DD256721A1 - METHOD FOR ACTIVATING CELLULOSE-CONTAINING SOLID CARBON SURFACES - Google Patents

Abstract

The invention relates to a method for activating hydroxyl-containing cellulosic Festkoerperoberflaechen with organosilanes and optionally homo- or heterobifunctional reagents for simple, gentle and stable binding of proteins, nucleic acids, low molecular weight ligands, cells, microorganisms and other biological materials. The invention finds application in the life sciences, biotechnology and medicine.

Description

Field of application of the invention

The invention relates to a method for the activation of hydroxyl-containing cellulosic solid surfaces, for the binding of proteins, lectins, enzymes, nucleic acids, low molecular weight ligands, cells, microorganisms and other biological materials in biotechnology, the life sciences and medicine for analytical and preparative purposes are used.

Characteristic of the known technical solutions

Cellulose-containing carrier materials have been used for years for binding proteins, nucleic acids, low molecular weight ligands, cells and other biologically active materials.

Such carrier-fixed proteins, nucleic acids, low molecular weight ligands, cells and other biologically active materials have i.a. For the separation and isolation of specific interaction partners, as enzyme reactors, for the qualitative and quantitative detection of biologically important compounds in the life sciences, biotechnology, but also increasingly in medicine, great importance. "

The prerequisite for this is in many cases the introduction of chemically reactive groups that allow chemical bonding of the corresponding ligands. The chemical modification referred to as activation depends in principle on the type of functional groups of the matrix and the ligand, but is also determined by the chemical stability of the matrix and the stability of the biological properties of the ligand. The choice of activation method is further characterized by the technological cost of activation and the associated costs of the activation process, the reactivity of the introduced chemical groups, the possibility of storage of the activated carrier, the toxicity and biocompatibility of the modifying reagents and the stability of the chemical bond between Carrier and ligand determined.

Given the large number of parameters and influencing factors to be observed and limited, it is not surprising that a number of activation processes for cellulosic solid surfaces have been described in recent years (overview:

J.F.Kennedy: Adv. Carbohyd. Chem. Biochem.29 [1974] 306), the z. also be used commercially.

By far the most common is the one by Axen et al. Activation by cyanogen bromide (R. Axen, J. Porath, S. Erckback:

Nature 214 [1967] 1302). Although this method undoubtedly led to the widespread use of carrier-fixed compounds in the life sciences and biotechnology, it is with the disadvantages of a relatively unstable chemical bond between the solid surface and ligand - especially at pH <5 and> 10, which does not The release of the bound ligand (leakage) can lead to an ineffectual release, which severely restricts the use, above all, of therapeutic in vivo measures in medicine, the high toxicity of cyanogen bromide, which makes the technology of activation costly, and the risk of linking the carrier molecules with each other (cross-linking) afflicted.

Other frequently used activation methods are activation by means of triazine derivatives (cyanuric chloride) (G. Kay, EMCrook: Nature 216 [1967] 514: H.-D.Hunger, C.Coutelle, J.Lampe, H.Schlechte, W.Schößler R.-M. Leiser, J. Schubert, G.Barchend: EP 134025) and epichlorohydrin (LSandberg, J.Porath: J. Chrom.90 [1974] 87). These compounds are also toxic. In addition, in the use of epoxy or cyanuric activated solid surfaces not infrequently an inactivation of sensitive biological materials can be observed, based on the low reactivity of the epoxy groups and thus long incubation times during the binding of the ligand and on the other attributed to binding to cyanuric chloride liberated hydrogen chloride. A significant disadvantage of cyanuric chloride-activated solid surfaces is the ease of hydrolyzability of such modified supports, which considerably limits practicability.

It has long been known (RA Messing, HH Weetall: US Patent 3,519,538; HH Weetall, NY Elmira: US Patent 3652761; HHWeetall: Methods in Enzymol .44 [1976] 134) that the OH groups on SiO ₂ surfaces, preferably glass, as targets for chemical agents, in particular organosilanes having different functional groups, are used for self wherein the organosilanes act as a bridge between the inorganic support ¹ and the organic compound.

