AU634960B2 - Process for immobilizing or depositing molecules or substances on a support - Google Patents

Abstract

In the process disclosed, the support is a structure consisting of at least one layer of molecules containing identical proteins extending along a flat, curved, cylindrical or vesicular surface, the molecules being arranged in the form of a crystal lattice with a lattice constant of 1 to 50 nm.

Description

OPI DATE 16/10/89 AOJP DATE 09/11/89 APPLN. I D 34357 89 PCT NUMBER PCT/AT89/00031 PcI INTERNATIONALE ANMELDUNG VEROFFENTLICHT NACH DEM VERTRAG UBER DIE INTERNATIONALE ZUSAMMENARBEIT AUF DEM GEBIET DES PATENTWESENS (PCT) (51) Internationale Patentklassifikation 4: G01N 33/544, C12N 11/02 G01N 33(553, C12Q 1/68 G01N 33/549, C07K 17/02 A6 IK 9/00 I I(11) Internationale Veriiffentlichungsnummer: WO 89/ 09406 Internationales Veroffentlichungsdatum: 5. Oktober 1989 (05.10.89) (21) Internationales Aktenzelchen: PCT/AT89/0003 I (22) Interuationales Anmeldedatuni: 28. Marz 1989 (28.03.89) (31) Priorititsaktenzeichen: (32) Priorltitsdatum: 174,127 28. M~Irz 1988 (28.03.88)1 SE (europ~isches Patent), US.

Verdffentlicht Mit internationalem Recherchenberich.

6 34i9 6 0 (33) Priorititsland: (71X72) Anmelderund Erfinder: SLEYTR, Uwe, B. [AT/ ATI; Parhamerplatz 10, A-I1170 Wien SARA, Margit [AT/AT]; Vorgartenstr. 90/2/24, A-1200 Wien

(AT).

(74) Anwilte: ITZE, Peter usw,; Amerlingstrasse 8, A- 1061 Wien (AT).

(81) Bestimmungsstaaten: AT (europaisches Patent), AU, BE (europ~isches Patent), CH (europdisches Patent), DE (europ~isches Patent), FR (europdisches Patent), GB (europ~isches Patent), IT (europaisches Patent), J P, LU (europ~isches Patent), NL (europaisches Patent), (54) Title: PROCESS FOR IMMOBILIZING OR DEPOSITING MOLECULES OR SUBSTANCES ON A SUPPORT (54) Bezeichnung: VERFAH-REN ZUR IMMOBILISIERUNG BZW. ABLAGERUNG VON MOLEKCJLEN BZW.

SUBSTANZEN AUF EINEM TRAGER (57) Abstract In the process disclosed, the support is a structure consisting of at least one layer of molecules containing identical proteins extending along a flat, curved, cylindrical or vesicular surface, the molecules being arranged in the form of a crystal lattice with a lattice constant of I to 50 nm.

(57) Zusanitenfassung Verfahren zur Immobilisierung bzw. Ablagerung von Molekfilen bzw. Substanzen auf einemn Trager, wobei als Trdger eine Struktur eingesetzt wird, welche wenigstens eine sich entlang ebener, gekrilmmter, zylindrischer oder vesikuldrer Fl~chen erstreckende Schicht identischer Proteine enthaltender Molek~le besteht, die in Form eines Kristallgitters mit einer Gitterkonstante von I bis 50 nm angeordnet sind.

Process for Immobilizing or Depositing Molecules or Substances on a Support The invention relates to a process for immobilizing or depositing molecules or substances on a support.

Until now polymers with sponge-like structure (gels) of different chemical structure (for example agarose, modified polyacrylamide) have been used for immobilizing macromolecules.

Due to the random arrangement of the polymer chains with different molecular weight in the gel body, functional groups present exhibit neither a defined position nor a defined orientation. The distribution of functional groups on the surface and in the interior of the gel matrix is therefore "accidental". If these groups are activated for immobilizing macromolecules, the arrangement of the foreign molecules will also take place in "accidental" distribution.

For the so-called test-strip technology (for example glucose test sticks used to determine the sugar content of blood or urine) quality papers ar'e impregnated with enzymes in dissolved form and dried. As in the previously described gel the foreign molecules are herein also present in statistical distribution in the relatively coarse fiber matrix of the paper. A further disadvantage is that the enzymes are not bound covalently so that during examinations in an aqueous environment enzyme losses can occur. Thereby quantifiability of the analyzing substance is no longer ensured.

To ensure, on the one hand, a covalent bonding of the foreign molecules, but to have on the other hand a thin support 2 film, protein films of serum albumin or collagen were created. By introducing glutaraldehyde cross-linkage of the protein film was accomplished as well as the simultaneous covalent bonding of foreign molecules. But with this method too it could not be achieved to bind foreign molecules in defined position and orientation in a monomolecular layer convalently to a support film.

According to a first embodiment, the present invention consists in a process for immobilizing or depositing molecules or substances on a support, characterized in bringing the molecules or substances in contact with a support being a structure having at least one membrane extending along flat, curved, cylindrical or vesicular surfaces comprising at least one layer of identical molecules containing proteins which are arranged in the form of a crystal lattice with a lattice constant of 1 to 50 nm.

According to a second embodiment, the present invention consists in a support having at least one membrane extending a long flat, curved, cylindrical or vesicular surfaces comprising at least one layer of identical molecules containing proteins which are arranged in the form of a crystal lattice with a lattice constant of 1 to 50 nm.

According to the invention a structure is employed as support which has at least one membrane extending along flat curved, cylindrical or vesicular surfaces comprising at least on layer of identical molecules 2a containing proteins arranged in the form of a crystal lattice with a lattice constant of 1 to 50 nm. It is thereby achieved that the immobilized molecules are always disposed in a spatially defined configuration on the surface connected to the support so that a coating of maximum density of the free surfaces with the molecules to be bound is possible. For the reactive groups of the proteins do not only assume a precisely defined position in the polypeptide chain but rather after the folding of the proteins in the crystal lattice also have a precisely defined position and orientation. This applies in the same manner also for carbohydrate fractions which are potentially still present on the membrane and their hydroxyl groups.

Advantageously a membrane can be used having pore sizes of 0.5 to 40 nm. Due to the presence of pores a treatment of the substrate can also be achieved thereby that this is slowly passed through the membrane wherein during the passing through or along the desired conversion or reaction through the molecules immobilized at the membrane takes place. Furthermore it 3 achieved that the membrane can be charged on both sides with the molecules or substances since due to the pores the substrate to be treated can reach both sides of the membrane and can subsequently enter into a reaction.

