NO160968B - BIO-COMPATIBLE POLYMERS WITH BIOLOGICAL COMPONENTS IF BINDING COMPLEMENTS ARE PATHOLOGICAL PROCESSORS. - Google Patents

Description

The present invention relates to biocompatible polymers

which have immobilized reactive biological components thereon

which bind selected specific pathological agents or specific groups of pathological agents associated with diseased body fluids.

The course of many disease states is often reflected

of elevated levels of specific blood proteins and other molecules. This phenomenon is typically used as a diagnostic tool to define the pathology and follow the course of clinical treatment. In many cases, these specific blood components are directly or indirectly responsible for the primary or secondary manifestations of the disease course. "Autoimmune" diseases can be described as diseases characterized by circulating antibodies to endogenous substrates and tissue proteins that are required by the body for normal growth and maintenance. "Neoplastic" diseases are typically charac-

serted by uncontrolled growth of an undifferentiated transferred cell line that avoids or endangers the body's natural defense mechanism by eliciting immunosuppressive blockers

end factors, surface antigen-masking components and/or growth regulator components. Specific partitioning of these pathological agents on a biocompatible substrate is consistent with the restoration of the "normal" body

function by removing the pathological triggers of the disease process.

The main function of the organs, cells and molecules that

includes the immune system, is to recognize and eliminate foreign substances from the body. These foreign substances eli-

is mined by reaction between the foreign substances and anti-

substances that are formed in response to the substance. In general, this function is performed efficiently and without any harm to the patient. IN

in certain cases, however, disturbances can occur which can lead to pathogenic disturbances such as e.g. an uncontrolled response (allergic disease) or an abnormal response (auto-

immune disease). The pathogenesis of both of these diseases is directly or indirectly linked to the production of anti-

substances with cross-reactivities to either surrounding antigens

(allergens) or self-antigens.

An autoimmune disease is a pathological condition that occurs when a patient responds immunologically with the production of antibodies with reactivity towards a self-antigen. Autoimmunity can affect virtually every part of the body and generally involves a reaction between a self-antigen and an immunoglobulin (IgM or IgG) antibody. Representative autoimmune diseases may include the thyroid gland, kidneys, pancreas, neurons, gastric mucosa, adrenal glands, skin, red cells and synovial membranes as well as thyroglobulin, insulin, deoxyribonucleic acids and immunoglobulins.

For certain types of autoimmune and neoplastic diseases, non-specific immunosuppressive treatment such as whole-body X-ray irradiation or administration of cytotoxic drugs has been used with limited success. The disadvantages of such treatment include the toxicity of the agents used and the increased frequency of various cancers, especially lymphomas and reticulum cell sarcomas, which follow such treatment. In addition, the use of non-specific agents for chronic cellular suppression will greatly increase the patient's susceptibility to serious infection from ambient fungi, bacteria and viruses that would not normally cause any problems. The invention described here is specific in that it removes only the pathological agent or those groups of pathological agents which are related to and responsible for manifestations of a particular disease.

If one considers the state of the art, one finds that in recent times there have generally been two possibilities for the therapeutic treatment of autoimmune and/or neoplastic diseases.

The first of these is to introduce a material into the patient

which causes a specific type of immunological tolerance to be induced. This suppression of the antibody response will then produce a tolerance to the provocative antigen. A typical example of this type of opportunity is described in

US patent 4,222,907. According to this publication, a sick patient is given a therapeutic treatment consisting in the introduction of conjugates of an antigen chained to a D-glutamic acid: D-lysine copolymer.

The other possibility has been via the extracorporeal route. The procedures generally include removal of whole blood, separation of cellular and soluble blood substances. substitution or treatment of blood plasma and recombination infusion of the treated whole blood. The first example of this solution would be plasma substitution or exchange with salt, sugar and/or protein solutions, and this is described by McCullough et al, "Therapeutlc Plasma Exchange", Lab. With. 12(12), pp. 745 (1981). Plasma exchange is a relatively crude technique that requires a large volume of replacement solution. Another example of this possibility involves physical and/or biochemical modification of the plasma portion of the whole blood. A typical example of the prior art with respect to this therapeutic treatment is Terman et al's article "Extra-corporeal Immunoadsorption: Initial Experience in Human Systemic Lupus Erythematosus", The Lancet, October 20, 1979, pp. 824-826. This article describes a hemodialysis type system that uses two mechanical filters with a DNA collodion bone charcoal filter between the two mechanical filters. However, typical of this known technique is that the adsorbent column is only semi-specific for immune components because the bone-charcoal substrate will not specifically eliminate many vital low-molecular components from the treated plasma. A second application of this possibility can be illustrated by the article by Terman et al "Specific Removal of Circulated Antigen by

Means of Immunoadsorption", FÉBS Letters, vol. 61, no. 1, January 1976, pp. 59-62. In this publication the specific removal of radiolabeled antigen by antibody-treated cellulose membranes is described. However, the author shows that control membranes have a significant capacity to non-specifically adsorb proteins.

A third application of this possibility is illustrated in the article by Bansal et al "Ex vivo Removal of Serum IgG in a Patient With Colon Carcinoma", Cancer, 42(1), pp.1-18

(1978). This publication describes the semi-specific adsorption of immunoglobulin by ex vivo treatment of plasma with formalin and heat-killed Staphylococcus aureas. The biological activity of certain strains of S. aureas is attributed to a molecule present on the cell wall, called protein A, which interacts with and binds to the Fc portion of mammalian IgG. Because this interacts with the Fc part, this treatment does not distinguish between normal and pathological IgG components, and trials have shown the possibility of significant side effects.

A fourth application of this possibility can be illustrated by the article by Malchesky et al "On-line Separation of Macromolecules by Membrane Filtration With Cryogelation", Artif. Organs 4:205, 1980. This publication describes the semispecific removal of cryoglobulin substances from plasma by a combination of filtration and cold treatment chambers. The occurrence and composition of cryoglobular deposits are not necessarily consistent with, or any indication of, many autoimmune or neoplastic diseases.

Another problem associated with the existing known

technique is that without systems that make use of mechanical filtration, the specific pathological agents that one wishes to remove have not been removed in large enough quantities to bring about any improvement for the sick patient in that the columns do not specifically absorb only the desired specific pathological evokes.

It has now been found that high specificity in the removal of pathological effectors can be achieved by treating blood and/or plasma in an economical manner using the present biospecific polymer.