Object of the invention

It is an object of the invention to develop a simple, inexpensive, non-toxic activation process for hydroxyl-containing cellulosic solid surfaces which results in stable activated celluloses to which proteins, nucleic acids, low molecular ligands, cells, microorganisms and other high yielding biological materials can be bound stable and biocompatible.

Explanation of the essence of the invention

The object of the invention is to activate hydroxyl-containing cellulosic solid surfaces with compounds which are usually used for binding organic substances to inorganic substances, wherein a high degree of activation and a stable and biocompatible chemical modification of the cellulose surfaces should be achieved.

According to the invention the object is achieved in that the located on the cellulosic solid surfaces hydroxyl groups with an organosilane of the formula

(XRM _n SIrUn. '

where X is an amino, carbonyl, carboxy, epoxy, isocyano, diazo, isothiocyano, nitroso, sulfhydryl or halocarbonyl group and R 'is an alkyl, alkylphenyl or phenyl group and η 'can be between 0 and 20 and η can be between 1 and 3

 Common organosilanes can be represented by the following formula:

Y _n SiR ₄ -H (H)

where Y is an amino, carbonyl, carboxy or sulfhydryl group and R is an alkoxy, phenoxy and halogen group and η can take the value between 1-3. In practice, widely used and often used organosilanes have the general composition

XCH ₂ CH ₂ CH ₂ -Si (OR) ₃ (IM)

where X is the reactive organic group corresponding to formula I and R is a methyl or ethyl group. The conversion is also carried out with at least two organosilanes of the formula I in a mixture or in succession. Preferred environments for the organosilanes are the liquid or the gaseous phase. Subsequently, if appropriate, a further treatment with homo- or heterobifunctional reagents takes place.

Cellulose-containing solids in the context of this invention have on their surface hydroxyl groups, which are responsible for the hydrophilic properties substantially. Although only a fraction of the OH groups present in the cellulose molecule should be sterically accessible on the surface, it has surprisingly been found that organosilanes which have hitherto been reacted exclusively on inorganic solid surfaces react with at least the same reactivity with cellulosic solid surfaces. While the silicofunctional group reacts with the accessible hydroxyl groups of the solid, the organofunctional group faces the usual chemical reactions using amino, carbonyl, carboxy, isocyano, diazo, isothiocyano, sulfhydryl, nitroso, or halocarbonyl Groups so that these compounds act as a bridge between the cellulose and the organic / biological compound.

The cellulosic solid surfaces activated in this way can now be treated in the following directly via the corresponding functional group or else by mediation of homo- or heterobifunctional reagents in a manner known per se with the proteins, enzymes, lectins, nucleic acids, low molecular ligands, cells, Microorganisms and other biological materials are implemented. This is done, for example, according to the invention so that the hydroxyl-containing cellulosic solid surfaces are reacted with aminopropyltriethoxysilane (X = NH ₂ -; R '= (-CH ₂ ) ₃ ; n' = 1; R = -OC ₂ H ₅ ; η = 1 in formula I). The amino group now on the solid surface can be subsequently reacted with glutaraldehyde or N-succinimidyl 3- (2-pyridyldithio) propionate (SDPD). The binding of the proteins or other ligands is in these cases to an aldehyde group via amino groups - preferably located in the ε-position amino group of the lysine or disulfide bonds. Analogously, the cellulosic solid surfaces can be reacted with glycidoxypropyltriethoxysilane. In this variant, the biological materials to be bound react directly with the epoxy group of the solid surface. The use of mercaptopropyltrimethoxysilane opens up the possibility of reversible binding of biological materials, since the resulting disulfide bond can be reversibly cleaved by the use of suitable reducing agents. The reversible ligand coupling is particularly advantageous in those cases in which the elution of the specifically bound interaction partner carries the risk of denaturation, so that the elution of the protein

Ligand complex is preferable.

