molecule has a vertical J-shaped shape, then the side is 115 squares. Nges) Low J1, with a volume of 1.5 x 106 cubic angstroms, 111’1. It means to get L. Average molecular weight of 443,000, avocado From the number of ports and the volume assumed above, the density is 0.49 g/cc, which is 1 la. I can do it. Similarly, 897 cpm covalently bound by FDAC (7) maximum The value will be 0.25g/cc. Polysaccharide Hri.

t of up to several times the dry volume of the solution is a well-known phenomenon, and the observed results This is an explanation. However, a combination of swelling and flattening was observed on the surface. It is a coating.

It is possible to implement the present invention using the table if+i, which is almost completely covered with the AECM ficoll. I can't do it. The reason for this is as shown by some of the other beautiful examples. , antibody mounds can be coupled to the surface with either cyanogen chloride or EDAC. Because you can't. U.S. Pat. No. 4,264°766 discloses that proteins are The complex oxidation, bonding and reduction processes for However, this method is not easy for users to handle, and the borohydride required to carry out the present invention is Not all proteins retain their biological activity after redox.

The present invention aims to transfer proteins, antibodies, etc. attached to microspheres to other regions covered with polysaccharides on the surface of the microspheres. Provided with most or all of Share proteins before or after polysaccharide coating Alternatively, embodiments permit non-covalent binding. A produced by a number of methods The materials of the present invention having a partial layer of ECM Ficoll have been routinely observed to last for more than one year. It has storage stability at 4°C for a period of time. By simply mixing this material with proteins, It easily binds to the protein, which binds the user's own protein and provides long-term I want shelf life. This is a significant advantage for the user. Once bound to a protein, the reagent can be stored for more than 6 months. Has a shelf life.

Support - difference - [ Appeasement Pigeon AECM) Cor Nothing 400 mics of phosphate buffered saline (PBS) with p) I = 7 in each of 20 test tubes. Lolitre and 100 microliters of carboxylated polystyrene imitation pellets ( 0.5 micron in diameter, 0.74% by weight in distilled water). These test tubes can be easily Simply sonicate 5 microliters of EDAC at a rate of 10 mg/mu. Added. The test tube was again sonicated and incubated at room temperature for 2 hours. Leave at constant temperature Afterwards, the microsphere suspension was washed twice with PBS by centrifugation and diluted with 500 microliters. The cells were resuspended in PBS and briefly sonicated.

Various amounts of T-15 antibody were added to test tubes at a ratio of 1 mg/1 m PBS. vinegar That is, test tube 1.5.9.13. 'i? takes 50 microliters of T-15 received. Test tubes 2, 6, to, 14.18 are 25 microliters I received a T-15. Test tubes 3, 7, 11, 15.19 are 10 microphones I received Lorittl's T-15. Test tubes 4, 8, 12°18.20 are 5 mi Chloritol T-15 was received. Briefly sonicate all test tubes and The mixture was incubated at room temperature for 1 hour.

After incubation, the microsphere suspensions in test tubes 1 to 4 were washed twice with PBS by centrifugation. , resuspended in 500 microliters of PBS with sonication.

The contents of test tubes 5-8 were combined with 1% bovine serum albumin and 0.1% sodium azide. Washed twice with PBS containing protein buffer (protein buffer) and incubated for 50 min with sonication. Resuspend in 0 microliters of protein buffer. These were heated at 4°C for 48 hours. held for a while. Wash the microspheres in test tubes 9 to 20 twice with distilled water and do not sonicate. was resuspended in 500 microliters of distilled water.

Add 10 microliters of non-radioactive AECM Phyco to the microspheres in test tubes 13-20. was added.

Test tubes 9 to 20 were incubated at room temperature for 1 hour and then at 4°C for 48 hours.

Wash the contents of test tubes 9 to 12 three times with PBS by centrifugation to wash each test tube. The waste liquid was collected. The accumulated cleaning waste liquid and the washed microspheres are collected in trillions of liters each. Put it in a scintillation bottle and add 20 Kawabun's scintillation liquid (which fluoresces when exposed to radiation). Standard solution) was added to the container and counted.

In this way, the following results were obtained.

Stalk counterfeit production 1 factory call preemptive 1 nanabe lees 9 5 [i9 1831 10 512 2038 +1 413 1933 12 4]? 1948 10 microliters of C14AECM Ficolu 2120cp river These results A nearly constant amount of radiolabeled AECM Ficoll was used in test tubes 9-12. This shows that the monoclonal antibody was bound despite a 10-fold reduction. use The highest antibody concentration tested (tube 9) was found to be able to completely coat the microspheres in the sample in a single layer. Indicated. This is shown by similar amounts of C14-AECM ficoll binding. This problem occurs due to the difficulty for negatively charged antibodies to approach negatively charged microspheres. It is an indicator of