The present invention thus relates to a biospecific polymer, which is characterized by the fact that it consists of

(a) a biocompatible polymer carrier selected from a hydrogel or modified cellulose, which biocompatible polymer carrier is bonded to a mechanically stable support member selected from purified cellulose, polyester fiber, microporous polyolefins, cotton cloth, polystyrene, polycarbonate, polyphenylene oxide, reticulated polyurethanes and combinations thereof; (b) optionally a spacer compound covalently bonded to indicated biocompatible polymer carrier; and (c) a biological substance or substances immobilized on specified spacer compound or on specified polymer carrier, and whereby the biological substance or substances retain their reactivity for binding specific pathological agents or specific groups of pathological agents.

Also described is a system for the therapeutic treatment of autoimmune diseases in which a sick patient's blood, plasma or other body fluid is passed over a biospecific polymer that has immobilized reactive biological substances, whereby the desired pathological agents are removed from the patient's blood or plasma after which the blood is returned to the patient.

These and other aspects of the invention are described in detail in the following description and claims.

I. Biocompatible polymer carrier

The biocompatible polymer carriers which can be used according to the invention are materials which are not likely to cause harmful effects when they come into contact with body fluids such as e.g. plasma or whole blood, while at the same time maintaining a reactive but immobilized biological substance oriented so that the biological substance protrudes from the surface of the polymer carrier. The materials that are suitable are those that can be molded into films and other physical forms while being able to have specified biological substances chemically bound to them without harming either themselves or the biological substances bound thereto. The types of materials which are generally considered to be suitable are those which are known in the art as hydrogels and which can either be

copolymers or homopolymers.

Modified cellulose and cellulose derivatives, especially cellulose acetate, have also found use as biocompatible carriers which are applicable according to the invention. By modified cellulose derivatives is meant that the cellulose polymer is surface-modified by covalent binding of attached biocompatible surface groups to the cellulose substrate polymer, which makes it more biocompatible. Such surface groups are well known and do not need; is described in more detail here, but as an example, albumin has shown particular utility as a modifying group. Methods for binding such groups are described below.

Homopolymers can also be used as suitable biocompatible polymer carriers according to the invention. However, it must

it is stated that when homopolymers are considered, these may include materials which can also be identified as weakly cross-linked homopolymers. That is, that they contain a relatively small amount of another component which is either present in the material during the manufacture of the monomer or which is added for the purpose of

ensure sufficient cross-linking to protect the homopolymer from slow dissolution in an aqueous medium such as blood. An example of this type of homopolymer, which is often weakly cross-linked, is hydroxyethyl methacrylate (HEMA).

In the case of the hydrogels, suitable polymers can either

be regular homopolymers which essentially do not contain

some other material holds their matrices, or they can be copolymers made from two or more monomers such as e.g. styrene and vinyl acetate. In certain cases, this type of composition of the copolymers with different monomers can increase the desired properties of the biocompatible polymer carrier material. Examples of suitable monomers that can be copolymerized include e.g. hydroxyethyl methacrylate and glycidyl methacrylate.

Also useful are terpolymers which are a subclass of copolymers containing three monomers that are polymerized. An example of a suitable terpolymer is glycidyl methacrylate/N-vinylpyrrolidone/hydroxyethyl methacrylate (GMA/NVP/HEMA).

In addition to the specific copolymers and homopolymers indicated above, copolymers, prepared with or without various additional monomers, and homopolymers suitable according to the invention can be polymerized from the following monomers: hydroxyalkyl acrylates and hydroxyalkyl methacrylates, e.g., hydroxyethyl acrylate, hydroxypropyl acrylate bg hydroxy -butyl methacrylate; epoxy acrylates and epoxy methacrylates such as e.g. glycidyl methacrylate; aminoalkyl acrylates and amino-alkyl methacrylates; N-vinyl compounds such as e.g. N-vinylpyrrolidone, N-vinylcarbazole, N-vinylacetamide and N-vinylsuccinimide; aminostyrenes; polyvinyl alcohols and polyvinyl amines which must be prepared from suitable polymeric precursors; polyacrylamides and various substituted polyacrylamides; vinylpyridine; vinyl sulfonate and polyvinyl sulfate; vinylene carbonate; vinyl acetic acid and vinyl crotonic acid; allylamine and allyl alcohol; vinyl glycidyl ether and allyl glycidyl ether. Methods and procedures for the production of copolymers and/or homopolymers from the above-mentioned monomers are well known in the art. These parameters are not critical for the invention provided that the final copolymer and/or homopolymer is not toxic to animals, including humans.

The method used for casting these materials in a form suitable for use according to the invention is not of critical importance. A preferred method is spin casting, and this is exemplified in examples 2, 3 and 4.

II. Biological substances

As indicated herein, a biological substance and/or substances may be defined as a chemical compound that exhibits the ability to covalently bind to the biocompatible polymer carrier or spacer compound (defined hereinafter) while retaining an activity to bind a desired constituent that causes a pathological condition. It should also be understood that, in addition, the biological substance or substances used must be of such a size that they are covalently bound to the surface of the polymer carrier and are not sufficiently small to penetrate the porous matrix of the polymer carrier so that they can be chemically bound on the inside of or in the interior of the carrier material. In such a circumstance, a spacer compound can be used to ensure that the reactive site of the biological substance that remains and is able to bind to the desired pathological component can really be presented to this component, i.e. that it is kept out from the carrier so that it comes into contact with the body fluid flowing over the carrier. It is of course obvious that the reactivity for binding the desired pathological component is really retained after immobilization of the biological substance or substances on the biocompatible polymer carrier. Examples of materials that can be used as biological substances include e.g. acetylcholine receptor proteins, histocompatibility antigens, ribonucleic acids, basement membrane proteins, immunoglobulin classes and terminal classes, myeloma protein receptors, complement components, myelin proteins and various hormones, vitamins and their receptor components. Particular examples are e.g. binding insulin to a biocompatible polymer carrier to remove anti-insulin antibody associated with the autoimmune disease insulin resistance; binding of anti-Clq and/or Clq to a biocompatible polymer carrier to remove immune complexes associated with connective tissue and cell division diseases such as e.g. rheumatoid arthritis and carcinoma.

Any generally known method of chemical attachment will be sufficient to bind the biological substances to the biocompatible polymer carrier provided that the biological substance still has at least one active site for the particular autoimmune disease-related component. In general, the methods used for chemical bonding will fall under three classes or methods of bonding. These three methods are, 1) spontaneous binding, 2) chemical activation of functional end groups, and 3) coupling reagent binding. Spontaneous covalent binding of biological substances to the polymer carrier surface takes place via chemically reactive groups that protrude from the polymer carrier. Reactive groups such as e.g. aldehyde and epoxy protruding from the polymer carrier easily bind biological substances containing available hydroxyl, amino or thiol groups. Free aldehyde groups on the polymer carrier also connect via acetal bonds to hydroxyl-containing biological substances and via imide chains to amino-containing molecules. In addition, for example, free oxime groups can be linked via alkylamine, ether and thioether bonds to biological substances containing amine, hydroxyl and thio groups. For expedient reasons, all bonds and links are defined here as immobilizations.