It is important that the reaction with the organosilanes, which are non-toxic and are produced industrially to a considerable extent, by simply contacting or dipping, wherein the activation in the swollen

or non-swollen state of the solid can take place or takes place in the gas phase. Of great importance here is that the reaction with organosilanes in the liquid phase with organic solvents such as acetone, toluene, dioxane, methanol, ethanol, etc., solvent mixtures such as methanol / ethanol, and in an aqueous medium or water-solvent mixtures such as ethanol / Water and methanol / water, can be done, so that in contrast to many other activation method of the technological effort is low. The activation in gaseous phase should be particularly advantageous by using aerosols or by negative pressure.

A further advantage is that by choosing different organosilanes and optionally bifunctional coupling reagents virtually all possible reaction possibilities can be realized, since using the underlying principle and inventive solid surfaces with a variety of functional groups (X in formula I and III) can be obtained.

In addition, due to the spacer effect of the organosilanes (R '= 0-20) and / or the bifunctional coupling reagents (eg glutaraldehyde), the bound biological materials retain their biological activity almost without exception. , ,

The use of mixtures of organosilanes of different functional groups is particularly advantageous since they can bind biological materials in high yields via different mechanisms. Thus, mixtures of aminopropyltriethoxysilane and glycidoxypropyltriethoxysilane as well as aminopropyltriethoxysilane and mercaptopropyltrimethoxysilane prove to be particularly suitable because these biological materials can bind by reaction via different functional groups with very high yield. In these cases, the binding of the ligands takes place via aldehyde and epoxyoxy groups or via aldehyde and mercapto groups.

The resulting by reaction of organosilanes with the hydroxyl groups of the cellulose-containing solids bonds are very stable, so that so activated solid can be stored in the dry or wet state for a long time and that bound biologically active ligands stable for long periods even under extreme conditions stay tied. The nonspecific bonds to so-activated hydroxyl-containing cellulosic solid surfaces are extremely low. Organosilanes are characterized by good biocompatibility.

Suitable cellulose-containing solids are all celluloses which have a sufficient number of sterically accessible hydroxyl groups on their surface, the composition of which is not decisive for the activation process according to the invention. The cellulose-containing solids activated in this manner are in the form of shaped articles, and the shaped articles may have a spherical, fibrous or angular shape, which may be joined to column packings, sheet carriers or other higher structures by packing, interweaving or by way of binders. Spherical cellulose-containing solids (eg, perlcellulose), which are used primarily for chromatographic methods and processes, and sheet-like carriers ("activated paper"), which have gained widespread use in analytics and diagnostics, are of paramount importance Of these are also thin layers, which are as films or paints on other solids, which are not involved in the reaction according to the invention are located, wherein the inert for the activation reaction according to the invention support materials may be homogeneous or non-homogeneous and formed or not formed.

The thus activated cellulosic solid surfaces are used for chemical binding of proteins such as antigens, antibodies, enzymes and lectins, nucleic acids, low molecular weight ligands, cells, microorganisms and other biological materials. Such bound to solid surfaces ligands can be used for separation and isolation of biospecific interaction partners by affinity chromatographic methods, as enzyme reactors, for the qualitative and quantitative detection of biologically important compounds in the life sciences, biotechnology, but also medicine

 In the following the invention will be explained with some examples.

Example 1:

1 g of perlcellulose with an exclusion volume of 5000000 D and a particle size of 80-200 / Am and a dry matter content of 64 mg / ml with 5% aminopropyltriethoxysilane (NB 1114, VEB Chemiewerk Nünchritz, DDR) in ethanol / water (1: 1), pH 2.5,6 h incubated at 60 ° C and then washed twice with 5 ml of ethanol / water and five times with 5 ml of 0.1 M phosphate buffer, pH 6.8. Subsequently, the degree of activation is determined by determination of the amino groups on the solid surface according to G. Antoni (Anal. Biochem., 129 [1983] 60). The degree of activation can be varied over a wide range by selecting the concentration of the organosilanes and / or the incubation times. As a rule, one obtains characteristic degrees of activation of 10-15μ.ΜοΙ amino groups per ml of perlcellulose.