For changing the surface properties of the membrane, protein and/or peptide molecules can be immobilized on it. Thereby inter alia a surface enlargement is achieved whereby additional space is created for immobilizing further molecules which due to the defined arrangement of the proteins and/or peptide molecules likewise has precisely defined spatial structure. Thereby it can be achieved further that the surface becomes either hydrophilic or hydrophobic. Through such changes of the surface properties is also achieved inter alia that the membranes have a low nonspecific adsorption. Idential properties can also be achieved in that on the membrane glycoprotein and/or glycopeptide molecules are immobilized. Likewise, on the membrane polysaccharides and/or oligosaccharides and/or sugars can be immobilized wherein all the listed molecules can be used depending on the subsequent application purpose.

However, for achieving these properties lipid molecules can also be immobilized wherein these molecules additionally can serve for the application of monomolecular lipid layers, for example Langmuir-Blodget films or as support for other polymer layers, in particular hydrophobic membranes. For specific purposes also can be immobilized on the membrane lipopolysaccharides which also can bring about the stated effects.

For use in enzymatic conversion on the membrane enzymes and/or coenzymes can also be immobilized. If the membrane according to the invention is to be used for biotechnological purposes then on the membrane can also be immobilized a substance of the group containing antigens, antibodies, lectins, biotin, avidin, protein A and haptenes. For particular purposes nucleic acids can also be immobilized which are suitable inter alia for hybridization tests and sounding molecules in gene technology.

If on the membrane dyes are immobilized in particular fluorescent dyes, then the membrane according to the invention can act as test membrane for example as indicator membrane or as a membrane which is suitable in particular for optoelectronic evaluation (optic sensors). For the evaluation membrane groups can be used of which every membrane has a different dye so that different reactions can be measured simultaneously.

It is possible to immobilize or deposit on the membrane if necessary conducting materials such as metals and/or metal compounds and/or carbon and/or silicon oxide and/or synthetic materials. Thereby the possibility is given to use the membranes as sensors and specifically for example as biological field effect transistors or chemical field effect transistors since the created structures are ion-selective and hence can be used as socalled ion-selective field effect transistors (ISFET). For the control of the structure or the physical properties and manufacture of alloys on the membrane mixtures of the materials or separate layers of several materials can be deposited.

To achieve a controlled electron transport or ion derivation from the medium, mediator or transmitter molecules can be immobilized on the membrane. To achieve ready recovery of the used enzymes in enzyme-controlled processes a membrane with enzymes and/or coenzymes immobilized thereon can be used as an enzyme membrane. Subsequently the substrate is brought into contact with the enzyme membrane or if the membrane is vesicular these vesicles are suspended in the substrate whereupon then after completion of the reaction the substrate is separated from the membrane or the vesicle, for example through filtration.

In particularly preferred manner a membrane can be used as ELISA membranes with antigens, antibodies, lectins, biotin, avidin, protein A or haptenes immobilized thereon. For carrying out diagnostic reactions membranes can be used having immobilized thereon enzymes or coenzymes, antigens, antibodies, biotin, avidin, lectins, protein A, haptenes, nucleic acids or mediators or transmitter molecules as diagnostic reagent. In particular with enzymes a bioluminescence reaction is of interest, because thereby a light-optical evaluation of reactions is made possible.

A membrane with enzymes or coenzymes, antigens, antibodies, biotin, avidin, lectins, protein A, haptenes, nucleic acids or mediator or transmitter molecules immobilized thereon can also be used as a sensor membrane which allows a direct application for the testing of substances on present components. For particular forms of signal derivations, in particular for oxidationreduction systems, the substances immobilized on the membrane and the membrane itself can be provided with a conducting layer, for example metal or conducting synthetic material. In particularly simple manner the conducting layers can be applied by sputtering.

For particular materials it can however be advisable that the conducting layer is applied by vapor deposition. Lastly the synthetic layer can be applied by plasma-polymerization.

For a controlled freeing of the deposited or immobilized substances, in particular with pharmaceutical agents, these 6 substances can be enclosed by the membrane.

The invention will be explained below in greater detail in conjunction with examples.

Example 1: Immobilization of poly-L-lysine on crystalline protein membranes (S layers) Vesicular structures consisting of crystalline protein membranes of clostridium thermohydrosulfuricum Lll-09 are crosslinked with glutaraldehyde for stabilization. To this end 0.5 g of moist pellet of the vesicular structures (obtained through centrifugation at 20,000 x g) suspended in 50 ml 0.1 M sodium cacodylate buffer, pH 7.2, and after the addition of 0.5 ml glutaraldehyde incubated for 20 minutes at 200C. After the reaction is stopped through the addition of ethanolamine the suspension is centrifuged at 20.000 x g, washed three times with distilled water and again pelletized. For the chemical activation of the carboxyl groups 0.2 g of the obtained pellets are suspended in 8 ml of distilled water, 60 mg of l-ethyl-3,3' dimethyl(aminopropyl)carbadiimide (EDC) are added, the pH-value of the suspension is adjusted to 4.63 by adding 0.1 N NaOH or 0.1 N HCL and held constant during the reaction lasting 80 minutes.

After centrifugation at 20,000 x g the pellet is washed with icecold distilled water and after repeated centrifugation at 20,000 x g suspended in a solution of poly-L-lysine (MG 30,000, 2 mg/ml distilled water). After 4 hours of incubation at centrifugation takes place to remove the poly-L-lysine not covalently bound, distillation with distilled water takes place, and subsequently washing with 0.1 M potassium phosphate buffer (pH 7 Through the immobilization of poly-L-lysine due to the free amino groups of the lysine residue present in the polymer the surface properties of the vesicular structures (protein fragments) can be specifically changed and a positive net charge can be generated. Furthermore, the present amino groups can be activated for an immobilization of further foreign molecules.

Example 2: Immobilization of phosphatidylethanolamine Vesicular structure as described above are for chemical stabilization cross-linked with glutaraldehyde and activated carboxyl groups present at the crystalline matrix are activated with EDC. After an activation time of 80 minutes the suspension is centrifuged at 20,000 x g, the pellets are washed with dioxan at a temperature of 46 C and again allowed to settle.

Subsequently the pellets are dissolved in a solution of phosphatidylethanolamine (PE; 3 mg/ml dioxan) and incubated for hours at 200C. To remove the PE not covalently bound the vesicular structures are washed five times with dioxan after centrifugation and subsequently washed with a mixture of chloroform-methanol (50/50). Through the reaction of the free amino groups of PE with the carboxyl groups of the S layer protein it is accomplished to bind the PE so to the crystalline matrix that the hydrophilic portion is oriented to the membrane surface while the hydrophobic residue remain exposed toward the outside. The so created hydrophobic surface function for the deposition of monolayers of tensides (for example arachinic acid) which now in turn bind with their hydrophobic portion while the hydrophilic portion is exposed toward the outside. Now a further 8 deposition of a monolayer of arachinic acid is possible wherein now the molecules bind with their hydrophilic side. According this principle several monolayers of tenside molecules can be deposited on the surface of a crystalline-configured vesicular structure.