Microspheres in test tubes 13 to 16 were washed twice with PBS and incubated for 5 minutes with simple sonication. 00 microliters of PBS. Test tube 17-20 microspheres Wash twice with white matter buffer and add 8 ml of protein buffer to 500 microliters of protein buffer. Resuspended and briefly sonicated. On the other hand, flexible U-shaped bottom pvc (polyvinyl chloride) ) 48 in 86-well microttle plate (Guinatequin Corporation) For the wells, add 50 microliters of T-15 at a rate of 0.1 mg per 1 ml of PBS. Add 50 microliters of phosphorus choline to each of the first 16 wells. Maglobulin conjugate (PCCBCG, specific binding partner of T-15 antibody) Add PBS to each of the next 18 wells at a ratio of 0.1 mg/mM and incubate for 50 min. Add Chloritol's protein buffer to each of the remaining 16 wells and keep them at room temperature. The mixture was prepared by incubating at a temperature of 1 hour for 1 hour. The contents of these wells are removed by aspiration. by removing, filling the top with protein buffer, and aspiring the contents. The wells were then washed three times with protein buffer. Then test tubes 1-8 and 1 Place 50 microliters of the microspheres prepared in steps 3 to 20 into each well. The combination was incubated at room temperature for 1 hour, aspired, and then the wells were incubated with protein. The cells were washed three times with quality buffer and then twice with distilled water by aspiration.

Dry these wells and visually inspect them using 4.3, 2.1 and a negative number (-1). The relative number of microspheres bound to the wells was evaluated.

The results are as follows.

PCBGG 444444444 4 4 4 4 4 4 4 mild type 1 liquid 11 121111-1-1-1-1-1-1-1-IPCBGG is a characteristic of T-15 antibody. is a heterozygous binding partner, and any sealing of microspheres in the absence of PCBGG (in this example (all T-15 binding) indicates non-specific binding. All microspheres are PCBG Shows specific binding to G plate coat. Microspheres with AECM Ficoll (test tube) 13-20) when binding was carried out in buffer in the absence of protein (test tubes 13-1 Even G) showed no detectable non-specific binding. AECM without ficoll Microspheres are non-specific in all cases, especially in the absence of protein in the ladle solution (tubes 1-4). It showed a specific combination.

This example calculates the ratio of specific binding to non-specific binding to the portion of the microsphere surface that is not bound to the antibody. We provide clear evidence that by covering with AECM Ficoll. Also, non-resistant Even when using excess antibody and covalent bonding, the amount of surface binding is This shows that it is equivalent to a small ball.

Branch blood M EDAC/AEC Town 7 5 Le/CTC' Ha (7) Leeno As mentioned above, Green fluorescent diameter 0.72 micron carboxylated polystyrene microspheres prepared regularly A partial surface coating of covalently bonded AECM Ficoll is formed on the surface by the method described in F. did. 708 44.5+C9- suspension containing 3.75g of microspheres M! l (') Added to H20, then 0.41g c7) AECM Fico 39 nl of wood was added containing 100 ml of wood. The resulting suspension was thoroughly combined, 30I119 while stirring and sonicating. of 75 mg dissolved in water. EDAC was added. p)I at this point was 5 or less. This hang/! i'l 'Jlrj was left at room temperature for 2 days and washed 3 times with distilled water. No ultra f1 wave processing After re-snoring and making the water cloudy, the microspheres were passed through Whatman #1 paper. suction The mixture was filtered to remove any aggregates and centrifuged again to remove the clear liquid. then , the microspheres were suspended in 186 m of cold water until a single microsphere was obtained. Ultrasonicated. Add 0.36 g of cyanogen chloride to this suspension and 36 m of ETOH. and 76 m of the melt dissolved in cold water were added. The resulting suspension is subjected to ultra-hair wave treatment. The mixture was kept in a water bath for 30 minutes and then centrifuged at 4°C. 1-, remove the clear liquid The microspheres were then washed three times with cold water by centrifugation. Do not ultrasonicate these microspheres. resuspended in 320 mfl H2O containing 1.07% solids by weight. I had it. 10 microliters of microspheres to 5 microliters of I125-T -15 antibody (17, incubated at 148 cpm for minutes, protein buffered by centrifugation) 2+10 cpm of antibody was bound to the microspheres by washing three times with solution.

Fruit"1"■ A microscopic example of binding to an iLI label and a label in tissue culture. A part of the sphere was suspended in 100m standard @ salt water at room temperature by ultrasonication 1 0ml with 0, 1mg of My-1, (kappa) with 1g of mouse monoclonal antibody. The microspheres were bound to MY-1 by incubation. The microspheres are then cooled. Pellet for 10 minutes at 10,0OOX g and 4°C in a storage centrifuge, and remove the supernatant. The liquid was removed.

I00 containing 4 fJ of 10% newborn calf hem while sonicating the pellet. Resuspend in +nu RPMI 1840 cell culture medium and pellet as described above. The supernatant was removed. After repeating this operation again, add 10% new 20M RPMI If containing live bovine serum (resuspended in 40M cell culture medium, It was sonicated and stored at 4°C under sterile conditions.

Such MY-1 bound microspheres do not maintain sufficient activity for more than 6 months. It was done.