Example 2:

5 ml of perlcellulose according to Example 1 is incubated with 10% aminopropyltriethoxysilane in ethanol / water (1: 1), pH 2.5, 6 hours at 60 ⁰ C and then twice with 30 ml of ethanol / water (1: 1) or five times each with 20 ml of 0.1 M phosphate buffer, pH 6.8, washed. The thus activated gel is subsequently reacted with 5% glutaraldehyde for 2 h at 37 ° C. in 0.1 M phosphate buffer, pH 6.8, and subsequently washed again five times with 20 ml phosphate buffer each time. Subsequently, the protein is bound by simply adding the protein solution to the gel activated in this manner. For this purpose, the perl cellulose is mixed with 10 ml of human immunoglobulin G in a concentration of 35.5 mg / ml in a 0.1 M phosphate buffer, pH 6.8, 2 h at room temperature. After sucking off the unbound supernatant, the determination of the bound protein amount is carried out by differential measurement of the protein concentration of the protein solution used and of the supernatant. In this way, 12mg IgG / ml perl cellulose could be bound.

Example 3:

1 ml of perlcellulose is activated as described in Example 2 and reacted with glutaraldehyde in a concentration of 3%. Subsequently, the so-activated carrier is added with ¹²⁵ J-IgG (rabbit, directed against the enzyme alkaline phosphatase) in a concentration of 41.3 mg / ml and incubated for 1 h at room temperature. After aspirating the excess amount of protein and subsequent intensive washing with 5 ml of phosphate buffer, the bound protein amount is determined by measuring the radioactivity with the introduction of the protein solution used as standard. In this experiment 11.4 g of ¹²⁵ μg IgG / ml of perlcellulose could be bound.

Example 4:

5 ml of perlcellulose according to Example 1 is incubated with 5% glycidoxypropyltriethoxysilane (NB 1115, VEB Chemiewerk Nünchritz, DDR) in ethanol / water (1: 1), pH 2.5,6h at 60 ° C and then at 120 ⁰ C for a further 6 h heated. After intensive washing with 20 ml each of Etlianol / water or 0.1 M phosphate buffer as in Example 2, the percellulose thus activated is mixed with 10 ml of human immunoglobulin G in a concentration of 35.5 mg / ml in a 0.1 M phosphate Buffer, pH 6,8,2 h at room temperature. The bound amount of protein, determined as described in Example 2, was 7.2 mg IgG / ml percellulose.

Example 5:

5ml pearl cellulose according to Example 1 with 5% mercaptopropyltrimethoxysilane (Serva, FRG) in ethanol / water (1: 1), 4 h at 60 ^c C and subsequently incubated with in each case 5 ml of ethanol / water and five times with 5 ml 0.1 ml of phosphate twice Buffer, pH 6.8. The cellulose is then treated with 10 mg of human IgG, previously activated with N-succinimidyl-3- (3-pyridyldithio) propionate (SDPD, Pharmacia, Sweden) according to the manufacturer's instructions. 2-thione can be traced photometrically at 343 nm, and this bound IgG can be used to recover and isolate rabbit anti-human IgG antibodies. After affinity chromatography, the disulfide-bonded IgG can be replaced by mercaptoethanol or dithiothreitol (50 mM). be cleaved again, so that the activated matrix for a new binding of a ligand is available.

Example 6:

5 ml of perlcellulose according to Example 1 is brought into contact with a mixture of aminopropyltriethoxysilane and glycidoxypropyltriethoxysilane (adhesion promoter 6130, VEB Chemiewerk Nünchritz, DDR) and shaken for 2 hours at room temperature. After suctioning off the excess reagent and drying in vacuo and subsequent intensive washing with 0.1M phosphate buffer, pH 6.8, the support is reacted with 3% glutaraldehyde and then ¹²⁵ J-IgG, as described in Example 3, bound , It was possible to bind 26.4 mg of ¹²⁵ I-IgG of pearl cellulose.

