JPS62223206A - Activated polymer solid and its production - 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 The present invention is a chemically modified and activated surface of a hydroxyl group-containing polymeric solid, natural or synthetic, which can be used in biomanufacturing technology, bioscience and medicine for analytical and fA production purposes. Proteins, nucleic acids, small molecule ligands,
This article relates to products used for binding cells, microorganisms, and other biological substances, and their production methods.

Biologically active substances immobilized on supports have been used for several years for the separation and isolation of unique interacting partners (Perspectives: H,
D, 0rth, 11 Briimer: An
gew.

Chem, 8S (1972) 319; W, B.
Jakob, M. filch-ek mMethod
e in Enzymol 34 (1974), “On the immobilization of enzymes and their use as enzyme reactors” CK,
Mo5bach 1m Methods in En
zymol, 44 (1976)) and widely used in bioscience, biomanufacturing technology and increasingly in medicine for the qualitative and quantitative detection of biologically important compounds. The following requirements are imposed on: - Bath-proofness - Macroporosity - Mechanical and chemical stability - Special shape - Hydrophilicity - Slight non-specific binding - Resistance to the influence of microorganisms and enzymes - Chemical There is no such ideal matrix that meets all requirements for the presence of functional groups for modification and activation and is therefore universally usable. Therefore, in recent years a large number of carrier materials have been described and offered on the market, ranging in diversity from natural polymers (polysaccharides) such as dextrans and agaroses to synthetic polymers such as polyvinyl alcohol, acrylic #1 derivatives, vinyl polymers, and agaroses. and polyacrylamide, as well as natural and synthetic copolymers. All of these carriers have more or less hydrophilic groups on their surface which give them an essentially hydrophilic character and therefore also account for a small amount of non-specific binding.

Conducive conditions for the immobilization of biologically active substances such as proteins, lectins, oxygen, nucleic acids, small molecule ligands, cells and other biological substances are often chemical This is the introduction of reactive groups. The chemical modification called activation is primarily determined by the type of functional groups in the matrix and the ligand, but also by the chemical stability of the matrix and the stability of the biological properties of the ligand. . The selection of the activation method also depends on the production technology burden during activation, the associated activation method, the reactivity of the introduced chemical group, the storability of the activated carrier, and the modification reaction. The cost is determined by the toxicity and biocompatibility of the agent and the stability of the chemical bond between the solid surface and the compound.

There are therefore a large number of parameters and factors to be taken into account and to limit, so it is no wonder that a large number of activation methods have been described in combination with a large number of carrier materials.

Activation methods using geltaraldehyde or other homologous or heterotrifunctional reagents instead, CNBr r-activation,
Activation using hydrazine, bisepoxylane, divinyl sulfone, epichlorohydrin, benzoquinone, carbonylimidacil, triazine derivatives, tosyl chloride as well as periodate oxidation and diazotization (Perspective:
J., M., Egly, H., Bo3chettl.
jtN Practical Guidelines for Use in Kachromatography and Related Techniques, IBF-LJC 8, 1983) By far the most widespread is the Bromsian activation introduced by Axen et al.
, J., Porath, 8. Ernback
: Nature214 (1967) 1302
). Although this method has indisputably led to widespread application of substances immobilized on supports in bioscience and biomanufacturing techniques, it is important to note that the relatively unstable chemistry between the solid surface and the ligands At pH values less than 5 and greater than 10, this can lead to considerable release (leakage) of the immobilized ligand and therefore also makes its application particularly difficult for in vivo therapy in medicine. disadvantages such as the high toxicity of bromysia (which makes the activation production technique expensive) and the risk of formation of canon groups on the support surface. @ The disadvantages in the case of hydrazide/activation are technical problems such as a low bonding yield with polymeric ligands.

In contrast, other activation methods involve high toxicity of the reactants (divinyl sulfone, triacyl chloride, epichlorohydrin) and/or high cost of the process (divinyl sulfone, tosyl chloride, tresyl chloride).
Unfavorable environmental conditions during binding, and therefore also risk of inactivation of biological substances (bisepoxiranes, triazine derivatives, epichlorohydrin), unstable immobilization at high pH values (divinylsulfo/, carbonyldiimidazole, diazonium compounds), long activation and fixation times for the ligands (ebichlorohydrin/bisepoxylan),
Due to manufacturing technology issues (triazine derivatives, tosyl chloride, carbonyldiimidazole, diazotization), low binding yields (periodate oxidation) as well as relatively strong single charge-transfer interactions that also affect the cost of the process. Attributable one non-specificity fp! It is plagued with drawbacks such as N (benzoquinone, 4 triazine derivatives, diazonium compounds).

For a while now (R, A, Messing, H, H,
Weetall: US Patent K 3519538; H,
H, Weetall eN, Y, E1mirl US Pat. No. 3,652,761; H, H, Weetall m
Methods in Enzymol 44 (19
76) 134) 5in2 especially O on the surface of glass
It is known that the H-group can be used as a point of attack for chemical agents, especially organosilanes with various functional groups. Indeed, these organosilanes have traditionally been used mostly for inorganic supports and solid surfaces.