■ 1 'n fly, 9 sitting] □ 'E group → HL-60 cultured cells in the cell Separately separate each cell suspension (10 m) of DAUDI and DAUDI human white tissue cultured cells. 5m 2 Histopachy of 3 sentences in the centrifuge tube, 107? Cell stratification guy? (Sigma Chemical (trade name) -1-, and centrifuge at 850 x g and room temperature for 30 minutes. I let go. - Remove the clear water and remove the cell-containing interfacial layer with 10% fetal bovine serum (FCS). ) was washed twice with RPMI 1840 cell medium containing )IL-[0 and DAUDI cells were incubated with 0.1% BSA (blood albumin) in each centrifuge tube. ) and HBSS containing 0.1% sodium azide (bank buffered salt solution) ) to 1. The cells were resuspended at a rate of Xl06 cells/m. 0.5 m cell suspension Aliquots were placed into each 2 m well of a 24 well tissue culture plate. Prepared above Add 20 microliters of MYl-1 antibody reagent to both cell solutions and It was about right. This plate was centrifuged for 9 minutes at +50Xg and 4°C. It was left at constant temperature for 1 hour. Hanging p: Combine 4M of Jm and separate 12x75mm glass opening Layer on top of 1111 FCS and centrifuge for 10 minutes at 250 x g and room temperature. I let go. All free microspheres remained in the supernatant on the FCS and were removed. Ta. The cell plates were then gently resuspended in O,l+n RPMI 1640. . Regarding My-1 antibody microscopic activity on cells, 20 microliters of both cells Hanging 8M was examined by fluorescence microscopy.

3 1 DAUDI 3.8 My-1 antibody is a cell surface antibody against the surface of human granulocytes and )It,-ffO cells. It is a marker.

This marker is absent on DAUDI and other human white blood cells. Similar results The labeled cells were obtained for human white blood cells, which are granular leukocytes.

1 gonko 1 Microspheres that match the AECM standard 2 Ratio by - method Aliquots taken from three different patches of carboxylated polystyrene microspheres. were treated in two different ways to obtain two different reactive surfaces. Then these were used in the experiments to show different surface properties.

Microspheres: A) 0.99 microns in diameter, containing fluorescent blue dye B) 0.54 in diameter Micron, containing fluorescent blue dye C) 1.01 micron in diameter, undyed These microspheres were brought to p)I=7 with distilled water.

"Viper" Covalently bonded using carbodiimide - 1.25 g of microspheres A, B and C Label the volumes containing 220 m of distilled water as A1 and BI C1, respectively. Microspheres were placed in three beakers containing water and treated with ultrasonic waves to produce microspheres. 73 (15) Dispersed. Next, first, 0.825% by weight of AEC:M (the surface of the microsphere was Add 15 m of ETOH solution (in excess of the amount needed to completely cover) and add another 0.0 m Add 25g of EDAC (carbodiimide) ETOH solution 10+nJ2 to each beaker. - added to.

After addition, the suspension was thoroughly sonicated and incubated at room temperature for 12 hours. Microsphere The hearts were washed three times with distilled water by separation and resuspended in 200 m of distilled water. then , the suspension was filtered through Whatman #1 filter paper. Microsphere suspension A1, B1 and and C1 were tested for each weight percentage and necessary adjustments were made for residual microspheres. child Change A1 to 1.47%, B1 to 0.80%, and C1 to 1.5% as follows. The total volume is IEi 5m for A, 200m for B, and 100m for C. It was a sentence.

4-ben” Microspheres A with a difference of 2.94 g per person due to ion r halogen hisyanide, 1. Each volume A2 contains 6 g of microspheres B and 2.98 g of microspheres C. , B2, and C2 and placed in a beaker containing 80 ml of distilled water. 0. Add 10 m of a solution of 885% by weight AECM Ficoll to each beaker and make sure Dispersed by ultrasonication. After incubating at room temperature for about 15 minutes, centrifuge the suspension. Washed twice with distilled water, sonicated, and resuspended in 125 m distilled water. , sonicated and filtered through Whatman #1 filter paper. Distill each microsphere suspension I made 180 sentences with water. From this, remove the aliquot and refill it as in the process. By adjusting the product, A2 is 1.47% by weight, 197nlj, B2 is 0.80% by weight, C2 was set to 1.5% by weight and 193II was set to 1 sentence.

Each microsphere suspension was added to 70+n volumes of distilled water and thoroughly sonicated. 0. Dissolve 13g of cyanogen chloride in 10ml of ETOH and dilute with 20ml of distilled water. The solution was added to each of the three suspensions and dispersed by thorough sonication. . After incubation for 1/2 hour, the suspension was washed 3 times with distilled water by centrifugation, and Sonicate, resuspend in 190+n of distilled water, and thoroughly sonicate. Is this it? Take an aliquot from A and calculate its volume and weight percentage as follows: , B was adjusted to 199m sentences, 0.80%, and C was adjusted to 183m sentences, 1.5% (upper (See Processing ■).