Example 7:

The perl cellulose reacted according to Examples 3 and 6 with ¹²⁵ I-IgG is washed three times each with 20 ml of PBS buffer, pH 7.4, containing dioxane and subsequently the bound radioactivity is determined. The protein levels remaining on the perlcellulose are unchanged at 11.4 mg / ml (Example 3) and 26.6 mg / ml (Example 6).

Example 8:

The immunoglobulin G used in Examples 3, 6 and 7 was obtained by immunizing a rabbit with the enzyme alkaline phosphatase. Thus, it is possible to use the ¹²⁵ I-IgG using the appropriate antibody activities to purify and isolate the enzyme alkaline phosphatase. For this purpose, alkaline phosphatase pre-purified by n-butanol extraction is bound to the perl cellulose reacted with ¹²⁵ I-IgG in accordance with Example 3 and subsequently eluted with triethylamine, pH 11.4. The thus purified enzyme has a specific activity of 1000 IU / mg, the yield being about 80%. In polyacrylamide electrophoresis, no accompanying proteins are detectable.

Example 9:

Human immunoglobulin G is bound to perl cellulose as described in Example 2 and used for the isolation of rabbit antihuman IgG antibodies. For this purpose, the carrier loaded with the IgG is filled into a chromatography column which is loaded with a specific antiserum against human IgG. The elution is carried out first with PBS buffer and then with a 3 m KSCN solution. Following immediate dialysis of the antibody fractions, detection of specific antibodies was performed with an enzyme immunoassay and double radial immunodiffusion (Ouchterlony technique). The test for non-specific binding is performed by applying a corresponding rabbit normal serum and testing all antibody-containing fractions with polyacrylamide disc electrophoresis.

Example 10:

Planar cellulose supports (Whatman paper 540 and FN 4 paper) are activated with 3% aminopropyltriethoxysilane or glycidoxypropylsilane as described in Examples 2 and 4 and then washed with ¹³¹ J human serum albumin ( ¹³¹ J-HSA) in 0.1 M phosphate. Buffer, pH 6.8, in the concentrations 10 / ig / ml and 100 / tg / m I added. After intensive washing, the amount of bound protein was determined by measuring the radioactivity with a standard. The results obtained are summarized in Tab. 1.

, Table 1

bound ¹³¹ J-HSA

Aminopropyltriethoxysilane Glycidoxypropyltriethoxysilane

used protein conc. 10 ^ g / ml 100 / xg / ml 10, ug / ml 100> g / ml

FN 4 192ng / cm ² 1036ng / cm ² 75ng / cm ² , 343ng / cm ²

Whatman 540 61 ng / cm ² 486ng / cm ² 15ng / cm ² 76ng / cm ²

Example 11: · ..

The paper supports loaded in Example 10 with ¹³¹ J-HSA are used below in the enzyme immunoassay. For this purpose, defined areas of these carriers are added after prior blocking of possibly free reactive groups with ethanolamine with a rabbit anti-human albumin antiserum in a suitable dilution and incubated at room temperature overnight. After washing three times to remove unbound antibody, the paper support with a goat anti-rabbit IgG conjugate are (Enzyme: alkaline phosphatase) 4h at 37 ⁰ C and washed again three times. The enzyme activity is determined by hydrolysis of 4-nitrophenyl phosphate in 0.5 ml diethanolamine buffer, pH 9.8, and photometric measurement of the enzyme product at 405 nm.