Example 3: Immobilization of glucose oxidase on crystalline vesicular structures For immobilizing glucose oxidase vesicular structures as described aboce are. cross-linked with glutaraldehyde, and subsequently carboxyl groups present in the crystal lattice activated with EDC. After washing with ice water and renewed centrifugation at 20,000 x g the activated pellet from vesicular structures suspended in a solution of glucose oxidase (3 mg/ml distilled water) and incubated for 6 hours at 200 C. The suspension is subsequently centrifuged and, as described in Example 1, washed to remove the material that is not covalently bound. Using this method it is possible to bind covalently 1 mg of glucose oxidase per mg of S-layer protein. This means that per unit of S layer one glucose oxidase molecule can be immobilized which corresponds to the densest possible packing of a foreign molecule in a crystalline matrix. Immobilized glucose oxidase in comparison to the native enzyme shows an activity of t 1 0 j i 4I 9 Example 4: Coating of crystalline S layers with a metal layer Membranes (S layers) with a crystalline arrangement are suspended in distilled water and under a pressure of 2 bars in a 10 ml ultrafiltration cell (microfiltration membrane of polycarbonate of the firm Nuclepore: pore size 0.1 pm) deposited on a microporous support. The individual membrane fragments are subsequently cross-linked with glutaraldehyde in 0.1 M sodium cacodylate buffer, pH After an incubation time of 20 minutes excess reagent is removed by washing with distilled water, the microfiltration membrane with the deposited protein fragments removed from the ultrafiltration cell and dried in air. Subsequently the structure is placed into a Sputter Coater installation (Polaron Instruments) and evacuated to a vacuum of 5 x 10- 2 Torr. By applying a voltage of 2000 V at a current flow of approximately 20 mA gold is now sputtered onto the surface of the crystalline membrane fragments, whereby a continuous metal layer with a thickness of approximately 40 nm is generated. After removal from the Sputter Coater installation the structure is now carefully trensferred into a beaker filled with chloroform and so placed that the polycarbonate support membrane comes directly in contact with the liquid, whereby the polymer present is dissolved. Now an extremely thin structure remains on the liquid surface comprising the gold layer and the inferior S-layer fragments. This structure can now be applied to any surfaces by careful lifting. Through sputtering of gold on a Smembrane fragment having crystalline configuration it is possible to achieve a direct contact between metal and the support.

Example Immobilization ot fluorescent dyes on structures having crystalline configuration Vesicular structures consisting of protein subunits in a crystalline configuration are used for the covalent binding of the fluorescent dyes 27?-hydroxy-cumarin-3-carboxylic acid (HCC) representing a pH-sensitive dye. 50 mg of the dye are dissolved in distilled water, the pH-value adjusted to 4.75 through the addition of 0.1 N HC1 or 0.1 N NaOH and subsequently 20 ing ot EDC are added. After an activation of the carboxyl groups of HCC during a reaction time of 80 minutes this preparation is mixed with 0.5 g of moist pellet of vesicular structures (obtained after centrifugation at 20,000 x g) and the suspension is incubated for 2 hours t 200 C. During this time the EDCactivated carboxyl groups of HCC can react with Eree amino groups of the S-layer proteins and the dye can be covalently bound to the crystalline matrix. To remove excess reagent the suspension is transferred to a presoftened dialysis tube and dialyzed for 48 hours at a temperature of 40 C against distilled water. Lowmolecular compounds such as HCC or EDC can be removed duri) the dialysis. The suspension is subsequently centrifuged, washed with distilled water and I N NaCl to remove non-specifically adsorbed material. The fluorescent dye immobilized at the vesicular structures reacts to changes of the pH-value in the appropriate environment by a change in the wave length of the emitted fluorescence, o0 Example 6: Immobilization of glucose oxidase on crystalline protein fragments and coating with a metal layer The immobilization of glucose oxidase takes place at protein fragments with crystalline configuration which were deposited on a microporous support (microfiltration membrane of nylon of the firm Pall; pore diameter 0.1 m The microfiltration membrane is placed into an ultrafiltration cell with a diameter of 25 mm, and 3 ml of a suspension containing the protein fragments are placed with a pipette onto the membrane surface.

After applying a pressure of 2 bars and deposition of the protein fragments on the porous support r3mbrane protein present is covalently cross-linked through the addition of glutaraldehyde (0.5 in 0.1 M sodium cacodylate buffer, pH After washing the membrane with distilled water it is removed from the ultrafiltration cell an placed into a beaker containing 10 ml of distilled water. After adding 50 mg EDC to activate carboxyl groups the pH-value is kept constant at 4.65 during the following 80 minutes. Through the immersion of the membrane in ice water excess reagent can be removed. The membrane is now placed in the ultrafiltration cell so that the side coated with protein fragments is directed upward.

Subsequently 2 ml of a glucose oxidase solution (2 mg/ml) are added and incubated for 2 hours at 200 C for the covalent binding of the enzyme. Enzyme which is not covalently bound can be removed by washing with distilled water and buffer (see above).

'After the removal of the membrane from the ultrafiltration cell it is dried in air, placed in a Sputter Coater installation (Polaron Instruments) and at a pressure of 5 x 10 2 Torr sputtered with platinum at a current strength of 20 mA during a length of time of 2 minutes. The metal layer which has a thickness of 30 nm is directly in contact with the enzyme bound in densest possible packing to a crystalline matrix. Thereby it is possible that the electrons originating on the enzyme during the enzymatic reaction are directly drawn off from the platinum layer.

Example 7: Fabrication of diagnostic membranes using antibodies As starting material protein fragments bound to microporous supports are used. Carboxyl groups present on the surface of the protein fragments are activated with EDC (method, see above examples). Subsequently the activated membrane is placed into an ultrafiltration cell with a diameter of 25 mm and 2 ml of an antibody solution (0.4 mg/ml) (antibody A generated against virus protein placed with a pipette onto the membrane surface and incubated for 4 hours at 200 C. After decanting the antibody solution the membrane is removed from the ultrafiltration cell and for 2 hours washed with 0.2 M potassium phosphate buffer, pH 7.2. The antibody molecules were bound in the process of the immobilization in the form of a monolayer in the densest possible packing to the surface bf the crystalline matrix. The membrane charged with antibodies can therefore be used for demonstrating the presence of virus protein. To this end the membrane is brought into contact with a liquid containing virus and incubated for 2 hours at 200 C. Subsequently for removing excess antigen washing with 0.2 M phosphate buffer, pH 7.0 and with distilled water. For the quantitative evaluation of the amount of bound virus protein the membrane is now placed into a further solution containing biotinylated antibody A (0.1 mg/ml) (0.1 N sodium hydrogen carbonate) and incubated for 30 minutes.