More in-depth discussions of these reactions can e.g. found in "Chemical Procedures for Enzyme Immobilization of Porous Cellulose Beads", Chen, L. F. et al, Biotechnology and Bioengineering, vol. XIX, pp. 1463-1473 (1977) and "Epoxy Activated Sepharose", 6B, Pharmacia Fine Chemicals, Affinity Chromatography, pp. 27-32 (1979).

Chemical activation of functional end groups can be carried out by activation of functional polymer surface groups by chemical modification of their end components. This method can, for example, be oxidation of epoxy end functions with periodic acid during the formation of active aldehyde groups.

This method is further exemplified in e.g. "Immobilization of Amyloglucosidase on Poly [(Glycidyl Methacrylate) Co (Ethylene Dimethacrylate)] Carrier and Its Derivatives", Svec, F. et al, Biotechnology and Bioengineering, vol. XX,

pp. 1319-1328 (1979). Immobilization of the biological substances proceeds as described above. Condensation reactions can be carried out between the free carboxyl and amine groups via carbodiimide activation of the carboxy groups as described in e.g. "New Approaches to Non-Thrombogenic Materials",

Hoffman et al, Coagulation - Current Research and Clinical Applications, Academic Press, N.Y. (1973). Briefly stated, the immobilization of the biological substances can be carried out by carbodiimide activation of either the polymer's or the biological substance's carboxyl groups and the condensation with a free amine to form a stable peptide bond. The final orientation of the biological substance is generally determined by whether an amine- or a carboxyl-containing polymer is used.

Coupling reagent binding can be performed during use

of a number of coupling agents during the formation of covalent bridges between polymer and biological substances. Free hydroxyl- and/or amine-containing polymers and biological substances are covalently linked here with reagents such as e.g. cyanogen bromide, diisocyanates, dialdehydes and trichloro-s-triazine. An in-depth discussion of this technique can e.g. found in the above cited article by Chen et al.

The preferred method for immobilizing a reactive biological substance on a biocompatible polymer substrate in a given case is generally determined by the molecular localization of the reactive binding portion of the biological substance and the functional groups on the biological substance and the polymer substrate that can be covalently combined . When it comes to polymer substrates containing hydroxy end functions, it is preferred, e.g. currently to activate by treatment with an alkaline solution of cyanogen bromide (10 to 20% w/v).

Typically, the reaction mixture is maintained at room temperature

(20 to 25°C) for approx. 30 minutes. The pH of the solution is maintained in a range of from 10 to 12, by adding alkaline material, e.g. KOH or NaOH. The polymer is thoroughly washed with physiological saline (0.9 g%) and incubated with solutions of a purified biological substance dissolved in a weakly alkaline buffer solution for 12 to 16 hours at 2 to 8°C. The polymer is rinsed thoroughly with physiological saline to remove unbound or non-specifically bound biological components.

Biological substances are immobilized on glycidyl-containing polymers via ether, thioether or alkylamine bonds. Epoxy-activated polymer substrates are rinsed and swollen with aqueous neutral buffer solutions at room temperature. Purified biological substances, dissolved borate, carbonate or phosphate buffer solutions are incubated with the glycidyl polymer substrate for 12 to 20 hours at 4 to 30°C. Excess non-specifically bound biological substance is removed by rinsing the polymer with saline, acetic acid (0.2 to 1.0M) and phosphate-buffered

(pH = 7.2 - 0.2) salt solutions. Activation of amine- and carboxyl-containing polymer matrices is carried out by treatment with purified biological substances dissolved in weakly acidic (pH 4.5 to

6.5) buffer solutions of a water-soluble carbodiimide. Biological substances are covalently linked to the polymer carrier substrates by incubating the polymer carrier, biological substance and carbodiimide reactants for 12 to 16 hours at 2 to 8°C. Polymer-biological substance conjugates are washed alternatively in acidic and then alkaline rinse water until the washing solutions are free of biological substance and carbodiimide reactants.

To determine the specific binding characteristics of polymer-immobilized biological substances, physiological serum solutions of complementary biomolecules were treated with activated membranes. The amounts of biomolecules were measured radiochemically. Significant reduction of specific biomolecules took place after short-term exposure to the biologically modified polymer substrates.

III. Distance connections

A distance connection can be defined

as a molecule or compound capable of binding to the surface of a biospecific polymer support, which is large enough to protrude from the surface of said support and which is capable of immobilizing a biological substance and/or biological substances. The spacer connection ensures that the active site of the biological substance is held outwardly away from the carrier in order to contact the body fluid more effectively. From the above it is obvious that the reactivity for binding with the desired diseased complex is indeed retained after immobilization of the biological substance or substances on the spacer compound and therefore to the biocompatible polymer carrier.

The spacers are derived from organic molecules that have at least two reactive functional groups that are generally located at opposite ends of the molecule. Such groups serve as binding carriers capable of connecting the spacer to the polymer carrier and to the biological substance. The reactive functional groups on the spacer compound may be the same or different provided that they react with functional groups along the surface of the polymer carrier and the functional groups protruding from the biological substance forming covalent bonds. Any known method for carrying out such coupling reactions will suffice. For example, the coupling methods described above for binding the biological substance directly to the polymer carrier can also be used.

Suitable examples of spacer compounds that can be used according to the invention, where the reactive functional groups are the same, include e.g. 1,6-diaminohexane, glutaraldehyde, 1,4-cyclohexane-dicarboxylic acid, ethylenediamine-tetraacetic acid, triethylene glycol, 1,4-butanediol-diglycidyl ether, methylene-p-phenyl-diisocyanate and succinic anhydride. Examples of distance compounds in which the reactive functional groups are not the same include e.g. 6-aminocaproic acid, p-nitrobenzoyl chloride, 1,2-epoxy-3-(p-nitrophenoxy)-propane, aminopropyltriethoxysilane and homocysteine thiolactone.

Polypeptides and proteins can also be used as spacers according to the invention. Albumin, a low-affinity protein, has e.g. successfully used as a distance connection. In addition, albumin and other natural proteins act to make the polymer carrier more biocompatible.

Finally, it should be understood that certain materials can act simultaneously as a spacer compound and as an activator in the reaction used to combine the spacer compound and the biocompatible carrier. Examples of this type of connection include e.g. glutaraldehyde and 1,4-butanediol diglycidyl ether.