The object of the present invention is to provide a simple, inexpensive, and non-toxic activation method for natural or synthetic hydroxyl group-containing polymeric solid surfaces, which can be used to stabilize proteins, nucleic acids, small molecule ligands, cells, microorganisms, etc. The objective is to develop activated and stable solid surfaces capable of immobilizing biological substances with high yield and biocompatibility.

The present invention provides natural and synthetic human af-cyl group-containing boIJs.
- Activating the solid surface with compounds normally used to blend organic to inorganic materials to produce highly activated, stable, biocompatible polymeric solids. is the issue. The present invention solves this problem when the hydroxyl group present on the surface of a natural or synthetic solid has the general formula / 7 no, nitroso-, sulfhydryl- or halocarbonyl-&, R' ff, alkyl-, alkylphinyl- or phenyl group, while R represents γ-lkoxy, phenoxy- or halogen group, nl is 0 to and n can have a value from 1 to 3). Typical organic compounds can be represented by the following formula: 5iRn4-n (II) (wherein Y represents an amino-, carbonyl-, carbonyl-, carbonyl-, or sulfhydryl group, and R is an alkyl group. represents a pheasant, phenoxy, and halogen group, and n can have a value of 1 to 3.Organosilanes that are widely distributed and often used in practice generally have a composition of
CH2CH2CH2-81(OR)3(lfi). In the formula, X is a reactive organic group corresponding to Formula 1, and R is a methyl or ethyl group =sbf.

The reaction can also be carried out with at least two organosilanes of formula 1 in a mixture or sequentially. The preferred medium for organosilanes is liquid or gas phase, and activation can be carried out in aqueous, water-solvent or organic solvents. Subsequently, the optionally modified solid surface is further treated with homogeneous or heterogeneous bifunctional reagents.

The activated polymer solid produced according to the method according to the invention is an organic polymer containing OH- groups, on the surface of which there are groups of the general formula %() and OH- groups; R1 is alkyl-, alkyltzenyl or phenyl group, x is yt/-, -h#
M-nyl-, carboxyl-, ethoxy, incyano-
, diazo-, isothiocya/-, =)roso, sulfhydryl, or hanosc1 carbonyl group, nl is a value of 0 to addition, and n is a value of 1 to 3. Foundation ■ and OH
The ratio of numbers to groups is 1:9 to 9:1.

Natural and synthetic polymers have OH groups on their surfaces, as already mentioned, which enhance the hydrophilic properties of the solid surface and thus also contribute to the reduction of non-specific or undesired interactions.

Surprisingly, the highly reactive organosilay
It has been discovered that solid surfaces of this type react with high yields. Sili: x functional group is solid, accessible human c
While the organic functional group reacts with the amino-,
Carbonyl, carboxyl, isocyano, diazo, inthiocyano, sulfhydryl, nitroso
Or they can be used in conventional chemical reactions using halocarbonyl-groups to enable these compounds to act as bridges between solid surfaces and organic/biological compounds. The thus activated natural or synthetic solid surface can then be directly immobilized via the corresponding functional groups or by methods known per se, either directly via the corresponding functional groups or via the intervention of homogeneous or heterogeneous bifunctional reagents, and thus the protein to be immobilized. It can be reacted with lectins, # elements, nucleic acids, low molecular weight ligands, cells, microorganisms, and other biological substances. This means, for example, that according to the invention, the surface of a hydroxyl group-containing solid is coated with γ-aminoglobiltriethoxysilane (formula 1
In X:NH2-1R1=(-CH2)3,N'
-4, R=-QC' 2H5, n=1). The atno group now present on the solid surface is subsequently replaced by geltal dialdehyde or N-succinimidyl 3-(2-pyridyldithio)propiona) (5p
PD). The immobilization of proteins or other ligands in these cases involves amino groups - preferably lysine (
The aldehyde group is formed through an amino group at the Lysin position or through a disulfide bond. In exactly the same way, hydroxyl group-containing solid surfaces can be reacted with glycidoxyprobyltriethoxysila/. In this variant, the biological substance to be immobilized reacts directly with the epoxy groups on the solid surface. What is important in this case is that the reaction with organosilas, which are non-toxic and are produced quite extensively in large-scale industry, is carried out simply by contact or immersion, and the activation is carried out by swelling or immersion of the solid. It can be carried out in the unswollen state or in the gas phase.

In this case, it is important that the reaction with the organosilane in the liquid phase occurs in organic solvents such as acetate, doluol, dioxa/, methanol, ethanol, catalytic solvent mixtures such as methanol/ethanol, and aqueous media or water-based media such as ethanol/water and methanol/water. - It can be carried out in a solvent mixture and, unlike many other activation methods, it is less burdensome on manufacturing technology. Activation could be carried out particularly advantageously in the gas phase by means of an aerosol or by means of negative pressure. Furthermore, the basic principles of the present invention can be utilized to obtain solid surfaces with a wide variety of functional groups (X in Formula 1), so that a wide variety of organic and possibly bifunctional bonding reagents can be obtained. It is advantageous that by choice virtually all reaction possibilities in question can be realized. In addition, organic silane (R'=0-20) and/or
or due to the spacer effect of bifunctional binding reagents (e.g. geltaraldehyde), immobilized biological substances almost invariably retain their biological activity.
Particularly advantageous is the use of mixtures of organosilanes of various functional groups. This is because these can fix biological substances at a high yield through various mechanisms. Mixtures of aminopropyltriethoxysilane and glycidoxyprobyltriethoxysilane and of aminopropyltriethoxysilane and mercatoprobyltrimethoxysilane therefore prove particularly suitable.