In history Microsphere suspensions A1, B1, CI (treatment ■) and A2, B2.02 (treatment ■) Six samples were prepared as described above. 500 in each of 6 test tubes Add microliters of phosphate buffered saline (PBS) and then add 6 microsphere suspensions. 50 microliters each of suspensions (AI, B1, C1, A2, B2 and 02) was placed in each test tube, and the test tubes were briefly sonicated. 4mg/m sentence T- Add 15 antibodies 25+n to each test tube, sonicate, and incubate at room temperature for 1 hour. did. Next, the microsphere suspension was mixed with 1% newborn calf serum and 0.1% azide (protein laxative). The cells were washed 3 times by centrifugation with PBS containing buffer solution) and washed 3 times by ultrasonication. Reconstituted u%A in 00 microliters of protein buffer.

On the other hand, a flexible U-shaped bottom PvC 88-well micro-little plate (Guinatech Co., Ltd.) at 0.1 mg/m in each of the first 6 wells. It was prepared by adding 50 microliters of a PBS solution of Bun T-15.

Each of the next 6 wells contains O, 1 mH/m of phosphorus choline-bovine gamma globulin conjugate. Add 50 microliters of PBS solution of PCBCG to each of the next 6 wells. Each well contained 50 microliters of protein buffer. Coat these boards at room temperature. Incubate for 1 hour, remove the contents of the wells by aspiration, and then add to the top of the wells. Protein buffer solution is obtained by filling the protein buffer solution and aspirating the contents. The wells were washed three times.

After that, the microspheres AI, B1. 50 miles each for CI, A2, B2, C2 Add Chloritol to the corresponding wells and add T-15, PCBCG and protein buffer. The wells were treated with the solution. After incubating for 1 hour, remove the contents from the wells. spirate and then wash the wells three times with protein buffer and twice with distilled water. Purified. Dry the board and visually test using 4.3.2, ■ and a negative number (-1). The number of microspheres bound to the wells was evaluated by observation.

Test #AI BI CI A2 82 C2PCCBCG -1-1-143 4 White matter buffer -1-1-1-1-1-1 treatment ■ (partial AEC coated with Ficoll) Microspheres prepared according to shows heterozygous binding, but in treatment I (surface completely covered with AECM ficolls). Therefore, the above-mentioned fact shows that the prepared microspheres do not bind to PCBGG or others. I'll be learning from the test.

That is, microspheres completely covered with AECM Ficoll have a high resistance to biologically active materials. It has no binding ability.

Shigoko y Ant that was reduced to 1 with EDAC/AECM fucol l'c and its Hisian. Coat the raw g carboxylated polystyrene imitation globule suspension with Dl, El and and Fl in bottles of distilled water labeled as Fl.

Old: 1.3 microns in diameter, 1.9% by weight, contains fluorescent green dye El, 0.98 micron diameter, 1.5% by weight, contains fluorescent red dye Fl: diameter 0.7 microns, 1.04% by weight, containing fluorescent red dye Three batches of these microspheres were processed using a processing method as in Example ① to form microspheres. The sphere surface was completely covered with AECM Ficoll.

Samples of 0.825 g each of microspheres DI', El and Fl were added to D2, F2 and Ultrasound in a 250mM Ersinmeyer flask labeled with F2. It was suspended in 35 mL of distilled water during processing. This suspension was cooled with water and 0.06 5g of cyanogen chloride was dissolved in 5ml of ETOH and diluted with distilled water to 10ml of cold water. added something. The resulting suspension was sonicated and incubated on ice for 30 min. . These were washed three times with cold distilled water by centrifugation, sonicated, and distilled water (30 ml (D2), 30m sentence (F2) and 50m sentence (F2)). , its exact volume and solid molecular weight % D2 is 40ml 1.1.8%, F2 is 4 F2 was adjusted to 1.04% at 100m.

50 microliter aliquots of each from bottles D1, El and Fl were phosphate buffered. Wash once with salt water (PH1) and add 500 microliters of P while sonicating. BS was given %A again. These were labeled D3, F3 and F3, respectively. . 50 microliters of each sample from flasks D2, F2 and F2 were added to D4, F2. 4 and F4, each with 500 microliters of PB. into a test tube containing S. This test tube was sonicated and Lmg/ml of goat 25 microliters of a PBS solution of hostile murine antibody was added. Flask D2, 50 microliters of each sample of F2 and F2 was added to D5, F5 and F5. Each sample contained 500 microliters of PBS, each containing 500 microliters of PBS. I put it in a test tube.

\ 0 This test tube was sonicated and a PBS solution of IB/ml of goat antagonistic mouse antibody 2 was added. 5 microliters were added. Samples 5 each from flasks D2, F2 and F2 0 microliters to 500 microliters labeled D5, F5 and F5 respectively. Chloritol was placed in a test tube containing PBS.