IV. Support part

Most, if not all, of the suitable biocompatible polymer carriers have very low mechanical stability. Most of these materials are in reality gels or gel-like in contrast to materials that have high mechanical stability, such as e.g. sheets of polypropylene. In most embodiments that make use of the present invention, a support member that is mechanically stable is necessary. This support member enables large surface areas to be used to ensure rapid and medically acceptable, as well as commercially acceptable, levels of removal of the immune disease associated component. In addition to being mechanically stable, the support part must also be cheap and must be sterilizable in order for it to be compatible for use in a system in which the blood from a sick patient is treated according to the invention. Examples of materials that are suitable according to the invention as support parts include e.g. filter paper, polyester fibers, polycarbonates, reticulated polyurethanes, Noryl®, a polyphenylene oxide polymer manufactured by General Electric Company, microporous polyolefins such as polypropylene, and cotton cloth.

Many methods for attaching the activated or biocompatible polymer carrier with biological substances chemically bound thereto can be used. Thus, e.g. methods such as spin coating, horizontal casting, vacuum impregnation, dip coating, dip coating with subsequent crosslinking, and solution copolymerization are used. Specific examples of these methods can be found in the following examples.

V. Therapeutic system

Generally stated, the therapeutic system under consideration is the therapeutic treatment of autoimmune and other diseases whereby the blood or plasma of a sick patient is exposed to a biospecific polymer that has immobilized reactive biological substances, whereby the specific pathological agents are removed from the patient's blood or plasma, after which the blood is then returned to the patient. This therapeutic treatment may or may not necessitate the use of blood separation techniques. Thus, it is taken into account that the treatment is carried out in a similar way to a dialysis treatment with the advantage that total blood separation is not necessary and that there is very little, if any, physical damage to normal blood components.

It is of course also possible to utilize the invention in the treatment of plasma.

Plasma can be obtained from whole blood by any of the currently known and used methods. Thus, e.g. plasma is separated from a patient's blood by known methods, can then be processed and recombined with the other blood components and returned to the patient using commonly known procedures. In addition, plasma used in known medical treatment can be used according to the invention to treat plasma before it is administered to a patient in need of plasma from, for example, a blood bank. Of course, whole blood from a blood bank can also be processed and benefit from the present invention.

It should also be understood that the invention can also be applied to other body fluids to effect the removal of pathological agents.

Because of the above stated advantages obtained according to the invention, as well as others, it will be clear to those skilled in the art that many types of disease states are taken into account when it comes to response to the invention used in a therapeutic system. Generally speaking, six groups of disease states can be treated with advantage. These six disease categories are diseases of immune components, drug overdoses, toxin exposure, imbalance in body substances, infections and neoplastic conditions. Many diseases are currently treated using plasmaphoresis and cytophoresis where the desired result is the removal of a specific

substance. Present invention

will be able to be applied to these diseases as for

the time is treated with plasmaphoresis and cytophoresis.

Examples of immune complex diseases that can be treated are e.g. any disease state involving antibody,

antigen, antibody-antigen, antigen-antigen and antibody-antibody interactions, cell surface complexes, cytoplasmic complexes, etc.

Examples of drug overdoses that can be treated are e.g. overdoses of iron, dioxin, aspirin, tylenol, methotrexate and other tricyclic compounds.

Examples of poisons and toxins against which the invention is Sealed are e.g. lead, aluminium, fungi (Anatoxin) and organic phosphates.

When body substances are present in excess, these can lead to disease. Examples of such that can be eliminated during use, of the invention, include e.g. cholesterol, uric acid, immunoglobulins, sickle cells, uremic toxins, bilirubin, porphyrin, cortisol and prostaglandins.

Some examples of infectious agents that can be treated are e.g. viral diseases such as cytomegalovirus; protozoal diseases such as malaria, trypanosomes and leishmanias; bacterial infections such as streptococci; fungal infections such as tinea versicolor; mycoplasma such as pleuro-pneumonia-like organisms, rickettsial diseases such as typhoid and spotted typhus; spirochetes such as syphilis and chlamydia agents in the psittacosls lympho-granuloa-trachoma disease group.

Neoplasmic diseases that can be treated using the present invention include e.g. lymphomas, sarcomas, carcinomas and leukaemia. These can be removed by specific removal of a cell line, inhibitors, initiators of the disease and combinations thereof.

Further examples of disease states that can be treated using the invention include e.g. following: Infections such as: post-streptococcal glomerulonephritis, subacute bacterial endocarditis, secondary syphilis, pneumococcal sepsis, lepromatous leprosy, ventricular shunt infection, infectious mononucleosis, typhoid fever, subacute sclerosing encephalitis, Landry-Guillain-Barre syndrome, hepatitis B infection, Quartan malaria, Schistosomiasis and Trypanosomiasis.

Neoplasmic diseases such as: hepatoma, lymphoma

and Hodgkin's disease, acute leukemia, hypernephroma, carcinoma of the colon, bronchogenic carcinoma and Burkitt's lymphoma.

Connective tissue diseases such as Periarthritis nodosa, chronic glomerulonephritis, acute or subacute thyroiditis, vinyl chloride poisoning, chronic liver disease, mixed cryo-globulinemias, Berger's disease or Iga nephropathy, rapidly progressive glomerulonephritis and sickle cell anemia.

Hematological diseases such as thrombotic thrombocytopenic purpura, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, idiopathic neutropenia, cold hemagglutinin disease, Paroxysmal cold.hemoglobinuria, circulating anticoagulants, congenital hemophilia, leukemia, lymphomas, Erythroblastosis fetalis, pernicious anemia and Rh diseases.

Neurological diseases such as acute demyelinating encephalitis, multiple sclerosis, Landry's palsy, Guillain-Barre syndrome, peripheral neuritis and Myasthenia gravis.

Collagen diseases such as Raynaud's, Lupus Erythematosus, Polyarthritis nodosa, Scleroderma, dermatomyositis, Sjøgren's syndrome, rheumatoid arthritis, rheumatic fever and Erythema nodosa.

Endocrine diseases such as e.g. Cushing's syndrome and disease, Thyrolditis, Thyrotoxicosis, Addison's disease and Aspermatogenesis.

Gastrointestinal diseases such as portal cirrhosis, acute hepatitis, chronic active hepatitis, lupoid hepatitis, biliary cirrhosis, ulcerative colitis, regional enteritis and pancreatitis.

Various diseases such as e.g. hypercholesterolemia, glomerulonephritis, basal membrane disease, psychogenic conditions - drugs, postaortic valve prosthesis - hemolytic anemia, exfoliative dermatitis, Id reaction, psoriasis, Behcet's syndrome, carcinoma, subacute bacterial endocarditis, hypertension, asthma, hereditary angioneurotic edema, meningococcemia, Crohn's disease, hepatic encephalopathy and Raynaud's disease.