This is because these biological substances can be fixed at extremely high yields through reactions via various functional groups.

In these cases, the fixation of the ligands takes place via aldehyde and epoxy groups or via aldehyde and mercarbut groups.

The use of mercabutoglobil trimethoxysilane opens up the possibility of reversible binding of biological substances. This is because the resulting disulfide bonds can be reversibly separated by use of a suitable reducing agent. Reversible ligand binding is particularly advantageous when the separation of the heterotically fixed interaction partners poses a risk of denaturation and the release of the protein-ligand complex is preferred. Prove.

The chemical bonds formed by the reaction of the hydroxyl groups of natural and synthetic polymers with organosilanes are extremely stable and the thus activated solids can be stored for long periods of time in dry or wet conditions and are immobilized by immobilized organisms. So much so that chemically active ligands remain stably immobilized for long periods of time even under extreme conditions. Nonspecific binding to the thus activated natural or synthetic polymer solid surface is extremely low. Organic sila/ is distinguished by good biological compatibility.

As natural or synthetic polymeric solid surfaces, all polymers in which a sufficient number of sterically accessible hydroxyl groups are available on their surface come into consideration; the composition of the solid is not critical for the activation method according to the invention. do not have. A natural polymer of cross-linked dextran substrate (8ephadex■
) or agarose (Sepharose (!5) or cellulose substrate polysaccharides are in widespread use. Common synthetic polymers include cross-linked polyvinyl alcohols, copolymers of glycidyl methacrylate and alcohols ( Fractogel”)
and N-acryloyl-2-amino-2-hydroxymethyl-1,s-7'lovandiol (Tr-1s
It is a copolymer derived from acryl. However, natural and synthetic polymers, such as copolymers of agarose and polyacrylamide (Ul trogel AcA@), which can also be activated according to the invention, are often used. and droxyethyl methacrylate,
Ethylene glycol dimethacrylate tocobolimers as well as cellulose-containing solids for which a sufficient number of sterically accessible hydroΦ-syl groups are available (which can be activated using the method according to the invention) are the same.

The solids thus activated are advantageously present as shaped bodies, these shaped bodies having a spherical shape.

It can be fibrous or prismatic and can be attached to column packings, flat supports or other high grade structures by packing, weaving or by means of binders. Of particular importance in this case are the spherical cellulose-containing solids (e.g. granular cellulose) used in chromatographic methods and processes, and in analytical chemistry and chromatography.
It is a flat carrier (activated paper) that has come into use.

Included among these are also four layers which can be present as films or lacquers on other solid surfaces which are not incorporated into the reaction according to the invention; the support material inert for the activation reaction according to the invention is homogeneous. It may or may not be molded.

Activated solid surfaces are used for chemical immobilization of nucleic acids, small molecule ligands, cells, microorganisms, and other biological materials, such as proteins, antigens, antibodies, enzymes, and lectins. This cypress ligand, which is immobilized on a solid surface, can be used as an enzyme reactor in bioscience, biomanufacturing technology or for the separation and isolation of biologically specific interaction partners by affinity chromatography. It is used in medicine for the qualitative and constant detection of biologically important compounds.

The present invention will now be described with reference to some embodiments.

Example 1: Crosslinked dextran (Sweden Pharm-E
lepharo8e 4B), Trisacr
yl” (Sweden LKB) and Fracto
1 g each of gelo (West Germany Me-rCk) and 5% amnoprobyl triethoxysilane (East Germany 1!: B Ch
emiewerk N5-nchritz NB111
4) in ethanol/water (1:1) pH, s for 6
Incubate at 0°C for 6 hours, followed by ethanol/water 5
- twice with 0, 1 m phosphate buffer pH 6, 8, 5
- Wash 5 times. Then G, Antoni (An
The degree of activation is determined by measuring the amino groups on the solid surface according to Al, BIochem Tan Liao (1983) 60). The degree of activation can be varied over a wide range by selecting the organosilane concentration and/or incubation time. Typically, a typical degree of activation of 10 to 1 μmol of amino groups per gram of carrier is obtained.

Example 2: Trisacryl oGF 200 5. WoLO%7
Mi/7'.

Pyrtriethoxysilane (East Germany WEB Chemie
-werk Nunchritz NB 1114)
and ethanol/water (1:1) at pH 2.5 at 600 for 6 hours, shaken, and ethanol/water (1:1)
2 to 3 layers of 1 m phosphate buffer pH 6,
Wash 5 times at 820m each. The thus activated gel was then reacted with 5% gel taraldehyde in 0.1 m phosphorus salt buffer pH 6.8 for 2 hours at 37°C and washed again five times with 2 parts of phosphorus salt buffer. Immobilization of proteins is carried out by simply adding protein bath to the thus activated gel. Therefore, the carrier is 0.1 m
Mix with 35.5 by 4 pre-specified human immunoglobulin G 10- in M@salt buffer pH 6,8 for 2 hours at room temperature. After absorbing the unfixed residue, the amount of protein immobilized on the carrier is measured by differential measurement of the protein solution used and the remaining protein. By this method, IgG49.7In9 could be immobilized on Trisacrylic.