This test tube was sonicated and 1 mg/+IIQ of UPC10 (murine myeloma protein Add 25 microliters of PBS solution of white matter to test tubes D4, F4 and F4. Each was added. These test tubes D4, F4, F4, D5, F5 and F5 at room temperature. It was left at constant temperature for 1 hour. These microspheres in the test tube were centrifuged to produce 1% of newborn calves. 3 in PBS containing serum and 0.1% sulfurium azide (protein buffer). Washed twice and resuspended. These were each treated with 500 microns while being treated with ultrasonic waves. Resuspend in a liter of protein buffer.

Next, a flexible U-bottom PVC 98-well micro-little plate (Guinatech 0.1 mg/mu of goat and mouse Place 50 microliters of antibody in PBS into each of the first 9 wells and add 0.1 Add 50 microliters of mg/nlj UPC 10 to each of the following 9 wells or Add 50 microlins of protein buffer to each of the last 9 wells. It was prepared from These wells were incubated at room temperature for 1 hour, and the liquid was aspirated from the wells. Viration, then fill the top of the well with protein buffer and aspirate the contents. Each well was washed three times with protein buffer by piping.

Next, test tubes D3, D4, D5, E3, E4. From E5, F3, F4, F5 (50 microliters) into the appropriate wells of the microliter plate. It was then left to stand at room temperature for 1 hour. These were then distilled three times with protein buffer. Washed twice with water and dried. All the results showed negative (-1) binding.

Test tube # D3 E3 F304 E4 F4 D5 E5 F5 goat hostile rat Antibody -1-1-1-1-1-1-1-1-IUPC10-1-1-1-1- 1-1-1-1-1 protein buffer -1-1-1-1-1-1-1-1-IPB The microspheres Di, El, and Fl in S are the protein particles on the plate even after cyanogen chloride treatment. after polysaccharide coating as in the treatment process of Example II. Indicates inability to bind to proteins.

Branch farm J Carpoxyhypolis of 1.0400 in diameter, 0.7 micron in diameter As for Chiren's I, there is one made in EDAG/AECM fucol. Various concentrations of EAD to find the optimal concentration for binding proteins to microspheres. We compared C. T-15 antibody was at three different concentrations.

50 microliter microspheres numbered 1, 3, 5, 7, 9 and 500 microliters Each test tube contained Lolitol's phosphate buffered saline.

These test tubes were treated with ultrasound to disperse the microspheres. 0.05mg T-15 Add antibody and 0-05 mg of EADC to tube 1, add 0.05+a,g of T -15 antibody and 0-005 mg of EAIIC were added to tube 3 and 0.005 mg of EAIIC was added to tube 3. Add 0.0 mg of T-15 antibody and 0.05 mg of EADC to tube 5; Add 0.05 mg of T-15 antibody and 0.005 mg of EADC to test tube 7; 0.05 mg of T-15 antibody was added to test tube 9 and no EADC was added. . Each tube was sonicated briefly after addition of protein and after addition of EADC.

The microsphere suspension was incubated at room temperature for 1 hour, and diluted with 1% newborn calf serum and centrifuged. and 3 times with PBS containing 0-1% sodium azide and do not sonicate. The cells were resuspended in 500 microliters of protein buffer.

Next, a flexible U-bottom PVC 98-well micro-little plate (Guinatech 15 wells of T-15 antibody at 9.1 mg/31 Add 50 microliters of BS solution to each of the first 5 wells at 0.1 mg/II Bun's phosphorus choline bovine gamma globulin (PCCBCG) (7) PBS solution 50 mic Add 50 microliters of protein buffer to each of the next 5 wells. into each of the last 5 wells. Leave at room temperature for 1 hour Afterwards, the contents of the wells are removed by aspiration, and the tops of the wells are filled with protein buffer. Wash the wells three times with protein buffer by aspirating the contents. Ta.

Next, add 50 microliters each of T-15/microspheres 1, 3, 5, 7 and 9. It was placed in the appropriate well and incubated at room temperature for 1 hour. Aspirate the contents of the well. Fill the wells and aspirate three times with protein buffer. , with distilled water 2. [1 lil was washed. Dry the board and visually check 4.3.2. The number of microspheres bound to the well wall was evaluated by observing and using a negative number (-1). .

#13579 PCBGG 1 1 1 1 -1 Protein buffer -1-1-1-1-1 A few microspheres in wells 1, 3.5 and 7 with pcBcic coats are young. This indicates that specific binding occurs but is not sufficient for reliable results.

Support group J mouth In this example, a flexible U-bottom polyvinyl chloride 96-well microttle plate is used. (Guinatetsu Quin Corporation) was used. Also, transparent PVC 4 The strips can be used in place of the wells of a microliter plate, in which case the reagent The strip was placed in the reagent instead of being placed in the well.

0.7 micron diameter microspheres containing fluorescent green dye were prepared and AECM Phyco Partial coating as in Example ■, Treatment ■ with Le/cyafcyan chloride. Obtained. Place 100 microliters of microspheres in a test tube containing 1 ml of PBS. and sonicated. 4+*g/50 microns of recommended T-15 antibody PBS solution liter and mixed by sonication. After standing at room temperature for 1 hour, The microsphere suspension was washed three times with protein buffer by centrifugation, and then sonicated. and resuspended. Resuspend the microspheres in IIIL protein buffer and thoroughly 3. Each was prepared in tubes numbered 4 and 5. The concentration in the test tube is 0 ．． lff1g/+s, and the content in test tube 2 was 0.01mg/m.