The biotinylated antibody now forms a bond with the still free haptene of the virus protein. After removing the membrane from the antibody solution this is again washed for 20 minutes with 0.1 M phosphate buffer, pH 8.0. For the quantitative evaluation now a peroxidase avidin conjugate is used. Avidin characteristically forms a bond with biotin, and consequently with the particular biotinylated antibodies. After an incubation time of 10 minutes the membrane is removed, washed again for minutes with phosphate buffer and lastly 1 ml of a 0.01 hydrogen peroxide solution containing 1 mg of the dye odianisidine placed dropwise on the membrane. After 10 minutes the reaction is stopped by the addition of 5 N HC1 and the dye formed determined quantitatively by measuring at a wave length of 400 nm. Through an appropriately established calibration curve the quantity of the bound virus protein can be measured.

Protein fragments with crystalline configuration offer the advantage that the antibody A can be bound in the densest possible packing to a defined surface and that thereby the detection limit for the protein to be tested is correspondingly lower.

Example 8: Immobilization of biotinylated proteins on vesicular structures Vesicular structures consisting of protein crystals are chemically cross-linked according to the above listed specification and the carboxyl groups present on the surface are 14 activated with EDC. 0.1 g of the moist EDC-activated pellet-are mixed with 2 ml of a solution of biotinylated ovalbumin (1 mg/ml of distilled water) and incubated for 2 hours at 20 0 C. Subsequently centrifugation at 20,000 x g takes place and the pellet is washed five times with 0.1 M potassium phosphate buffer, pH 7.0. 2 molecules of biotinylated ovalbumin could be bound per protein subunit of the crystalline matrix, which again corresponds to the densest possible binding density of a foreign molecule to a crystalline matrix. Immobilized biotinylated ovalbumin can now be detected by adding avidin-peroxidase conjugate and by measuring subsequently the formed dye through peroxidase activity. To this end 50 mg of the moist pellet of vesicular structures are suspended in 1 ml of 0.1 M sodium hydrogen carbonate, pH 8.5 and 200 1p of 0.2% avidin-peroxidase conjugate added. After an incubation time of 20 minutes at 20°C centrifugation takes place, the pellet is washed five times with 0.2 M potassium phosphate buffer (pH 7.0) and 20 mg of the pellet (consisting of vesicular structures, biotinylated ovalbumin and avidin-peroxidase conjugate) mixed with 200 pl of a solution of 0.01% hydrogen peroxide containing 1 mg 0-dianisidine. The developed dye is measured as described above.

Example 9: Immobilization of poly-L-lysine on crystalline protein membranes used as vesicular structures Whole cells from Clostridium thermohydrosulfuricum 4) 15 L110-69 are broken by ultrasonication (Branson Ultrasonics) for 3 min at maximum output. During this procedure the suspension is kept at 4 0 C. After centrifugation at 20,000 x g at 4 0 C for 20 min the supernatant is discarded, the pellet resuspended in 30 ml mM K-phosphate buffer, pH 7.2, and again centrifuged at 20,000 x g (conditions as described before). This procedure is repeated for two times. For removing plasm membrane residues the pellet consisting of cell wall fragments is then treated with the mild detergent TRITON-X-100 in 50 mM phosphate buffer, pH 7.0) for min at 20 0 C. Then the suspension is centrifuged at 20,000 x g. For removing residual detergent the pellet is wahed for three times with phosphate buffer. The obtained material consisting of an outer S-layer, an inner S-layer and the peptidoglycan between them is called "vesicular structures" or "vesicles" in the following. 0.5 g of the moist pellet of the vesicles are suspended in 50 ml 0.1 M sodium cacodylate buffer, pH 7.2, and after the addition of 0.5 ml glutaraldehyde incubated for 20 min at 20 0 C. Then the reaction is stopped through the addition of 500 mg ethanolamine, the suspension is centrifuged at 20,000 x g, washed three times with distilled water and again pelletized. For the chemical activation of the carboxyl groups 0.2 g of the obtained pellets are suspended in 8 ml of distilled water, 60 mg l-ethyl-3,3-dimethyl (aminopropyl) carbodiimide (EDC) are added, the pH value of the 16 suspension is adjusted to 4.63 by adding 0.1 N NaOH or 0.1 N HC1 and held constant during the reaction lasting min. After centrifugation at 20,000 x g the pellet is washed with ice-cold distilled water and after repeated centrifugation at 20,000 x g resuspended in a solution of poly-L-lysine (MG 35,000; 2mg/ml distilled water). After 4 h of incubation at 20 0 C centrifugation takes place to remove the poly-L-lysine not covalently bound.

Subsequently, the pellet is washed with 0.1 M phosphate buffer, pH 7.2, for at least five times. Through the immobilization of poly-L-lysine due to the free amino groups of the lysine residue present at the polymer the surface properties of the vesicular structures can be specifically changed and a positive net charge can be generated. This is confirmed by determining the free amino groups before and after covalent attachment of poly-L-lysine. Each P-membrane subunit on the vesicular structure reveals still 30 free amino groups after crosslinking with glutaraldehyde which is increased to 52 free amino groups after poly-L-lysine binding.

Furthermore, the present amino groups can be activated for immobilization of further foreign protein molecules.

Example 10: Immobilization of phosphatidylethanolamine Vesicular structures from Bacillus polvmyxa CCM1459 are prepared as described in example 9. After crosslinking the P-membrane protein with glutaraldehyde the free carboxyl groups are activated with EDC as described before. After this the suspension is centrifuged at 17 20,000-x g. Then the pellets are washed with dioxane at 4 C and again allowed to settle. Subsequently the pellets are dissolved in a solution of phosphatidylethanolamine (PE; 3 mg/ml dioxane) and incubated for 20 h at 20 0 C. To remove the PE not covalently bound the vesicles are washed for five times with dioxane after centrifugation and subsequently washed with a mixture of chloroforme-methanol (50/50). Through the reaction of the free amino groups of PE with the carboxyl groups of the P-membrane protein it is accomplished to bind the PE so to the crystalline matrix that the hydrophilic portion is oriented to the membrane surface while the hydrophobic residue remain exposed toward the outside. By this procedure a hydrophobic surface is generated. In the following the vesicles can be suspended in a solution of arachinic acid which is dissolved in chloroform-methanol (50/50). Arachinic acid has a hydrophilic and a hydrophobic part. 0.2 g vesicles (wet pellet as obtained by centrifugation at 20,000 x g) are resuspended in 5 ml of arachinic acid solution and stirred for 1 h at 20 0 C. Since the vesicles reveal a hydrophobic surface now the first monolayer of arachinic acid will bind with its hydrophobic end due to hydrophobic interactions. Now the hydrophilic end is exposed which enables binding of a second monolayer of arachinic acid through its hydrophilic end. According to this principle several monolayers of tenside molecules can be deposited on the surface of the P-membrane after 18 covalent attachment of PE.