Diseases characterized by antibodies against nuclear antigens, cytoplasmic antigens, cell surface antigens and subclasses, can be treated according to the invention. Suitable examples include e.g. antibodies to natural DNA (double-stranded) or single- and double-stranded, antibodies to SS DNA, antibodies to deoxyribo-nucleoprotein, antibodies to histone, antibodies to Sm, antibodies to RNP, antibodies to Sc 1-1 - scleroderma, antibodies to SS- A - Sjøgren's syndrome, Sicca complex, antibodies to RAP - rheumatoid arthritis, Sjøgren's syndrome, antibodies to PM-1 - polymyositis-dermatomyositis, and antibodies to nucleolar-systemic sclerosis, Sjøgren's syndrome.

Also antibodies associated with specific autoimmune diseases such as antibodies to smooth muscle - chronic hepatitis, antibodies to acetylcholine receptors - Myasthenia gravis, antibodies to basement membrane at the dermal-epidermal junction - vesicular pemphigoid, antibodies to mucopolysaccharide protein complex or intracellular cement substance - Pemphigus, antibodies to immunoglobulins - rheumatoid arthritis, antibodies to glomerular basement membrane - Glomerulonephritis, Goodpasture's syndrome, idiopathic primary hemasiderosis, antibodies to erythrocytes - autoimmune hemolytic anemia, antibodies to thyroid - Hashimoto's disease, antibodies to borderline factor - pernicious anemia, antibodies to platelets - idiopathic thrombocytopenic purpura, alloimmunization, antibodies against mitochondria - primary biliary cirrhosis, antibodies against salivary duct cells - Sjøgren's syndrome, antibodies against the adrenal glands - idiopathic adrenal gland eatropathy, antibodies to thyroid microsomal - Graves' disease, antibodies to thyroglobulin - Addison's disease and antibodies to islet cells - Diabetes mellitus.

Paraproteinemia such as multiple myeloma, macroglobulinemia, cryoglobulinemia and light chain disease,

hyperlipidemia such as primary biliary cirrhosis and familial hypercholesterolemia.

Endocrinopathies such as Graves' disease and Diabetes mellitus.

Alloimmunization such as hemolytic disease of the newborn and renal homotransplantation.

Also suitable for treatment using the present invention are e.g. post-transfusion purpura and auto-antibody diseases such as Goodpasture's syndrome, Myasthenia gravis, Pemphigus vulgaris, haematological diseases, idiopathic (autoimmune) thrombocytopenic purpura, autoimmune haemolytic anaemia, factor VIII inhibitor and poly-radiculopathy/Guillain-Barre syndrome.

Immune complex diseases can also be treated and include e.g. systemic Lupus Erythematosus, Polyarthritis nodosa, cutan vasculitis, rheumatoid arthritis, Glomerulonephritis and Dermatomyositis i.

While not adhering to any particular theory over any other, a review of the likely course of autoimmune pathology suggests that the pathological sequence is likely initiated by a free antigen challenge, followed by antibody development and complexation, and ending with antibody excess and complement binding of formed complexes. The correct choice of the biospecific polymer formulation and the provision of the correct effect will necessitate preliminary diagnostic procedures to determine the predominant form of the autoimmune trigger. E<J>, an illustrative example of this is described below for the treatment of rheumatoid disease. Briefly, rheumatoid disease can be characterized as following the course from a) free RF antigen (atypical Ig) (rheumatoid condition), b) free RF antibody development and RF complex formation and finally c) antibody excess and complement-activated RF complex binding. Treatment of rheumatoid disease in its early development can thus be determined by the detection of atypical immunoglobulins with monoclonal rheumatoid factor (m-RF) antibodies. Treatment at this stage would best be carried out with m-RF-activated biospecific polymers to remove the provocative antigen and thus prevent the development of endogenous RF (e-RF) antibodies. Diagnostic evidence of e-RF will indicate the use of biospecific polymers that have both m-RF and aggregated gammaglobulin-active biological substances (RF antigen). Alternatively, two biospecific polymers in series can be used, each of which has one type of active biological substance. In each case, this combination of m-RF and aggregated gamma globulin will adsorb both the provocative anti-

gene and antibody molecules to stop the course of the disease.

In the case where significant levels of RF antigen-anti-substance complex are detected, biospecific polymers containing Clq and/or collagen effector molecules can be used. If finally the disease process has developed into complement binding of formed immune complexes, an effective bio-specific polymer can contain one or more anti-complement antibodies such as e.g. anti-Clq, anti-C3 or anti-C4. Again, the biological substances - if more than one is desired, can be immobilized on a single biocompatible carrier or each can be on a separate carrier and connected in series in relation to the blood or plasma flow.

As suggested above, effective use of the invention is achieved through thorough definition of the dynamics and degree of immune response to achieve effective disease treatment.

Today, plasmapheresis and cytopheresis are the disease treatments used to remove harmful substances or cells from the blood. It is currently believed that any disease that is treated with plasmapheresis and/or cytopheresis, where the desired result is the removal of a specific substance, can be advantageously used with the product according to the invention.

More specifically, a therapeutic system currently being considered for whole blood can be illustrated as follows: a) a vascular access is provided which will enable:

b) a blood flow of from 30 ml/min to 200 ml/min,

c) an anticoagulant is administered to the blood, and

d) a pumping device is provided,

e) the blood is brought into contact with the product according to the invention, f) depending on the anticoagulant used, further medication may be necessary or desired to neutralize the anticoagulant effect on the treated blood, g) the treated blood is returned to the patient.

The time frame currently considered for the system illustrated above is from 2 hours to 4 hours. It is of course taken into account that such a time frame can either be shortened or extended depending on the situation.

A therapeutic system currently being considered for plasma can be illustrated as follows: a) a vascular access is provided which will enable

b) a blood flow of from 30 ml/min to 200 ml/min,

c) an anticoagulant is administered to the blood,

and

d) a pumping device is provided,

e) separation devices to provide a plasma-rich blood component are provided, f) plasma is brought into contact with the product according to the invention, g) filtration is carried out through a 0.2 µm filter to remove any microemboli, bacteria and/or fungi, h ) the treated plasma and the formed blood components are recombined,

i) depending on the anticoagulant used, additional medication may be necessary or desirable to neutralize the anticoagulant effect on the treated blood,

j) the treated blood is returned to the patient.

The vascular access can be provided using techniques and procedures well known in medicine. Thus, e.g. an inherently large bore cannula is used intravenously or arterially. Examples of suitable veins and arteries include the antecubital vein, the clavicle vein, and the brachial or radial arteries. It should also be understood that an arteriovenous shunt or fistula (AV shunt) can also be used. In this case, the heart is the pumping device. If an AV shunt or fistula is not used, the preferred pumping device during venous access is a peristaltic pump capable of providing a flow rate of from 30 ml/min to 200 ml/min.