Example 12:

Planar cellulose supports (Whatman 540, Filtrak 1389, Filtrak 390, FN 3) are activated as described in Example 6 with a mixture of aminopropyltriethoxysilane and glycidoxypropyltriethoxysilane and then with ¹²⁵ J-IgG (directed against the enzyme alkaline phosphatase) in two concentrations 0.1 M phosphate buffer, pH 6.8, incubated. After washing three times in a PBS buffer, the amount of bound protein is determined by measuring the radioactivity. Thereafter, the turf-like cellulose supports are again washed ten times with PBS buffer and the amount of bound protein, in turn, determined by measuring the radioactivity while carrying a standard. Finally, the cellulose supports are washed several times over a period of four weeks with HCI-glycerol buffer, pH 2.2.3m KSCN, carbonate bicarbonate buffer, pH 10, and PBS buffer, containing 20% dioxane and again for determination used the bound Proteihmenge. The results are shown in Tab.

Whatman 540

Table 2

bound ¹²⁵ J-IgG Filtrak 1389

Filtrak 380

bound protein amount after another 10 washes 10 / μg / ml 50μu.g / ml

1888ng / cm ² 5000ng / cm ²

1939ng / cm ² 5612 ng / cm ²

1786 ng / cm ² 5281 ng / cm ²

FN 3

bound protein amount 2092 ng / cm ² 2143 ng / cm ² 2015 ng / cm ² 2372 ng / cm ² after washing three times: 5536 ng / cm ² 6122 ng / cm ² 5867 ng / cm ² 5967 ng / cm ² ^ G / ml 50 ^ g / ml

2092 ng / cm ² 5459 ng / cm ²

bound protein amount 1913 ng / cm ² 1913 ng / cm ² 1786 ng / cm ² 1964 ng / cm ² after four weeks 5102 ng / cm ² 5612 ng / cm ² 5306 ng / cm ² 5281 ng / cm ² ^ G / ml 50 / ig / ml

Example 13:

A surface support with a paper-like matrix according to the invention is mixed with human immunoglobulin G, which is radioactively labeled with ¹²⁵ J, in different concentration and buffer systems and incubated at 37 ° C. for 16 h. After washing three times with PBS-Puffem, pH 7.4, containing 0.05% Tween 20, any remaining reactive groups are blocked with 1 M ethanolamine solution. After washing again with the above PBS-Puffem the bound protein amount is determined by measuring the radioactivity. To determine possible desorptions, the samples are then washed again seven times with PBS buffer. The so loaded surface carriers are then added to determine the immunoreactivity with a conjugate consisting of a rabbit anti-human IgG and the enzyme alkaline phosphatase and incubated overnight at room temperature. After renewed washing, the enzyme activity of the alkaline phosphatase is determined by hydrolysis of 4-nitrophenyl phosphate in 0.5 ml diethanolamine buffer pH 9.8 and photometric measurement of the enzyme product at 405 nm after stopping with 1 N NaOH or 1 M EDTA. After carrying out the enzyme immunoassay, the surface carriers are washed twice each with 5 ml of PBS buffer and subsequently in multiple determinations with one of the following dissociation reagents for 1 h at 4 ° C.

1) PBS buffer containing 2m NaCl, 5% dioxane, 2) 1m KSCN, 3) 3m KSCN 4) 4.5m MgCl ₂ 5) 1m NaJ 6) HCl glycine buffer pH 2.8. By all these agents, the bound antigen-antibody complexes are cleaved, so that the loaded with human IgG surface carriers can be used again in the enzyme immunoassay.

Example 14:

Surface shaped bodies according to the invention (F = 56 mm ² ) are activated as described in Example 6 and loaded with human factor VIII. These carriers are transferred to polystyrene microtitration plates or polystyrene tubes, followed by the enzyme immunoassay for determination of F VIII antigen (W. Schossler, M.Stepanauskas, Chr.Dittrich, H.Heine: Acta biol.med.germ. 41 [1982] 263, W. Schossler, M.Stepanauskas, Chr.Dittrich: Acta biol.med.germ.41 [1982] 695) in the manner described. For this purpose, the shaped bodies are mixed with an incubation mixture consisting of the plasma to be determined and an excess of rabbit anti-human factor VIII antibody, and incubated at 37 ° C. for 6 hours. After washing again with PBS buffer, pH 7.4, containing 0.05% Tween 20, 200 μΙ of a conjugate consisting of a mutant anti-rabbit IgG and an enzyme alkaline phosphatase are added and after several hours of incubation and subsequently washing the enzyme activity, as described in Example 13, determined. The cleavage of the bound antibodies is carried out with one of the dissociation reagents listed in Example 13, so that the carrier loaded in the factor VIII can be used again in the immunoassay.