Example 3: Practical example 1 of TrisacryloGF 20001-
and activated as described in the second description and 3% v! React with glutaraldehyde. Next, the thus activated m-body is 41.3m9/ld (7) J-I of F1k degree.
g() (from rabbit, directed against alkaline phosphatase) and incubated at room temperature for 2 hours. After absorbing excess protein and washing thoroughly with +';
The amount of protein immobilized is determined by measuring radioactivity while the solution is introduced together. In this experiment, 32.4 to 125 J-IgG could be immobilized per trisacrylic.

Example 4: TrisacrYl■C)F2000 1 mg in aminopropyltriethane and glycidoxyprobyltriethoxysilane (adhesion mediator 6130. GDR VKB)
Chemiewerk Njtnchr 2) and shaken for 2 hours at room temperature. After wicking off the excess reagents, drying and subsequently washing thoroughly with 0.1 m phosphate buffer pH 6.8, the support was reacted with germeraldehyde, followed by reaction as described in Example 3.
5J-IgG lk is fixed. to! JX7/
! J kwt Ata IJ 125J -IgG 32.7
I was able to fix 1v.

]0IiL on.

8epharose 4B ■ 1vt If actual Aa example 1st
41.3V9/
d once 125. + React with G-gG and keep warm at room temperature for 2 hours. As described in Example 3, Proty/
The amount of immobilization was measured, and 566 ml was obtained per gel.

"1 and 28epharoie 4 Bol-" were activated with 2fit different organosilanes and geltaraldehyde as described in Example 4, followed by reaction with t25J-IgG. In this experiment 15 IgG per gel
．． 21151 was fixed.

Example 7: The gel (Trlsacryl) reacted with 1255-gG as in Examples 3 and 4 was treated with dioxa 720.
% of PH1-buffer pH 7, 420 td each, followed by determination of the immobilized radioactivity.

The amount of protein remaining in Tris acrylic is 14.3
#/- is unchanged.

Example 8: 8eE) blrosa reacted with 1255-gG as in Examples 5 and 6 was thoroughly washed with a PH1-buffer containing 20% dioxane as in Example 7. The amount of protein, as verified by radioactivity measurements, was determined to be 5II9 (Example 5) to 142H19 (Example 6) per gel.

Example 9: The immunoglobulin G used in Examples 3 to 8 was obtained by immunization of rabbits using the enzyme alkaline phosphatase. It is therefore possible to use 12'J-IgG for the purification and isolation of the enzyme alkaline phosphatase while taking advantage of the corresponding antibody activity. For this reason, n-
Alkaline phosphatase prepurified by butanol extraction was prepared as described in Example 6 at 125. r ++ I g
The BephalIose reacted with o was fixed, and then [
1, which is eluted with triethylamine pH 11.4, thus purified # element has a specific activity of 1000 IU/ζ and a yield of about 80
%. Associated proteins are undetectable in polyacrylic oxide electrophoresis.

Example 10: Human immunoglobulin G is immobilized on Trisacryl' GF 2000 as described in Examples 1 and 2 and used for isolation of anti-Human IgG antibodies from eel.

For this purpose, the carrier loaded with IgG is packed into a chromatography column, and the column is loaded with a special J.
Loads with 4-sex anti-bloody emotions. Elution is carried out first with a pH 1-4 buffer and then with a 3 m rcscN-solution. Detection of specific antibodies was performed by enzyme immunoassay and double immunodiffusion (Ouchte) after immediate dialysis of the antibody fraction.
It was applied using the rlony technique. Calibration of non-specific fixation is carried out by plating the corresponding normal rabbit serum and testing the antibody-containing fractions on polyacrylamide discs using pneumophoresis.

Life 4. 'M j'1. '11 +-1 8epharose 4 B 1 i was incubated in 5% mercaptogprolmethoxysilane (5erva, West Germany) and ethanol/water (1:1) for 4 hours at ω°C, followed by ethanol/water 4- and 5 times each with 0.1 m phosphate buffer pH 6, 75 wd. The gel was then pretreated with N-succinimidyl-3-(
2-pyridyldithio)probionate (Swedish Ph
armacia 8DPD) and mixed with human IgG 1019. The pyridine-2-thio/ liberated during the reaction can be followed with a photometric needle at 343 nm. This IgG thus fixed was Example #
! Similar to 1o, it can be used to collect and isolate rabbit anti-IgG antibodies. After affinity chromatography, the IgG was immobilized via disulfide bridges or mercaptoethanol or dithiothreitol (50 m
With M), the activated matrix can be separated again and made available for fixing the compound.