Suitable anticoagulants which can be used in the method according to the invention include acid citrate (about 1 ml for every 8 ml of whole blood), heparin, heparin/acid citrate-dextrose mixtures (e.g. 1250 IU heparin in 125 ml acid citrate-dextrose/ L), and prostaglandin. When using anticoagulants such as heparin and prostaglandins, it should generally be understood that a counteracting medication must be administered to the treated blood or plasma before returning or administering said blood or plasma to a patient.

As regards the treatment of plasma, it is further to be understood that any conventional method of removing the formed blood components may be used. Suitable examples of methods for separating plasma from formed blood components include plasmapheresis, centrifugal cell separation and cell sedimentation in a plasma bag. Where possible, both continuous separation and batch separation may be suitable, the above stated methods of separation being independent of the invention and its use.

The shape of the product according to the invention is generally not critical. Thus, the invention can utilize a biocompatible carrier containing the biological substance in the form of sheets, hollow fibers, cylindrical fibers, networks, cylindrical or rectangular channels, beads and combinations thereof. Application of a fluidized bed can also be advantageous in some cases.

Example 1

This example describes a method for casting the biocompatible polymer carrier and a method for chemically binding a biological substance directly to the polymer carrier. This example is also used to describe the use of a system that has no mechanical carrier connected thereto. Absorption of anti-insulin antibodies using insulin-activated polyhydroxyethyl methacrylate (p-HEMA) membrane

A. Polymer casting

Monomer solutions were prepared by combining 15.0 g of 2-hydroxyethyl methacrylate, 15.0 g of ethylene glycol, 0.08 g of sodium bisulfite and 0.036 g of ammonium persulfate. The solution was stirred for 15 minutes at room temperature.' About. 5 ml of the solution was placed on a glass plate (12.7 x 12.7 x 0.952 cm) in the center of a polyethylene separator (10 mil thick) cut to form a gasket with a 10.16 x 10.16 window cm. A second glass slide was placed over the gasket and solution, clamped in place, and the entire assembly incubated at 60°C overnight. The clamps were removed, and the glass slides were carefully pulled apart and transferred to a deionized water bath for at least 24 h. The swollen polymer membrane was carefully removed from the glass plate and was rinse-hydrated for at least 3 days in fresh, deionized, deionized water (500 ml per day).

B. Polymer Action Research

Membrane slices (5 mm diameter) were cut from the polymer sheet for activation and analysis. A 10-20 g% cyanogen bromide solution was prepared by dissolving 1.69 g of finely divided BrCN crystals in 10 ml of 0.2 M Na2CO3 (pH 11.1) with continuous stirring at 4 C. The pH of the solution was maintained above 11 by dropwise addition of 5N NaOH until the crystals were dissolved and the pH was stabilised. 4 membrane discs were placed in a small sieve and rinsed with approx. 5 ml of 0.1N HCl and incubated for 15 minutes in the cyanogen bromide solution. The discs were each rinsed at least twice more with 5 ml portions of 0.1N HCl and were incubated overnight in 5.0 ml U-100 regular "Iletin" insulin injection solution which had been adjusted to pH 8.7 by the addition of IN NaOH. The membrane disks were rinsed with 5 ml of 0.5M NaCl, 0.1M Na2CO.j solution and 3 times in 5 ml samples of phosphate (0.05M) buffered saline (0.9 g%) solution (pH = 7.4 ).

C. Assessment of membrane adsorption of anti-inflammatory substances

A double co-current radioimmuno-antibody binding assay was performed by incubating 560 picograms of 125

I -labeled porcine insulin and serial dilutions (980 to 15 picograms) of unlabeled porcine insulin or p-HEMA membrane slides with 280 µg guinea pig anti-porcine insulin antibody in 0.5 ml of phosphate (0.05 M) buffer (pH 7.4 ) saline (0.9 g%) containing 1 g% bovine serum albumin for 2 hours at room temperature. The p-HEMA discs were removed from each test solution. A 0.1 ml sample of goat anti-guinea pig gamma globulin was added to each test tube. The test solutions were mixed and incubated for a further 2 hours at room temperature. A 1.0 ml sample of cold (2-4°C) phosphate buffered saline (pH 7.4) was added to each tube. Each test solution was mixed and centrifuged for 15 minutes at 4°C at 7500 G, and the supernatant was decanted into 20 ml scintillation vials. The supernatant was gelled with 5.0 ml of Aquasol® liquid scintillation fluid and counted in an Isocap 300

counting for 4.0 minutes. Insulin-treated membrane slices adsorbed 111 pg of anti-insulin antibody from the solution or 690 pg per cm 2 surface area.

Example 2

This example describes how an unstored bio-specific membrane can be produced. It also describes how 6-aminocaproic acid (with a chain of 6 carbon atoms) can be used as a spacer to attach insulin to the biocompatible polymer carrier used to remove insulin antibody and adsorption of anti-insulin antibody using the insulin-activated poly-hydroxyethyl methacrylate (p-HEMA) membrane.

A. Polymer casting

Monomer solutions were prepared by combining 15.0 g of 2-hydroxyethyl methacrylate, 15.0 g of ethylene glycol, 0.08 g of sodium bisulfite and 0.036 g of ammonium persulfate. The solution was stirred for 15 minutes at room temperature. About. 5 mL of the solution was placed on a glass plate (12.7 x 12.7 x 0.952 cm) in the center of a polyethylene spacer (10 mil thick) cut to form a gasket with a 10.16 x 10.16 window cm. A second glass slide was placed over the gasket and solution, clamped in place, and the entire assembly incubated at 60°C overnight. The clamps were removed and the glass plates were carefully pulled apart and transferred to an deionized water bath for at least 24 hours. The swollen polymer membrane was carefully removed from the glass plates and was rinse-hydrated for at least 3 days in fresh, deionized, deionized water (500 ml per day).