Example 11: Immobilization of glucose oxidase on self-assembly products from P-membranes from Bacillus sphaericus CCM 2120 Vesicular structures from Bacillus sphaericus CCM2120 DSM 28 are prepared as described in example 9. After this preparation procedure 0.5 g of the wet pellet is suspended in 5 ml guanidine hydrochloride (GHC1; 5 M in Aqua dest.) for 2 h at 20 0 C. In order to remove the extracted P-membrane subunits from the insoluble cell wall material the suspension is centrifuged at 40,000 x g for 20 min at 20 0 C. The clear supernatant containing the extracted P-membrane subunits is dialysed against a CaC1 solution (10 mM in Aqua dest.) for 48 h at 2 4 0 C. During this procedure the subunits reassemble into double-layer self-assembly products with a size of up to um. The double layer are arranged in this way that the outer face of the P-membrane is exposed to the external environment. (The inner P-membrane faces are oriented to each other). The self-assembly products are recovered by centrifugation for 10 min at 10,000 x g, resuspended in 2 ml distilled water and the protein content is determined. Then the suspension is diluted to a protein content of 2 mg/ml distilled water. 20 mg EDC are added to 1 ml self-assembly suspension which leads to an intermolecular crosslinking of the P-membrane subunits by reaction of activated carboxyl groups with free amino groups on adjacent subunits. Carboxyl groups which do Mo 19 not react with adjacent amino groups remain in the activated state. After 80 min the suspension is centrifuged at 10,000 x g, washed with ice-cold distilled water and subsequently incubated with a glucose oxidase solution (3mg/ml distilled water) for 6 h at 20 0 C. The suspension is centrifuged and as described in example 1- washed for at least three times with 0.1 M phosphate buffer, pH 7.2, to remove non covalently bound enzyme.

Using this method it is possible to bind 1 mg glucose oxidase per mg P-membrane protein. This means that one glucose oxidase molecule is covalently bound per P-membrane subunit. Since the molecular weight of the P-membrane subunits is 140,000 and the molecular weight of GOD is 150,000 a monolayer of GOD is formed on the crystalline immobilization matrix. For determining the retained enzymatic activity 1 mg of lyophilized vesicles corresponding to 350 ug immobilized GOD are resuspended in 1 ml 0.1 M phosphate buffer, pH 7.0, and the enzymatic activity of the immobilized GOD was compared with that of free enzyme. Since 350 ug GOD corresponded to an enzymatic activity obtained with 175 ug free GOD, the retained activity is 50%. The advantage of P-membrares as immobilization matrix is the formation of an enzyme monolayer on the outermost surface which enables rapid diffusion of the substrate to the site of enzymatic reaction.

Example 12: Coating P-membranes with a metal layer Self-assembly products obtained from P-membranes from 20 Bacillus sphaericus CCM2120 are prepared as described in example 11. After dialysis the protein content of the suspension is determined and the suspension diluted to a protein content of 1 mg/ml. 100 ul of this suspension are transferred to 50 ml Ca C12 solution (10 mM in Aqua dest.). 10 ml of this suspension corresponding to a protein amount of 20 ug are used for the production of P-membrane ultrafiltration membranes. For this purpose a microfiltration membrane (Nuclepore, pore size 100 nm) with a diameter of 62 mm is inserted into an ultrafiltration cell. 10 ml of the self-assembly suspension are transferred into the ultrafiltration cell. By applying a pressure of 2 bar the self-assembly products are deposited on the surface of the microfiltration membrane. After releaving the pressure ml of a glutaraldehyde solution in 0.1 M sodium cacodylate buffer, pH 7.0) are transferred into the ultrafiltration cell. By applying again a pressure of 2 bar the glutaraldehyde solution is forced through the self-assembly products and the microfiltration membrane leading to a crosslinking of adjacent P-membrane subunits and adjacent self-assembly products. After obtaining ml filtrate the pressure is releaved again. The remaining 5 ml glutaraldehyde solution completely crosslink the P-membrane protein. After 20 min the glutaraldehyde solution is removed, and the P-membrane ultrafiltration membrane extensively washed with distilled water. The P-membrane ultrafiltration membrane 21 is subsequently used for the immobilization of alcohol oxidase which is followed by metal deposit. For activation of free carboxyl groups in the P-membrane protein 60 mg EDC are dissolved in 10 ml distilled water. The EDC-solution is transferred into the ultrafiltration cell. After 80 min the EDC-solution is discarded, the membrane surface rapidly washed with ice-cold distilled water and incubated with 5 ml alcohol oxidase solution (2 mg/ml distilled water) for 4 h at 20 0 C. Then the enzyme solution is discarded, the P-membrane ultrafiltration membrane removed from the ultrafiltration cell and washed with 0.2 M K-phosphate buffer, pH 7.2, for at least 3 h. Subsequently, the enzyme-loaded membrane is dried in a vacuum exsiccator, placed in a Sputter Coater installation (Polaron Instruments) and evacuated to 5 x 10-2 Torr. By applying a voltage of 2 000 V at a current flow of 20 mA gold is now sputtered onto the surface of the alcohol oxidase immobilized on P-membrane fragments. After 60 s a closed metal layer with a thickness of 10 nm is obtained. The obtained structure is now used as the biological component of a biosensor device. The electrons which are released during the oxidation of ethanol are directly transferred from the alcohol oxidase to the gold layer which is in contact with the electrode.

Example 13: Immobilization of a fluorescent dye on P-membranes Self-assembly products from P-membranes Clostridium

V

7 7 7 w 0' 22 thermosaccharolvticum D120-69 are prepared as described in example 11. The self-assembly products represent monolayer sheets which have a size of appr. 10 um. The self-assembly suspension obtained after dialysis is diluted to a protein content of 2 mg/ml. 1 ml 0.25 M triethanolamine HC1 buffer, pH 8.4, is added per ml self-assembly suspension. For introducing covalent bonds between adjacent subunits 5 mg dimethyladipimidate are dissolved per ml. The mixture is allowed to react for 3 h at 37 0 C. In the following the reaction is stopped by addition of 10 mg ethanolamine and the self-assembly products recovered by centrifugation at 10,000 x g for min. Dimethyladipimiadte introduces covalent bonds between free amino groups showing a distance of 0.9 nm.

This procedure leads to a complete crosslinking of the S-layer lattice, but still leaves approximately 20 free amino groups per P-membrane subunit which corresponds to crosslinking. In the following the free amino groups are used for the immobilization of the fluorescent dye 7-hydroxycumarin-3-carboxylic acid (HCC) which is pH sensitive. For this purpose the pellet is resuspended in ml distilled water, 2 mg of HCC and 10 mg EDC are added, the pH adjusted to 4.63, an the reaction allowed to proceed for 3 h at 20 0 C. EDC activates the carboxyl groups from HCC which can then be attached to the free amino groups in the P-membrane protein. The reaction is stopped by addition of 10 mg sodium acetate. After centrifugation at 20,000 x g, the pellet consisting of 23 the se-f-assembly products is carefully resuspended in distilled water and washed for at least five times with distilled water. The so treated self-assembly products can be used for coating fiber glass which is the light transmittor for optical sensors. The fluorescent dye reacts to changes of the pH value in the appropriate environment by a change in the wave length of the emitted fluorescence.