Example 12: Particle size within 15 to 5 μm! Table 1j1 Macroporous spherical 2-hydroxyethyl methacrylate with a product of about 70 per g of support and an exclusion limit of 2.16 Daltons.
Ethylene dimethacrylate copolymer (hereinafter referred to as 5epar
on ■ Hema 1000) II with ethanol/
10% aminopropyltriethquin silane in water (1:l), pH 2,5 (GDR VFiB ChemieW-e
rk Ntjnchr Mekosha NB 1114) in SOC for 6 hours, then at 120°C for 6 hours, followed by two 0.1 m incubations with 5 m each of ethanol-sand water.
Wash 5 times with phosphate buffer I) H6,85-. Subsequently, the degree of activation is determined by measuring the amino groups on the carrier. For this purpose, G-AntOj'li et al.
o-chem Jl (1983) 60) is promoted.

The degree of activation can be varied over a wide range by selecting the temperature and/or the incubation time. Usually, an activation level of 5 μMol per iil is achieved.

8eparon Hema with particle size 15-25 pm
10005J of 7minoprobyltriethoxysilane (￥jIL Germany VE) in ethanol/water (1:1) pH 2.5
B Chemie-werk Nlnchritz Company N
B 1114 ) at 10% ■C for 6 hours followed by washing twice with three portions of ethanol/water (1:1) and five times with 0.1 m phosphate buffer pH 6,820. The thus activated gel was prepared in 0.1 mm salt buffer p with 5% gel tar dialdehyde.
The reaction mixture was allowed to react for 2 hours in H6.8 at 10°C, followed by further washing 5 times with 20°C of phosphate buffer. Protein immobilization is carried out by simply adding a protein solution to the thus activated gel. Therefore, the activated 8epBon is 0
．． Mix with 10 m of human immunoglobulin (IgG) at a concentration of 18.619 m phosphate buffer and incubate for 2 hours at 37°C or overnight at 4°C. After absorbing the unimmobilized residue, the protein immobilized on the carrier is measured by measuring the protein difference between the protein solution used and the residue. In this way, the amount of protein supplied in the Tani experiment was small (95% of the protein was fixed. IgC provided in the second JA case) 186
183 # proti/ out of da are fixed, 5epa
36.719 IgG per r-on 9 are fixed.

``A'' The swollen 5eDaron lgo was activated as described in Sections J2 and 13 of the same publication and reacted with geltaraldehyde at a concentration of 3%, and the thus reacted support was subsequently reacted with a concentration of 35.5rNi/-. Human immunoglobulin 02m (=
7111g+ in 0.1 m phosphate buffer pH 6g for 2 hours at room temperature. Infection caused by absorbing excess protein 1. Read and measure protein fixation as described in Example 13. In this way, we were able to fix 23.6m9 of engineering gG per 5eparon Mt.

Example 15: Swollen searon 111t was mixed with disilane and glycidoxypropyltriate in aminopropyltriate. )−
1['Disilane (East German VgB Chemiewerk
N1jnc-hritz adhesion promoter 6130) and shaken for 2 hours at room temperature. Blotting off excess reactant and drying and subsequent 0.1 mm
After intensive washing with phosphate buffer PH 6,8, human IgG is subsequently immobilized as described in Example 14 by reaction with the carrier geltaraldehyde.

IgG2a and 8IQ were immobilized per 56 pafon ld.

[J's LfL] Human IgG was added as described in Examples 12 to 15.
epn (on) and used for the isolation of rabbit anti-human IgG antibodies ◎For this purpose, a carrier or chromatography column loaded with IgG is loaded with human IgG.
Load with specific antiserum against G. Elution begins at pH 8.
- Buffer followed by 3m K8ON Q solution. ) Immediately after dialyzing the antibody fraction, the enzyme was subjected to immunoassay and double radial immunodiffusion (Ouchterl
Detection of specific antibodies was performed using the ony technique). Testing for non-specific fixation is carried out by application of matched normal rabbit serum and testing of all antibody-containing Arax1 using polyacrylamide disc electrophoresis and immunoelectrophoresis.

Example 17: Excluded volume 5,000,000 D, grains Jj 80 to 20Q
pm and solid content -* 64 ky/- granulated cellulose II in ethanol/water (1:1) pH 2.5
5% aminopropyltriethoxysilane (GDR WE
B Cheml-ewerk NffnChritz
Incubate at 60c for 6 hours with company NB 1114), then wash twice with ethanol/water at 0.1m@5.
Wash 5 times with m phosphate buffer pH, 85 m each. , i4 and the activation degree is G, At1tOjli(Anal, B
iochem 129 (1983) 60) by measuring amino groups on the surface of a solid. The degree of activation can vary over a wide range depending on the concentration of the organosilane and/or the selection of the temperature during incubation. Normally, a characteristic degree of activation of 10 to 15 μMole of cellulose amino groups is obtained.

Once the granulated cellulose according to Example 17 was incubated in 10% 0% aminopropyltriethoxysilane in ethanol/water (1:1) pH 2.5 at 600°C for 6 hours, then The thus activated gel was then washed twice with 30° each of ethanol/water (1:1) and 5 times with 0.1 m phosphate buffer DH 6,820° each with 5% gel taraldehyde and 0.1 m phosphate buffer. salt 4 & titer solution pH 6, 3 in a
Mix for 2 hours at 7°C, followed by washing again 5 times with 20 g of g4d salt buffer. Immobilization of protein is carried out by simply adding a fixed amount of protein to the gel thus activated.