B. Polymer activation

Membrane disks were prepared as previously described in Example 1. A 10-20 g% cyanogen bromide solution was prepared by dissolving 1.69 g of finely divided BrCN crystals in 10 ml of 0.2 M Na 2 CO 3 (pH 11.1) with continuous stirring at 4°C . The pH of the solution was maintained above 11 by dropwise addition of 5N NaOH until the crystals had dissolved and the pH had stabilized. 4 membrane discs were placed in a small sieve and were rinsed with approx. 5 ml of 0.1N HCl and was incubated for 15 minutes in the cyanogen bromide solution. The slices were each rinsed at least twice more with 5 ml portions of 0.1N HCl and were incubated overnight in 10 ml of a 10 g% 6-amino-caproic acid solution (w/v), prepared in 0.1M Na 2 CO 3 , 0, 5M NaCl buffer solution, pH 8.6. The membrane disks were rinsed with 5 ml of 0.1M Na2CO3, 0.5M NaCl buffer and three 5 ml samples of phosphate-(0.05M) buffered (pH 7.4) saline solution (0.9 g%). The membrane disks were removed from the rinse solution, were activated by incubation in 10 ml of a 10% (w/v) 1-cyclohexane-3-(2-morpholinoethyl)-carbodiimide solution prepared in 0.1 M (2-[N- morpholino]-ethanesulfonic acid)-(MES) buffer (pH 6.0) for 30 minutes at room temperature, and each slice was rinsed in 5 ml of cold (4°C) phosphate-buffered saline. Duplicate membrane slices were incubated overnight in 5.0 ml of either U-100 regular "Iletin" insulin injection solution or regular porcine insulin "Iletin" solutions at 4°C. The membrane slices were removed from the protein solutions and rinsed three times in 5 ml of phos-

barrel-buffered saline solution.

C. Assessment of Membrane Adsorption of Anti-Insulin Antibody A double co-current radioimmuno-antibody binding-125 assay was performed by incubating 560 pg of I-labeled porcine insulin and serial dilutions (980 to 15 pg) of unlabeled porcine insulin or p-HEMA membrane slides. with 280 µg of guinea pig anti-porcine insulin antibody in 0.5 ml of phosphate (0.05 M) buffered (pH 7.4) saline (0.9 g%) containing 1 g% bovine serum albumin for 2 hours at room temperature. The p-HEMA discs were removed from each test solution. A 0.1 ml sample of goat anti-guinea pig gamma globulin was added to each test tube. The test solutions were mixed and incubated for a further 2 hours at room temperature. A 1.0 ml sample of cold (2-4°C) buffered saline (pH 7.4) was added to each tube. Each test solution was mixed and centrifuged for 15 minutes at 4°C at 7500 G, and the supernatant was decanted into 20 ml scintillation vials. The supernatant was gelled with 5.0 ml Aquasol^7 liquid scintillation fluid and counted in an Isocap 300 counter for 4.0 minutes. Insulin-treated membrane discs adsorbed 271 pg of anti-insulin antibody from the solution or 690 pg per cm <2>surface area.

Example 3

This example describes a method for molding biocompatible polymer carriers, both with and without mechanical support, via rotational molding. This example also describes another method for chemically binding the biological substance to the biocompatible polymer carrier.

Adsorption of anti-human immunoglobulin G(IgG) antibody (rheumatoid type 'factors') using immunoglobulin-activated polyhydroxyethyl methacrylate-co-glycidyl methacrylate-(p-HEGL) membranes

I. Polymer casting

The following example illustrates the preparation

of both stored and unstored polymer membranes by rota-

tion casting techniques.

A. Rotational molding device

The rotational molding device consisted of a closed aluminum drum with 6.35 mm thick walls. The inside dimensions of the drum were 10.16 cm in diameter and: 12.70 cm in length. The drum was connected to a motor which rotated the drum, and the drum's rotational speed per minute was measured with a stroboscope phototachometer. A heat gun heated the rotating drum, and thermocouples measured the internal drum temperature and the temperature of the air flowing over the drum. The drum was flushed with nitrogen before and during polymerization.

B. Preparation of stored membrane

Whatman hard filter paper of grade 50 was used as a substrate to provide mechanical strength for these rotational moldings. The paper was cut into rectangular sheets (12.53 x 31.59 cm) and then dipped in ethylene glycol (EG) for 30 minutes at room temperature. Excess glycol was removed from the paper by draining, and after draining the paper contained 2-4 g of EG. The conditioned paper was crumpled into a cylinder and placed inside the rota sea casting drum. The outer edge of the paper was pressed against the drum wall to expel air between the wall and the paper. When the paper is in place, it is preferred, but not necessary, that the ends of the paper butt against each other, as there may be some overlap. The paper backing was checked for air pockets and, if present, removed with a rubber bit.

For polymerizations that yield a highly adhesive polymer, the rotational molding cylinder may first be lined with a sheet of silicone release paper by placing the untreated side of the paper against the cylinder. The conditioned Whatman filter paper can then be placed against the slip paper carefully so as not to trap air.

C. Polymerization formulations

In what follows, representative polymerisation formulations which are currently used are indicated. In each case, the initiator was stirred with the reactive monomers at room temperature for 30 minutes or until the initiator was dissolved.

GMA-HEMA (50/50) copolymer

6.25 g 2-hydroxyethylmethanoxylate (HEMA)

6.25 g glycidyl methacrylate (GMA)

12.5 g ethylene glycol (EG)

0.02 g 2,2'-azobis-(2-amidinopropane) hydrochloride (ABAP)

GMA-NVP-HEMA (50/40/10) copolymer

6.25 g of GMA

1.25 g NVP

5.00 g HEMA

12.5 g EG<ft>

0.02g ABAP

The ethylene glycol weight includes 2-4 g of EG on the Whatman paper.

D. Rotational molding procedure

While the initiator was dissolved in the monomers,

the drum placed in the rotary casting assembly. The drum was operated at 1400 rpm at room temperature and was flushed with nitrogen for 15 minutes, after which the initiator monomer solution (25.0 ml) was injected into the drum with a hypodermic syringe with a flexible TefloiP^ tip. The nitrogen purge was repeated, and the drum speed was increased to 2900 rpm.

The fan (approx. 990 l/min) and heater on the spray gun were started and the drum was heated to 70-75°C for 90 minutes. The heat was then turned off, but the fan was left on to cool the drum until the internal drum temperature dropped to approx. 30°C. The cooled drum was removed from the rotational molding apparatus and filled with deionized water. After water treatment for 1 hour, the molded product was removed from the drum.

II. Polymer activation

Membrane slices were prepared as previously described

in Example 1. 14 individual discs were each incubated in 1.0 ml of 1.0 M hexanediamine solution for 72 hours at 4°C. The slices were removed from the hexanediamine solution and washed three times with 2 ml of phosphate buffered saline. A 4.0 g% human gamma globulin (HGG) solution was prepared by dissolving 4.0 g HGG in 100 ml 0.1 M MES buffer (pH 6.0) with gentle stirring at room temperature. After the protein was completely dissolved, serial dilutions were made with successive transfers of 1.0 ml of protein solution to 9.0 ml of MES solution, forming protein concentrations of 4 mg/ml, 400 µg/ml, 40 µg/ml and 4 µg/ml buffer. Two individual polymer disks were each incubated in 0.5 ml of the protein solutions and 0.5 ml of 0.25 M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide solution prepared in MES buffer for 72 hours at 4°C. Each membrane slice was removed from the protein solution and rinsed

3 times with 2 ml of cold (4°C) phosphate-buffered saline.