Next, place the flexible U-bottom PVC9Ei well microttle plate into the first 6 wells. Add 50% of Q, 1 mg/m T-15 antibody in phosphate buffered saline to each tube. Prepared. Well 7 contained Q, 1+*g/mu of phosphocholine bovine gamma globulin (P 50 microliters of PBS solution of CBCG) was added. Well 8 contains 1% bovine blood 50 mg containing clear albumin and 0.1% sodium azide (protein buffer) Microliters of PBS was added. These board coats were left at room temperature for 1 hour, Aspirate the well contents and fill the top of the well with protein buffer. The wells were washed three times with protein buffer by washing.

Place 50 microliters from test tubes 1-5 into wells 1-5, and add 50 microliters from test tubes 1-5. Tutle's protein buffer was placed in wells 6-8. After leaving it at room temperature for 1 hour, Aspirate the contents of the wells, fill and aspirate each well The cells were washed three times with protein buffer.

Fifty microliters of microspheres bound to T-15 antibody were placed in wells 1-8.

The plate was incubated for 1 hour at room temperature, the wells were aspirated, and the wells were incubated with protein buffer for 3 hours. and twice with distilled water. The amount of binding of microspheres is calculated from 4, 3, 2.1 and The following results were obtained by recording on a negative (-1) scale.

-Pipe #12345678 T-15+ PCBGG Diluent 4 3 2 -1 -1 -1 4 -IPCBGG B/m sentence 0. I Q, 010.0010.0OQJ Q, ■I This experiment was performed using T-15 board coating. binds the layer of PCBGG and then microspheres with T-15 antibody bind the plate coat. This indicates that the phosphorus choline component, which forms part of the coat, binds to the phosphorus choline component.

Shitokoshi 1 One test ′″ key Incubate clear PVC strips with antibody #l and add gelatin or bovine serum. Cover with albumin.

Next, put this in the urine suspected of pregnancy, leave it at a constant temperature for 1 to 60 minutes, and then Incubate the strip within microspheres bound to antibody #2. Such microspheres are cal Activated with bodiimide, washed, reacted with antibody #2, washed, AECM Phyco carboxylated polystyrene reacted with Antibodies #1 and #2 are human Purple membrane gonadotropin or the beta subunit of human purple membrane gonadotropin. It is an antibody that has the property of sometimes reacting with other antibodies. Namely, the human body's purple membrane gonadotropin Or a certain molecule of the beta subunit of the human purple membrane gonadotropin can be used at the same time. It can have antibody #1 and antibody #2 attached. Strips into microspheres After being left at a constant temperature for 1 to 60 minutes, it is taken out and washed. Positive test is microsphere indicated by a plastic strip with a cloudy appearance caused by the adhesion of . This assay can also be applied to purple membrane gonadotropins in the urine of species other than humans. Wear.

Industrial applicability According to an example of the invention, partially coated with a water-soluble polysaccharide, typically amine-functionalized, A novel composition having a water-insoluble surface partially attached to molecules of a biological substance is disclosed. provided. Molecules that serve as binding partners for biological substances and biological substances clear, accurate and significant information between binding partners and non-binding partners A highly selective separation can be obtained.

Although the present invention has been described in some detail through two consecutive examples for better understanding, It is clear that changes and modifications may be made within the scope of the invention.

Brief description of the drawing international search report

Claims (1)