Example 15:

A paper-like matrix surface support according to the invention is loaded with human factor VIII as described in Example 14. This carrier is used in the following as a model for screening tests for (monoclonal) antibodies. After carrying out the washing operations, which are particularly simple in this surface support, the substances to be examined and determined are applied in a punctiform manner with a suitable application stamp. In our case, suitable dilutions of a rabbit anti-human factor VIII anti-serum or of a rabbit serum served as negative control as antibody source. After incubation for 4-6 h at 37 ⁰ C of the carrier surface is washed again, and with a sheep anti-rabbit IgG, coupled with the enzyme peroxidase, 4 h at 37 ⁰ C or overnight at room temperature

incubated, washed several times and the enzyme activity with a suitable detection system, such. B. 4mM O-phenylenediamine and 1.5nM H ₂ O ₂ determined by visual evaluation of the resulting color. A positive staining clearly indicates an antibody.

Example 16:

³² P-labeled phosphoproteins contained in cardiac muscle membrane vesicles are detected in the phosphate-buffered sodium dodecylsulfate-polyacrylamide gel electrophoresis system of Weber et al. (K.Weber, J. Pringle, M.Osborn, Meth, Enzymol. 26, [1972] 3). Immediately after completion of the electrophoresis, the separated proteins on the invention activated paper in a triethanolamine and butyric acid-containing transfer system according to Kyhse-Andersen [J.Bioehem.Biophys. Meth. 10 [1984] 203) subjected to immunoblotting. After blotting, bath in phosphate buffered saline containing 0.1% gelatin and 0.05% Tween, then incubate with a first rabbit antiserum incubated with an anti-rabbit immunoglobulin horseradish peroxidase conjugate and finally the indicator action follows. In place of the peroxidase co-conjugate, an alkaline phosphatase conjugate can be used with equal success.

Example 17:

The proteins of intracellular membranes of various types of muscle cells are expressed in a dodecylsulfate-polyacrylamide gel electrophoresis system according to Lamliml. U.K. Laemmli, Nature 227, [1970] 680). After electrophoresis, the gel is bathed for 20-60 min in a buffer solution consisting of 25 mM triethanolamine hydrochloride, pH 8.4.0.1% sodium dodecyl sulfate. This is followed immediately by immunoblotting as described in Example 16.

Claims (3)

1. A process for the activation of hydroxyl-containing cellulosic solid surfaces, characterized in that the hydroxyl groups with an organosilane of the formula (XR ^) _n SiR ₄ -H '. (D where X is an amino, carbonyl, carboxy, epoxy, isocyano, diazo, isothiocyano, nitroso, sulfhydryl or halocarbonyl group, R 'is an alkyl, alkylphenyl or phenyl Group and R are an alkoxy, phenoxy or halogen group, wherein η 'the value between 0-20 and η have the value between 1-3, or that the reaction with at least two organosilanes of the formula I in admixture or in succession takes place in liquid or gaseous phase and that subsequently optionally carried out a further treatment with homo- or heterobifunctional reagents. 2. The method according to item 1, characterized in that the reaction of the hydroxyl groups with compounds of formula I in organic solvents, solvent mixtures, aqueous solutions or mixtures of water and organic solvents. 3. The method according to item 1, characterized in that the reaction of the hydroxyl groups in the gas phase is carried out with an aerosol or at a pressure below atmospheric pressure.