III. Assessment of membrane adsorption of anti-IgG antibody from physiological solutions

A radioimmunoassay was performed by incubating

125

individual membrane slices with 10 ng I goat anti-human IgG 1 1.0 ml PBS containing 1.0 g% human serum albumin in

2 hours at room temperature. The radioindicator solution was removed, and each membrane was rinsed three times with 2.0 ml of PBS solution. The membranes were incubated in the final rinse solution overnight at 4°C. Individual membranes were removed from the rinse solutions and counted in an Innotron hydragamma counter for 1 minute each. Stroke per minute was converted into disintegrations per minute (DPM) by dividing by the detector power. The amount of adsorbed antibody was determined by dividing the mean DPM by the specific radioindicator activity. The following results were obtained:

Example 4

This example shows the use of aminocaproic acid as a spacer compound for gamma globulin.

Adsorption of anti-human Immunoglobulin G (IgG) antibody ("rheumatoid-type" factors) using immunoglobulin-activated polyhydroxyethyl methacrylate-co-glycidyl methacrylate (p-HEGL) membranes.

A. Polymer casting

Rotational molded p-HEGL membranes were prepared as described in Example 2.

B. Polymer modification and activation

Membrane slices were prepared and processed as described

in example 2 with the exception that 1.0M 6-aminocaproic acid was used instead of hexanediamine as modifying and spacer compound.

C. Assessment of membrane adsorption of anti-IgG antibody from physiological solutions

A radioimmunoassay was performed as described in Example 3, and the following results were obtained:

Example 5

This example shows the use of albumin (molecular weight 67,000) as a spacer for folate. Folate is used to remove folic acid-binding protein.

Adsorption of folic acid-binding proteins (FABP) by folate-albumin-activated polyhydroxyethyl methacrylate (p-HEMA) membranes:

A. Polymer casting

Filter paper-stored p-HEMA polymer membranes were spin-cast as described in Example 2 using the following polymer formulation:

15.0 g of 2-hydroxyethyl methacrylate

15.0 g of ethylene glycol

0.08 g of sodium metabisulphite

0.036 g of ammonium persulfate

B. Polymer modification and activation

A folic acid-bovine serum-albumin complex was prepared by carbodiimide condensation of folate carboxyl groups with albumin-amine end groups. To achieve this, 200 mg of folic acid was dissolved in 8 ml of 0.1N NaOH, and 400 mg of 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide-metho-p-toluenesulfonate was dissolved in 2.0 ml of 0, 1M MES buffer (pH 6.0), and 1.0 g of bovine serum albumin (BSA) was dissolved in 40.0 ml of 0.1 M MES buffer. The solutions were combined, mixed and incubated for 72 hours at 4°C. The unreacted folate and carbodiimide were removed from the solution by treating 20 ml of the mixture with 20 ml of a BSA (2.5 g%) - bone charcoal (1.25 g%) suspension for 30 minutes at 4°C. The suspension was centrifuged for 15 minutes at 3400 G at 4°C, decanted and filtered through a 0.22 µm filter.

Membrane slices were prepared and treated with cyanogen bromide solution as previously described 1 example 1. After the slices were rinsed in cold saline solution, 8 slices were added to and incubated in 20 ml of either physiological saline, 160 mg% BSA or the previously prepared folate -albumin complex solution. The slices were incubated for 72 hours at 4°C. Each membrane set was removed from the solution, dried and placed in 20 ml saline solution at 4°C to be rinsed for at least 24 hours. Duplicate membranes were treated with 8 ml of 1% glutaraldehyde solution for 1 minute and rinsed overnight in 20 ml of phosphate buffered saline.

C. Assessment of membrane adsorption of folic acid-binding protein (FABP) from physiological solution

A competitive protein binding radioassay was performed by incubating 370 pg <3>H-pteroylglutamic acid (PGA) and standard dilutions (48 to 348 pg) of non-radioactive PGA or p-HEMA membrane slices with 234 pg binding activity of FABP (Kamen, B. A. and Caston, J.D., "Direct Radiochemical Assay for Serum Folate: Competition between <3>H-Folic Acid

and 5-Methyl-tetrahydrofolic Acid for a Folate Binder",

J. Lab. Clin. Med., 83, 164, 1974) in 1.0 ml of 0.05 M phosphate buffer (pH 7.6) containing 20 µl of folate free, normal human serum and 5 mg of sodium ascorbate. The radioassay tubes were mixed, incubated for 30 minutes at room temperature and 10 minutes at 4°C. Individual membrane slices were removed from the test solutions, and 0.5 ml of a cold (4°C) BSA (2.5 g%) - bone charcoal (1.25 g%) suspension was added to each tube.

All test solutions were incubated for 10 minutes at 4°C and were centrifuged at 2000 G for 15 minutes at 4°C. The supernatants were decanted into 20 ml scintillation vials. 12.0 ml liquid scintillation fluid was added to each vial.

The samples were counted in an Isocap 300 counter for 2 minutes each.

The following results were obtained:

Claims (5)

1. Biospecific polymer, characterized in that it consists of k (a) a biocompatible polymer carrier selected from a hydrogel or modified cellulose, which biocompatible polymer carrier is bonded to a mechanically stable support member selected from purified cellulose, polyester fiber, microporous polyolefins, cotton cloth, polystyrene, polycarbonate, polyphenylene oxide, reticulated polyurethanes and combinations thereof; (b) optionally a spacer compound covalently bonded to indicated biocompatible polymer carrier; and (c) a biological substance or substances immobilized on specified spacer compound or on specified polymer carrier, and whereby the biological substance or substances retain their reactivity for binding specific pathological agents or specific groups of pathological agents. 2. Biospecific hydrogel polymer according to claim 1, characterized in that specified biocompatible polymer carriers are selected from weakly cross-linked hydroxyethyl methacrylate homopolymer, hydroxyethyl methacrylate homopolymer, copolymerized hydroxyethyl methacrylate and glycidyl methacrylate or glycidyl methacrylate, N-vinylpyrrolidone, hydroxyethyl methacrylate terpolymer. 3. Biospecific polymer according to claim 1, characterized in that the biological substance is insulin used to remove anti-insulin antibody. 4. Biospecific polymer according to claim 1, characterized in that the biological substance is purified gamma globulin. 5. Biospecific polymer according to claim 1, characterized in that the spacer compound is selected from 6-aminocaproic acid and albumin.