Example 14: Immobilization of biotinylated invertase on P-membrane ultrafiltration membranes Vesicular structures from Bacillus alvei CCM 1463 are produced as described in example 1. 0.13 g wet pellet (obtained by centrifugation at 20,000 x g for 20 min at 4 0 C) are suspended in 50 ml distilled water. 6 ml of this suspension are used for the production of P-membrane ultrafiltration membranes which was done according to the procedure described in example 12. The P-membrane protein from B. alvei CCM 1463 is glycosylated. The carbohydrate chain contains mannose which has vicinal hydroxyl groups which further can be activated with cyanogen bromide. For this purpose the membranes were removed from the ultrafiltration cell, washed with 30 ml 0.1 M sodium bicarbonate, pH 8.4, and incubated in 20 ml of the same buffer. In the following 10 mg caynogen bromide is added which activates vicinal hydroxyl groups. After 8 min the membrane is removed from the reaction mixture and washed with ice-cold distilled water for at least three times. Then the membrane is inserted j 0 24 into a:reaction chamber in this way that only the face covered with vesicular structures is accessible. 5 ml of a solution containing 1 mg biotinylated ovalbumin per ml distilled water are transferred on the surface of the membrane and incubated for 2 h at 200C. During this time free amino groups from biotinylated invertase can react with cyanogen bromide activated hydroxyl groups.

Subsequently, the protein solution is discarded, the membrane removed from the chamber and washed with 0.2 M phosphate buffer, pH 7.5 for 20 min for removing loosely attached invertase molecules. Since invertase has a molecular weight of 200,000 the molecule is too large to enter the pore structure which allow free passage for molecules with molecular weights of only up to 30,000.

Covalent attachment is therefore restricted to the protein domains of the P-membranes. By applying this immobilization procedure 750 ug invertase can be bound per mg P-membrane protein. This corresponds to one invertase molecule per 2.5 P-membrane subunits. Since the molecular weight of the P-membrane subunits is 115,000, a monolayer of invertase molecules is generated. Immobilized biotinylated invertase can now be detected by adding an avidin-peroxidase conjugate and by measuring subsequently the formed dye through peroxidase activity. To this end the membrane is transferred in a beaker containing 10 ml of 0.1 sodium hydrogen carbonate, pH 8.5 and 200 ug of avidin peroxidase conjugate. After an incubation time of 20 min at 200C the membrane is 25 removed, washed for at least five times with 0.2 M phosphate buffer, pH 7.5 and transferred to a beaker containing 10 ml of a 0.1% hydrogen peroxide solution in which 6 mg o-dianisidine is dissolved. Peroxidase reacts with hydrogen peroxide and oxidizes o-dianisidine which gives a yellow-orange colour. After 3 min the reaction is stopped by addition of 1 ml HC1 and measured at 400 nm. By comparison with free avidin-peroxidase conjugate 340 ug could be bound per mg S-layer protein.

This means that one avidin-peroxidase molecule was bound per each biotinylated invertase molecule.

Example 15: Application of P-membrane ultrafiltration membranes in an immuno flow assay P-membrane ultrafiltration membranes from Clostridium thermohydrosulfuricum L110-69 are produced as described in example 11. Membranes with an diameter of 25 mm are punched and inserted into an ultrafiltration cell. Free carboxyl groups on the P-membrane subunits are activated with EDC as described before. After 80 min the P-membrane ultrafiltration membrane is washed with ice-cold distilled water for three times. 3 ml of a solution containing 2 mg humane serum albumin (HSA) per ml distilled water are transferred into the ultrafiltration cell and incubated for 3 h at 20 0

C.

After this the protein solution is pressed through the membrane by applying a pressure of 2 bar. Subsequently, the membrane is washed for 30 min with 0.2 M K-phosphate buffer, pH 7.0 for removing loosely adsorbed HSA.

A -N 26 Studies with P-membrane self-assembly products have shown that 500 ug HSA can be immobilized per mg P-membrane protein which corresponds to one HSA molecule per P-membrane subunit. 1 ml of anti-HSA IGg (from rabbit) containing 50 ug IgG per ml 50 mM phosphate buffer, pH is filled into the ultrafiltration cell. By applying again a pressure of 2 bar the buffer is forced through the membrane. IgG which is too large for passing through the pores and specifically directed to HSA will bind to the immobilized HSA molecules. By washing the membrane surface with buffer for at least 30 min all IgG which did not bind to the imobilized HSA molecules are removed. Then a further conjugate which is HSA-peroxidase is brought in contact with the ant-HSA IgG already bound to the immobilized HSA molecules. For this purpose 1 ml of a solution containing 100 ug of the HSA-peroxidase conjugate is forced through the P-membrane ultrafiltration membrane by applying a pressure of 2 bar. Subsequently the membrane surface is washed with phosphate buffer for removing excess conjugate. Finally, 1 ml 0.01% HO which contains 5 mg o-dianisidine 22 is brought on the membrane surface. After 2 min the reaction is stopped by addition of 50 ul 32% HC1 and the absorbance determined at 400 nm in a spectrophotometer.

Claims (21)

1. A process for immobilizing or depositing molecules or substances on a support, characterized in bringing the molecules or substances in contact with a support being a structure having at least one membrane extending along flat, curved, cylindrical or vesicular surfaces comprising at least one layer of identical molecules containing proteins which are arranged in the form of a crystal lattice with a lattice constant of 1 to 50 nm.

2. A process as stated in Claim 1, characterized in that a membrane is used having pores with a diameter of to 40 nm.

3. A process as stated in Claim 1 or 2, characterized in that protein and/or peptide molecules are immobilized on the membrane.

4. A process as stated in Claim 1 or 2, characterized in that glycoprotein and/or glycopeptide molecules are immobilized on the membrane. A process as stated in Claim 1 or 2, characterized in that polysaccharide and/or oligosaccharide and/or sugars are immobilized on the membrane. A6) A process as stated in Claim 1 or 2, characterized in that lipid moleculare are immobilized on the membrane.

7. A process as stated in Claim 1 or 2, characterized in that lipopolysaccharides are immobilized on the membrane.

8. A process as stated in Claim 1 or 2, characterized in that enzymes and/or coenzymes are immobilized on the 28 membrane.

9. A process as stated in Claim 1 or 2, characterized in that one of the substances of the group containing antigens, antibodies, lectins, biotin, avidin, protein A and haptenes are immobilized on the membrane. A process as stated in Claim 1 or 2, characterized in that nucleic acids are immobilized on the membrane.