[Claims] 1. A useful composition that specifically binds to a specific binding protein that becomes a specific binding partner for a biological substance when the specific binding protein is accompanied by other proteins, comprising: the specific binding protein and the other proteins; water-insoluble supports with a surface that can entrain proteins of a carrier, and a substance sufficient to substantially prevent proteins from binding to the first substantial surface portion of the surface. a polysaccharide coating covering said first substantial surface portion but not covering a second substantial surface portion of said surface. 2. The composition according to claim 1, wherein the support is made of latex. 3. The composition according to claim 1, wherein the biological substance is attached to substantially the entire surface of the second substantial surface portion. 4. The biological substance is covalently bonded to the second substantial surface portion, as set forth in claim 3. composition. 5. The composition according to claim 3, wherein the biological substance is hydrophobically adsorbed onto the second substantial surface portion. 6. The composition according to claim 1, wherein the polysaccharide coating is an amine polysaccharide coating and is cross-linked via a cyanogen halide component. 7. The support has a carboxyl functional group, and the polysaccharide is attached to the first substantial surface portion. A composition according to claim 1, which is covalently bonded via a boxyl functional group. 9 8. The composition of claim 7, wherein the covalent bonding is achieved through the use of a carbodiimide activator. 9. Claim 8, wherein a biological substance is covalently bonded to the second substantial surface portion. composition. ＼ ＼ 10. Claims that electrostatically hold the polysaccharide coating on the first substantial surface portion The composition according to item 1. 11. The composition according to claim 1, wherein the support is a particle, and the composition is labeled with a label that provides a detection signal. 12. The composition of claim 1, wherein the support is a macro-expandable surface. 13. The composition according to claim 12, wherein the giant expandable surface collapses the well. 14. The composition of claim 1, wherein the polysaccharide coating is in an amount of about 0.5 X 10-7 to 3 X I O-g per cm' of surface. 15. When a specific binding protein is accompanied by other proteins, In preparing the water-insoluble surface of the solid support that specifically binds to the specific binding protein as a heterospecific binding partner, the specific binding protein and other proteins are prepared. a solid support having a water-insoluble surface capable of entraining substances, a first substantial surface portion of said surface being coated with a polysaccharide coating, and a second substantial surface portion of said surface being coated with said polysaccharide coating. Water-insoluble solid support characterized in that it is not covered with Method of preparing sexual surfaces. 16. For coating with polysaccharides, the surface is nitrified to add nitro groups, the nitro groups are reduced to amine 7 groups, a cyanogen halide component is added to the amino groups, and the amine polysaccharide molecules are added to the first Substantive table 16. A force according to claim 15, comprising the step of electrostatically adhering to a surface portion. 17. The method of claim 16, further comprising the step of contacting a cyanogen halide with the electrostatically attached aminopolysaccharide molecules to cross-link adjacent amine polysaccharide molecules. 18. The method of claim 17, wherein the amount of amine polysaccharide deposited is about 0.5 x 10 -7 to -3 x 10 -7 grams per cm' of area of said surface. 19. The method according to claim 16, wherein the amount of the aminopolysaccharide deposited is about 0.5 x lo-7 to 3 x 10-7 g per 1 clT1' of the surface area. 20. The method of claim 16, further comprising the step of attaching a biological material to the second substantial surface portion. 21. Deposition of biological material involves electrostatic adsorption of the biological material onto the second substantial surface portion. 21. The method of claim 20, comprising: 22. Attachment of the biological material includes covalently bonding the biological material to the second substantial surface portion. 21. The method of claim 20, comprising: 23. To coat the polysaccharide, the surface is contacted with the aminopolysaccharide in an amount sufficient to cover the surface with a monomolecular layer of the amine polysaccharide, and the excess amine polysaccharide is slightly removed from the surface. Or almost neutral aqueous solution with no total heat salinity 16. The method according to claim 15, comprising the step of washing with a solution to leave a portion of the amine polysaccharide electrostatically bound to the surface and cross-linking a portion of the remaining amine polysaccharide. 24. Cross-linking is achieved by reacting a cyanogen halide with a portion of the remaining amine polysaccharide. 24. The method of claim 23, comprising simultaneously exposing the second substantial surface portion. 25. The method according to claim 24, wherein the cyanogen halide compound is a reaction product of cyanogen halide and ethanol. 26. The method of claim 23, wherein the amount of cross-linking of the amine polysaccharide is about 0.5×10 to 3×IOg per crrf of the surface area. 27. The method of claim 23, further comprising the step of attaching a biological material to the second substantial surface portion. 28. Deposition of biological material involves electrostatic absorption of the biological material onto the second parenchymal surface portion. 28. The method of claim 27, which comprises: 29. Attachment of the biological material involves covalently bonding the biological material to the second substantial surface portion. 28. The method of claim 27, comprising: 30. When the surface has an active group that can react with a water-soluble activator, the surface is 16. The method of claim 15, wherein the coating is carried out by contacting the amine polysaccharide compound and the water-soluble activator in solution. 31. The aqueous solution is almost neutral, contains almost no salt, and the amount of amine polysaccharide is 31. The method of claim 30, wherein the method is insufficient to completely cover the surface. 32. The method according to claim 30, wherein the activator is a water-soluble carbodiimide. Law. 33. The method according to claim 30, wherein the amount of the amine polysaccharide deposited is about 0.5 to 3XIO-7 g per crn' of the surface area. 34.7 The minopolysaccharide compound co-contacts the active groups on the first substantial surface portion by the contacting step. 31. The method of claim 30, when combined. 35. The method of claim 30, further comprising the step of attaching a biological material to the second substantial surface portion. 36. Attachment of the biological substance to the active group of the second substantial surface portion 1- 37. The method of claim 35fA, comprising covalently bonding said biological substance to a second substantial surface portion via reaction with a substance and an activating agent. 37. The method of claim 36, further comprising the step of washing away any excess activator prior to arrival of the activator. 