For this purpose, granulated cellulose was dissolved in 0.1 m phosphate buffer p
Mix with human immunoglobulin G 10- of 35.5 rIv/- in H6,8 for 2 hours at room temperature.

After absorbing the unfixed 1A portion, the amount of protein immobilized is determined by measuring the difference in protein concentration between the protein solution used and the remaining portion. In this way, it was possible to immobilize 12.9 IgG per granular cellulose.

Example 19: Granular cellulose 1- was activated as described in Example 18 with glutaraldehyde at a concentration of 3%.

Make it react. Subsequently, the thus activated carrier concentration 41
, 3 da/d of 1-IgG (from rabbit, directed against the enzyme alkaline phosphatase) and kept at room temperature for 1 hour. After absorbing excess protein and washing thoroughly with 5 portions of phosphate buffer, the amount of protein immobilized is determined by measuring radioactivity while introducing the protein solution used as a standard. In this experiment granular cellulose - 125 J-IgG per 1
It was possible to fix 1.4 da.

One large JLfl 恕'' - Granular cellulose 5sIt''! Am Example No. 17 Fujipropyltriethoxysilane in 5% glycidol in ethanol/water (1:1) pH 2,5 East Germany VEBChe
miewerk Nilnchritx NB 111
5) and kept warm for 6 hours at 120°C.
Heat for a further 6 hours at <0>C. The thus activated granular cellulose was added to a sickle which had been thoroughly washed in the same manner as in Example 18 with 20 parts of ethanol/water or 0.1 m phosphate #Jkm solution. once in lm phosphate buffer PH6,8,
Mix with 51n9/d human immunoglobulin G for 2 hours at room temperature. The protein/fixed amount measured as described in Example 18 was 7.2 per granular cellulose.
■It was.

Example 21: Granular cellulose 5- was mixed with 5% mercaptogproltrimethoxysilane (5erva, West Germany) as in Example 17.
4 at 0°C in ethanol/water (1:1) with
Incubate for 1 hour, then add 5 d each of ethanol/water for 0.
Wash 5 times each with 1 m phosphate/salt buffer pH 6,85Fn9. Cellulose was then pre-treated with N- as reported by the manufacturer.
Succini Z-dyl-5-(Z-pyridyldithio)probionate (Swede Pharmacia 8DPD
) and substituted with human IgGIOrI9. The pyridine-2-thione liberated during the reaction can be followed with a photometric needle at 343 nm.

This IgG thus immobilized can be used for recovery and isolation of rabbit anti-human IgG antibodies. At the end of the affinity chromatography, the molecules identified through disulfide bridges are separated again using mercaptoethanol or dithiothreitol (5QmM), and the activated matrix is used to immobilize the ligand again. can be made possible.

Example 22: Particulate cellulose 5- was mixed with disilane and glycidoxyprobyltriethoxysilane in aminopropyltriethane (GDR VIBChemiewerk Nl) as in Example g17.
jnchrItz adhesion promoter 61'30) and shaken for 2 hours at room temperature.

The excess reagent was blotted off and dried in vacuo, followed by 0.
After thorough washing with 1 m phosphate buffer pH 6, s, it was reacted with 3% geltaraldehyde as a carrier, followed by Example 1.
Fix 125 J-engine gG as described in 9. It was possible to immobilize 125 J-26.4 Da of IgG per granular cellulose.

125. As per the 19th and nth embodiments of Medical and Obstetrics and Ayu. - PBB buffer solution containing dioxane was added to the granular cellulose reacted with the cellulose.
Wash three times with H7,420- followed by determination of immobilized radioactivity. The amount of protein remaining on the granular cellulose was 11.4 da/- (Example 19) and zs, 6
yn9/dc Example 22) and remains unchanged.

The immunoglobulin G used in Example 19.4 and 2 was obtained by immunizing rabbits with the enzyme alkaline phosphatase. It is therefore possible to use 125J-IgG for the purification and isolation of alkaline phosphatase while taking advantage of the corresponding antibody activity. For this purpose, alkaline phosphatase prepurified by n-butanol extraction was prepared as shown in Example 19 in the middle 4 cases.125. - immobilization on granular cellulose reacted with ρ and subsequent elution with triethylamine pH 11.4; Thus fl
The purified enzyme has a specific activity of 1000 IU, 4 volumes, a yield of about 80%, and no accompanying proteins are detectable in polyacrylamide gel electrophoresis.

The same method described in Example 18 was used to immobilize Goon Purin G on granular cellulose and use it as a rabbit anti-IgG antibody. For this purpose, the IgG-loaded carrier is packed into a chromatography column, into which human I
Load with specific antiserum against gG. ＃First of all, P
B8-buffer followed by 3m K8ON bath solution.