11. A process as stated in Claim 1 or 2, characterized in that dyes, in particular fluorescent dyes or dye mixtures, are immobilized on the membrane.

12. A process as stated in Claim 1 or 2, characterized in that if necessary conducting materials such as metals and/or metal compounds and/or carbon and/or silicon oxide and/or synthetic materials are immobilized or deposited on the membrane.

13. A process as stated in Claim 12, characterized in that mixtures of the materials or separate layers of several materials are deposited.

14. A process as stated in Claim 1 or 2, characterized in that mediator or transmitter molecules are immobilized on the membrane. A process as stated in Claim 1, 2, and 9, characterized in that a membrane with antigens, antibodies, lectins, biotin, avidin, protein A or haptenes immobilized thereon is used as an ELISA membrane.

16. A process as stated in Claim 1 to 11 and 14, characterized in that a membrane with enzymes or coenzymes, antigens, antibodies, biotin, avidin, lectins, 29 protein A, haptenes, nucleic acids or mediator or transmitter molecules is used as diagnostic reagent.

17. A process as stated in Claims 1 to 11 and 14, characterized in that a membrane is used as a sensor membrane with enzymes or coenzymes, antigens, antibodies, biotin, avidin, lectins, protein A, haptenes, nucleic acids or mediator or transmitter molecules immobilized thereon.

18. A process as stated in Claim 17, characterized in that the substances immobilized on the menbrane and the membrane itself are provided with a conducting layer.

19. A process according to claim 18, characterized in that the conducting layer synthetic material. A process as stated characterized in that the sputtering.

21. A process as stated characterized in that the vapor deposition.

22. A process as stated characterized in that the plasma-polymerization.

23. A process as stated is metal or conducting in Claims 12, 13 or 17, conducting layer is applied by in Claims 12, 13 or 17, conducting layer is applied by in Claims 12, 13 or 17, synthetic layer is applied by in Claim 1 or 2, characterized in that the deposited or immobilized substances, in particular pharmaceutical agents, are enclosed by the membrane.

24. A support having at least one membrane extending a S' l.il CaI 30 long f-lat, curved, cylindrical or vesicular surfaces comprising at least one layer of identical molecules containing proteins which are arranged in the form of a crystal lattice with a lattice constant of 1 to 50 nm. A process for immobilizing or depositing molecules or substances on a support, substantially as herein described with reference to any one of Examples 1 to 8.

26. A support as defined in claim 24, substantially as herein described with reference to any one of Examples 1 to 8. DATED this 17th Day of August 1992 UWE B. SLEYTR AND MARGIT SARA Attorney: IAN T. ERNST Fellow Institute of Patent Attorneys of Australia of SHELSTON WATERS Abstract Process for immobilizing or depositing molecules or substances on a support wherein as support a structure is used having at least one layer of molecules containing identical proteins extending along flat, curved, cylindrical or vesicular surfaces which are arranged in the form of a crystal lattice with a lattice constant of 1 to 50 nm. INTERNATIONAL SEARCH REPORT International Application No PCT/AT 89/00031 1. CLASSIFICATION OF SUBJECT MATTER (i1 several classification symoolsappooy, indicate a~ll) According to international Patent Classification IIPCQ or to both National Classification ;ind IPC 4 G 01 N 33/544, C 12 N 11/02, G 01 N 337553, C 12 Q 1/68, Int. Cl. -G01 N 33/549, C07 K 17/02, A 61 K 9/00 1i. FIELDS SEARCHED Minimum Documentation SearchedI Classification System I Classification Symbolsi Int. C1. 4 G01 N, C12 N, B01D, C12 Q Documentation Searched other than Minimum Documentation to the Extent triat such Documents care Included In the Fields Searched'8