38. Where the surface contains an activator group capable of reacting with an activator compound The surface is cleaned of excess of the activator compound and any excess of other activator compounds, and an amount insufficient to react the biological material with all of the activator adducts is removed. attached to the base additionally covalently bonding the biological material to a second substantial surface portion, washing the surface, and contacting the surface with an aminopolysaccharide compound in the presence of water to cause reaction of the amino groups thereof. 16. The method of claim 15, wherein the coating comprises the step of covalently bonding the amine polysaccharide compound to the first substantial surface portion. 39. The method according to claim 38, wherein the activator is a water-soluble carbodiimide. Law. 40. If said surface contains active groups capable of reacting with an activating agent, attaching an amine polysaccharide to a first substantial surface portion and contacting said surface with a water-soluble activating agent to attach said active groups. forming an adduct onto a second substantial surface portion, and reacting the adduct with a biological material to covalently bond the biological material to the second substantial surface portion via the active group. 16. The method according to claim 15, which is carried out. 41. The method according to claim 40, wherein the activator is a carbodiimide activator. 42. The method according to claim 15, wherein the support comprises a plurality of water-suspended particles. 43. The method according to claim 39, wherein the support is labeled with a label capable of imparting a detection signal. 44. The method according to claim 43, wherein the label is a coloring agent and the signal is the color itself. 45. The method of claim 41, wherein the support is a macro-expandable support having a macro-expandable surface. 46. The method of claim 45, wherein the macro-expandable support comprises a well defining a macro-expandable surface. 47. Testing with increased specificity and sensitivity an aqueous sample solution containing, along with other proteins, a specific binding protein having a first binding site that is a specific binding partner for a first biological substance. In doing so, the specific binding protein may be accompanied by other proteins, a first parenchymal surface portion shielded by a polysaccharide coating, and a second parenchymal surface portion to which the first biological substance is attached. contacting a first solid support having a first water-insoluble surface comprising a first water-insoluble surface with the aqueous sample solution, and detecting the amount of specific binding protein bound to the attached first biological substance. Test method for sample aqueous solution. No. 48, the patent claim includes separating the aqueous sample solution from the first solid support before detection. The method according to item 47. 48. The method of claim 47, wherein the support comprises a plurality of particles. 50. The method according to claim 48, wherein the particles are labeled with a detectable label. 51. The method of claim 47, wherein the support is a macro-expandable support having a macro-expandable surface. 52. The method of claim 51, wherein the macro-expandable support contains wells that define the macro-expandable surface. 53. Polysaccharides are aminopolysaccharides that carry biological substances to the second parenchymal surface area. 48. The method of claim 47, wherein the attachment is via a xyl group. 54. The detection step can involve a specific binding protein having a second binding site that is a specific partner for the second biological substance, and other proteins. a second water-insoluble surface comprising an additional first real surface portion shielded by a similar coating and an additional second real surface portion to which the second biological substance is attached; contacting said giant expansible surface with an aqueous solution containing a second solid support composed of a plurality of particles, and observing the degree of adhesion of said particles to said giant expansible surface member. The method according to item 51. 55. The method according to claim 54, wherein the particles are labeled with a detectable label. 56, consisting mainly of a first parenchymal surface portion and a second parenchymal surface portion, having the ability to bind to a specific binding protein that is a specific binding partner for biological substances; In order to reduce the attachment of undesirable proteins to a water-insoluble surface having a strong force, the first stromal surface portion is shielded with a polysaccharide coating, and the biological material is transferred to the second stromal surface portion. A method for reducing adhesion of undesirable proteins, characterized by adhesion. 57. The method of claim 56, wherein the shielding step is performed before the deposition step. 58. The method of claim 56, wherein the shielding step and the adhering step are performed substantially simultaneously. 59. The method of claim 56, wherein the deposition step is performed before the shielding step. 80. The method of claim 56, wherein the surface is on a giant expandable support. 81. Specific binding having: a first binding site that is a specific binding partner for a first biological substance; and a second binding site that is a specific binding partner for a second biological substance Detect samples that potentially contain proteins together with other proteins. In the kit for determining the specific binding protein and other proteins, the polysaccharide coach may be present. a solid giant expansible support having a first water-insoluble surface consisting essentially of a first parenchymal surface portion shielded with a liquid and a second parenchymal surface portion to which the first biological substance is attached; the specific binding protein and other proteins, and the polysaccharide coated protein. a plurality of solid particles each having a second insoluble coated surface mainly consisting of a first substantial surface portion shielded by a coating and a second substantial surface portion to which the second biological substance is attached; A kit for assaying samples that will change. 62. The kit according to claim 61, wherein the particles are labeled with a label capable of imparting a detection signal. 63. The kit according to claim 62, wherein the label is a color (=j) and the signal is the color itself.64. joint part and a second binding site that is a specific binding partner for a second biological substance, along with other proteins. The specific binding protein can be accompanied by other proteins in the kit; a first water-insoluble substance mainly consisting of a first substantial surface portion shielded to prevent entrainment of the synthetic protein and other proteins; and a second substantial surface portion to which the first biological substance is attached. a giant expanded solid support having a surface capable of entraining the specific binding protein and other proteins, and a polysaccharide coated support; a plurality of solid particles, each having a second insoluble surface consisting essentially of a first substantial surface portion shielded by a chemical agent and a second substantial surface portion to which the second biological substance is attached; A kit for assaying samples that will change. 65. The kit according to claim 64, wherein the particles are labeled with a label capable of imparting a detection signal. 66. The kit according to claim 65, wherein the label is a coloring agent and the signal is the color itself.