After immediate dialysis of the antibody fraction, detection of % isomeric antibodies was performed by enzyme immunoassay and double radial immunodiffusion (
This was done using the 0uchterlon 3F technique). Testing for specificity fixation is carried out by application of matched normal eel serum and by testing all antibody-containing horns on polyacrylamide disk magnetophoresis.

] O11 mutually planar cellulose carrier (Whatman paper 560 and FN 4
Paper) Activation with 3% aminopropyltriethoxysilane or Grind Φsibrovalsilane as described in Example No. 19 and Addition, followed by concentration 10 μI/xt and 'c% 100 pV in 0.1 m phosphate buffer pH 6,8.
d J-1 nido serum 7/E/p i y (""J-
React with HEIA. After washing thoroughly, the amount of protein immobilized is measured by radioactivity measurement while introducing a standard. The results obtained here are summarized in Table 1.

Table 1 Fixed 131J -H8A FN 4 192rsFd 1036r&Ad
754M 343red Watsuto Beak 580 61ji
76-Example 27: In Example No. 1, paper carriers loaded with J-H8A are subsequently used in an enzyme immunoassay. If there are any free reactive groups, they are blocked in advance with ethanol, then replaced with appropriately diluted eel anti-E toalpmin antiserum and incubated overnight at room temperature. After washing three times to remove the antibodies, the paper was washed with a goat anti-Nagie ρ conjugate (enzyme: alkaline phosphatase) at 370 ml.
Keep warm for 4 hours and wash again 3 times. Enzyme activity is determined by hydrolysis of 4-nitrophenyl phosphate in jetanolamine buffer m9.8 odd and photometric measurement of the enzyme product at 405 nm.

Two flat cellulose carriers (Whatman 560, Filtruff 1389, Filtruff 390, FN3) were prepared as shown in Example 4 with a mixture of apropylenetriethoxysilane and dipropyltriethoxysilane in glycide. Activated with ``C0,1m phosphate mum p''
g 6.8chuno2 ffflnola[)125, +
Incubate with In(I# element) (directed against alkaline phosphatase). After washing three times with PSB buffer, the amount of protein immobilized is determined by radioactivity.

The plated cell carrier was washed again with PBi9-buffer solution 10 times, and the amount of protein immobilized was measured again by radioactivity measurement while introducing a standard. MCl-glycine buffer pH 2, 2, 3 m KBCN, carbonate-hydrogen carbonate-buffer OH 10 and PB containing dioxa 79% multiple times over a period of 4 weeks on cellulose carrier.
Wash with S-buffer and re-measure the amount of protein immobilized.

The results are shown in A2.

Table 2: Immobilized 125J-IgG Flat supports with paper-like matrices according to the invention were mixed with human immunoglobulin G radiolabeled with J and a buffer system of various degrees of solubility. Incubate at ℃ for 16 hours.

PBS-buffer pH containing Tween 200.95%
If excess reactive groups still remain after three washes with 7.4, block with 1 m ethanolamine solution. After washing again with the above PB8-buffer, the amount of protein immobilized is determined by radioactivity measurement. To determine the possible desorption, the samples are then washed again 7 times with PBS-buffer. The thus loaded flat carrier is then mixed with a conjugate consisting of rabbit anti-human Igf (rabbit anti-human Igf) and the enzyme alkaline phosphatase and kept at room temperature overnight for immunoreactivity measurement. After another wash, the alkaline phosphatase enzymatically active jetanolamine buffer pH 9,80,5-4-
Hydrolysis of nitrophenylphos-71 and tnN@OH
or photometric measurement of the enzyme product at 405 nm after stopping with In EDTA. After performing the enzyme immunoassay, the flat support is washed twice with 0.5-0.5 PBS-buffer and subsequently mixed for 1 hour at 4° C. with one of the following dissociation reagents in multiple measurements.

1) PBS-mild solution, 2m NaCl, containing 5% dioxane, 2) 1m KsCN, 3) 3m Ks
CN, 4) 4.5un Mgcl, 5) 1
m NaJ, 6) HC'-glycine buffer p
H2,8 All of these drugs reduce the bound antigen-
The antibody-conjugate is separated and the flat carrier loaded with human engineered IgG can be used again for enzyme immunoassay.

A flat molded body according to the invention (CF = 56-) was activated as described in Example @n and loaded with the human factor (2). Transfer these carriers into polystyrene microtiter plates or polystyrene tubules, followed by ? -■For antibody measurement (W, 5
ch85sler, M, 8t-epanauskas
Chr DittrichH, He1ne: Ac
ta b -1o1 med germ 41 (198
2) 263; W, ScMsslerM, 5t
epanauska@Chr Dittrich:
Acti blolmed germ 41 (19
82) Perform enzyme immunoassay as described in 695). For this purpose, the molded body is mixed with a warming mixture consisting of the blood glands to be measured and an excess of eel anti-human factor (1) antibody, and kept at 37 DEG C. for 6 hours.

PBS-di solution pH 7.4 (Tween 200
．． 05%), then wash with sheep anti-eel I
Conjugate 2 consisting of gG and the enzyme alkaline phosphatase
After several hours of incubation and subsequent washing, the enzyme activity is determined as described in Example 9. Separation of bound antibodies was performed using 1 liter of the dissociation reagents listed in Example 4.
The carrier loaded with factor ① can be used again for immunoassay.