111. DOCUMENTS CONSIDERtED TO 2E RELEVANTO Category 1 Citation of Document. 11 with Indication. where appropriate, of the relevanit pssages, 12 Relevant to claim No. 13 Y EP, A, 0189019 SUEYTR) 30 July 1986, see the 1-23 whole document Y EP, A, 0154620 SLIEYTR) 11 September 1985, see 1 1-23 the whole document y EP, A, 0173500 (PAuLL CORP.) 5 March 1986, see claim 1 1-23 Y -EP, A, 0184710 (BAYER AG) 18 June 1986, see pages 5-7 1-23 Y !US, A, 3979184 GIAEVER) 7 September 1976, see the 12,13,19-21 whole document A *EP, A, 0166233 i,.-ODECKE AG) 2 January 1986 *special categonse of cited documents: a0 later document Published after the international filing date documeont defining the general state at the art which is not or onority data end not in conflict with the application but considered t0 be of panfliar relevance cited to understand the principle of theory underlying the inventian sarlier document but published on or siter the international document of Particular relevance: the claimed invention filing date cannot be Considered novel or cannot bei coneidereod to documtentt which may throw doubts on priority claim(*) or Involve en inventive step which Ie cited to establish the Pubiication date of another document of particular roievance:' the claimed inventon Citation or other speciai reason t(as apecifled cannot be considered to involve en inventive step when the I"0" document referring to an oral disclosure, use. exhibition or document is combined with one or more otner such docu- other means ments. Such Combination being obvious to a person skilled document Published Prior to the international filing dala but in the art. later than the Priority date claimed document member of the same atent family IV. CERTIFICATION Date of the Actual Completion of the International Search Date of Mailing of this International Search Report 26 June 1989 (26.06.89) 20 July 1989 (20.07.89) International Searching Authority Signature of Authorized Officer EUROPEAN PATENT OFFICE Form PCT/ISA/210 (second sheet) iJornuary 1965) L ANNEX TO THE INTERNATIONAL SEARCH REPORT ON INTERNATIONAL PATENT APPLICATION NO. AT 8900031 SA 27565 This annex lists the patent"flamily members relating to the patent documents cited in the above-rmentioned international search report. The members are as contained in the European Patent Office EDP file on 11/07/89 The European Patent Office is in no way liable for these particulars which are merely given for the purpose of information. Patent document Publication Patent family Publication cited in search report date member(s) I date EP-A- 0189019 30-07-86 AT-A,B 382321 10-02-87 WO-A- 8603685 03-07-86 EP-A- 0207100 07-01-87 EP-A- 0154620 11-09-85 AT-A- 381463 27-10-86 WO-A- 8504111 26-09-85 JP-T- 61501619 07-08-86 US-A- 4752395 21-06-88 EP-A- 0173500 05-03-86 US-A- 4693985 15-09-87 CA-A- 1249781 07-02-89 EP-A- 0280840 07-09-88 GB-A- 2163434 26-02-86 GB-A- 2199327 06-07-88 JP-A- 61124868 12-06-86 EP-A- 0184710 18-06-86 OE-A- 3444939 12-06-86 JP-A- 61140523 27-06-86 US-A- 3979184 07-09-76 DE-A,C 2623100 0r-12-76 FR-A,B 2312224 24-12-76 GB-A- 1533286 22-11-78 JP-A- 51148015 18-12-76 EP-A- 0166233 02-01-86 DE-C- 3419782 14-11-85 JP-A- 61000095 06-01-86 Z For more details about this annex e Oicil Journal of the European nt Oie, o. 12/82 SFor more details about this annex :sce Official Journal of the European lPatent Office, No. 12(87 INTERNATIOINALER RECHERCHENBERICHT Internationales Akienzeichen PCT/ AT 89 /00031 1, KLASSIFIKATION DES ANMELDUNGSGEGENSTANDS (bel mehroren KlasalflkationssymIholon sind ails anzugobon1 6 Nach der Interostionalen Patentklaisfikation (IMP odor nach der nationalen Klassifikation und der IPC Iml d' G 017 N 33/544, C 12 N 11/02, G 01 N 33/553, C 12 Q 1/68, G 01.N 33/549, C 07 K 17/02, A 61 K 9/00 11. RECHERCHIERTE SACHGEBIETE Recherchierter Mindet~rIfSjoff 7 Klassifikationssystem_ Klassifikationssymbole Init.CI,4 G 01 N, C 12 N, B 01 D, C 12 Q Recherchierte nicht zum Mindesiprufstoff gehorende Veriaffentlichungen, sowvelt diese unter die rechtorchierten Sachgobiete fallen 8 MI. EINSCHLAGIGE VEROFFENTLICHUNGEN 9 Art' Kennzeichnung der Veroffentlichungl 1,sovveit erforderlich unter Angabo der mallgeblichen T61le0 2 Be. Anspruch N0A3 Y EP, A, 0189019 SLEYTR) 30. JUli 1986, 1-23 siehe das ganze Dokument Y EP, A, 0154620 SLEYTR) 11. September 1985, 1-23 siehe das garize Dokuxnent Y EP, A, 0173500 (PALL CORP.) 5. Mdrz 1986, 1-23 siehe Anspruch I Y EP, A, 0184710 (BAYER AG) 18. Juni 1986, 1-23 siehe Seiten 5-7 Y US, A, 3979184 GIAEVER) 7. September 1976, 12,13,19-21 siehe das ganze Dokument A EP, A, 0166233 (GODECKE AG) 2. Januar 1986 *Besondere Kategorien von angeponen Verofienflichungen 10 Veedffentlichung, die don ailgemeinen Stand der Technik Spaters Verciffentlichung, die nach dem internationalen An.- dofiniort, ebee nicht at% bosondors bodeuttam anzusehen Ist meldedlatum oder dem Prioritatscdatumn verotfentllcht woeden "E"iltr~ okuent di jeochera amodo nah dm itere- ist und mit der Anmeldlung nicht kollidiort, sondlorn nur zum tE"iolen Anlmetde osu eoffentlit a dr aden inere Veetindnis des der Erfindlung zuttrundiiiieglendeon Prinzips tionlenAnmededtumverbfenlict wodanistoder der ihr zugeundelieo~nden Theorist angogobon irt 'Li' Vordifentlichung, die goeognat ist, elnmn Pricirititsanspruch Veroffentlichung von besonderer Bedeoutung; die bomnspeuch- zweifolhaft eriheinen zu lessen, odor dlurch die das Verof- te Erfindlung kann nicht ala neu odor auf erfindariacher Titig- fentlichungatdatum amner andefon im Racheechenbericht go- keit beruhond bertrachtet warden nimitei, %Ardfentlichung beoegt warden soll oder die aus einam andaren bosonderen Grund angegeo~n ist Nwie gofUhrt) Verofientlichung von bosonderer Bedeutung; die benspruch- erdfenlicung diesic au em mtndlihe ffebirng, te Erfifidung kann nicht ala auf erfindariacher Taltigkoit be- eine Vobontiung, dne Auhhlusne odornderce Malenbamn ruhand botrachtot verdon. wenn die Vorotfemrlichung mit boigh atzug ieAneln dradr anh einer odor mehreren anderet, Veraffentlichungen dlieser Kate- boziehtgones in Verbindlung gebracht wird und dies. Varbindung fi~r Veroffentlichung, die vor dim internationalen Anmaldeda- amnen Factimann naheliegend !at tumn, aber nach dam beenspnuchten Priorititsdlatum verdffent- "V Veroffentlichung, die Mitglied deorselbon Patentfamilie ist licht worden ist IV. BESCHEINIGUNG Datum doe Abschlusses der internationalen Recherche 26. Juni 1989 Inernationale Recheechenbehoede L ~Europilaches Patentamrt Absendedatum des inteenationalen Recherchenbrichts Formblatt PCT/ISA/210 B Iat" 2) Ijanu or 1985) ANHANG ZUM INTERNATIONALEN RECHERCHENBERICHT tJBER DIE INTERNATIONALE PATENTANMELDUNG NR. AT 8900031 SA 27565 In diesemn Anhang sind die Mvitglieder der Patentfamilien der im obengenannren internationalen Recherchenbericht angefuhrten Patentdokumente angegeben. Die Angaben fiber die Familienmitglicder entsprechcn demn Stand der Datei des Europaischen Patentamts am 11/07/89 Diese Angaben dienten nur zur Lnterrictng und erfolgen oboe Gtwihr. Im Recherchenbericht Datum der Mitghied(er) der Datum der angefuhrtes Patentdokument Verolfentlichung Patentfamijie Veroffentiicbung EP-A- 0189019 30-07-86 AT-A,B 382321 10-02-87 WO-A- 8603685 03-07-86 EP-A- 0207100 07-01-87 EP-A- 0154620 11-09-85 AT-A- 381463 27-10-86 WO-A- 8504111 26-09-85 JP-T- 61501619 07-08-86 US-A- 4752395 21-06-88 EP-A- 0173500 05-03-86 US-A- 4693985 15-09-87 CA-A- 1249781 07-02-89 EP-A- 0280840 07-09-88 GB-A- 2163434 26-02-86 GB-A- 2199327 06-07-88 JP-A- 61124868 12-06-86 EP-A- 0184710 18-06-86 DE-A- 3444939 12-06-86 JP-A- 61140523 27-06-86 US-A- 3979184 07-09-76 OE-A,C 2623100 09-12-76 FR-A,B 2312224 24-12-76 GB-A- 1533286 22-11-78 ~JP-A- 51148015 18-12-76 EP-A- 0166233 02-01-86 OE-C- 3419782 14-11-85 JP-A- 61000095 06-01-86 Fuir nahere Einzelheiten zu diesem Anhang :siehe Amtsblatt des Europakschen Patentamts, Nr.12/82