Example 31: A flat carrier with a paper-like matrix according to the invention is loaded with human factor ① as described in Example 9. This carrier serves below as a model for screening studies for (monoconal) antibodies. After carrying out a particularly simple washing step on this flat carrier, the substance to be investigated and measured is applied in spots with the appropriate coating. In our case, dilutions of eel anti-human factor serum suitable for this purpose or, as a negative control, served as sources of antibodies. 4 at 37℃
After incubation for 6 to 6 hours, the flat carrier was washed again and incubated with sheep anti-rabbit IgG combined with the enzyme peroxidase for 4 hours at 37°C or overnight at room temperature, washed again several times, and the enzyme activity was detected using an appropriate detection system. t
j? 4 mu o-phenylenediamine and 1.5
mM H2O. It is measured by visual evaluation of the coloration produced using Positive staining clearly suggests antibodies.

Example 32: P-labeled phosphoproteins contained in membranes from wing heart muscle were analyzed in a phosphate-buffered sodium dodecyl sulfate-polyacrylamide gel electrophoresis system as described by Weber et al. , Weber, J.
, Pringle, M., 0sborn +Met
h 13: separated according to nzymol 26 (1972) 3).

Immediately after electrophoresis, the separated proteins were incubated with Kyllse-Ande on paper activated according to the present invention in a delivery system containing triethanolamine and butyric acid.
rlen (J, Biochem Biophys
Immunoplotting was performed according to Meth 10 (1984) 2o3). Plotting was followed by immersion in phosphate-buffered saline containing 0.1% gelatin and % Tween O,Os, followed by incubation with the first eel antiserum, followed by anti-eel immunoglobulin-wasabi radish. It is kept warm together with the peroxidase conjugate, and finally an indicator reaction is performed. Alkaline phosphatase conjugates can also be used in place of peroxidase conjugates with the same results.

Example 33: Proteins of the intracellular membranes of various types of muscle cells were analyzed in a dodecyl sulfate polyacrylamide-glue electrophoresis system.
Natul 227 (1970) 680) VC control. Electrophoresis Me, gel 25mM
)! jethanolamine-hydrochloride pH8.4
, immersed in a buffer consisting of 0.1% sodium dodecyl sulfate for m to ω minutes. Immediately after that, Example 32
Perform the discharge plotting as described.

Representative Patent Attorney Mamoru Fuji Yu 1 other person

Claims (1)

[Scope of Claims] 1. A method for activating the surface of natural and synthetic polymeric solids containing hydroxyl groups, in which the hydroxyl groups are substituted with the formula (XR^1_(n^1))_nSiR_4_-_n(
I) (wherein X is amino-, carbonyl-, carboxy-
, epoxy-, isocyano-, diazo-, isothiocyano-, nitroso-, sulfhydryl or halocarbonyl- group, R^1 is alkyl-1 alkylphenyl- or phenyl group, R is alkoxy-, phenoxy-1 or halogen group; , n^1 is a value from 0 to 20,
n having a value from 1 to 3) or carrying out the reaction with at least two organosilanes of the formula I in a mixture or sequentially in the liquid or gas phase and subsequently A method characterized in that optionally a further treatment is carried out with the same or different bifunctional reactants. 2. Process according to claim 1, characterized in that the reaction of the compound of formula I with the hydroxyl group is carried out in an organic solvent, a solvent mixture, an aqueous solution or a mixture of water and an organic solvent. 3. Process according to claim 1, characterized in that the reaction of the hydroxyl groups is carried out in the gas phase using an aerosol or at a pressure below atmospheric pressure. 4. The method according to claims 1 to 3, characterized in that the polymer is present as a molded body. 5. The method according to claim 4, wherein the molded body is spherical, fibrous, or prismatic. 6. The method according to claim 4, wherein the molded body is spherical. 7. Process according to claims 4 to 6, characterized in that the shaped body is assembled as a column packing or a flat material by filling, weaving or by means of a binder. 8. Process according to claim 4, characterized in that the shaped body is in the form of a thin layer which can be present on another carrier as a film or as a glue. 9 In an activated polymer molded body made of an OH-group-containing organic polymer (produced according to the Haji method described in claims 1 to 8), the surface of the polymer solid has the general formula ( -O-)_4_-_nSi(R^1_(n^1)X)
_n(IV) (wherein R^1 is an alkyl-, alkylphenyl or phenyl group, carbonyl group, n^1 is a value from 0 to 20, or n
has a value of 1 to 3) and an OH- group.
A polymer solid characterized in that the ratio of groups to groups IV is between 1:9 and 9:1. 10. Activated polymer solid according to claim 9, characterized in that the polymer solid is spherical, fibrous or prismatic. 11. Activated polymer solids according to claims 9 and 10, characterized in that the polymer solids have a predominantly two-dimensional extent in the bound and filled state. 12. Claim 1, characterized in that the bound polymeric solid is a nonwoven fabric, paper, foil, film or lacquer.
Activated polymer solid according to clause 1.