NO870391L - PROCEDURE AND ARTICLE FOR DETECTION OF IMMUNIC COMPLEXS - Google Patents

Abstract

Use of immunologically non-specific peptide-linked amino acid-containing compounds which have the ability to immobilize circulating immune complexes for the purpose of detection or removal from body fluids, e.g. serum or blood, such compounds including oligopeptides, modified oligopeptides, polypeptides, modified polypeptides, proteins, modified proteins and especially glycosylated polypeptides and proteins.

Description

Background for the invention

The immune system represents the body's main defense mechanism against infectious agents. There are two functional divisions of the vertebral immune system; humoral and cell mediated. Humoral immunity represents circulating components of the immune system that are produced by cells and secreted and thus can be found in serum. Cell-mediated immunity comes from the direct impact of various leukocyte cells on the targeted foreign substance. Planned action of these two interconnected systems provides protection against a wide variety of infection systems.

Under normal circumstances, invasion of the body by infectious agents elicits a response from the cells that are components of the humoral system. Specifically, B-lymphocytes synthesize specific proteins, i.e. antibodies, which bind to selected points on the invading organism. After the antibodies bind to the invading organisms, the invading organisms can then be destroyed by the action of the cell-mediated immune system, or the action of humoral fractions, or directly be inactivated by the antibody molecules themselves.

Generally, antibody molecules are not directed against the host's own or "self" agents. In certain individuals, however, the immune system mistakes self-components for foreign invaders. When this occurs, the body builds up an immune attack against it

itself, very much in the same way as it would do to a foreign invader. The results of such an autoimmune conflict are traumatic. Symptoms of such diseases include inability to utilize sugar (type I diabetes), destruction of joints (rheumatoid arthritis), kidney destruction (systemic lupus erythematosus, glomerulonephritis, and similar diseases), as well as destruction of the vascular system (vasculitis). Each autoimmune disorder leads to prolonged suffering along with elevated death rates.

Autoimmune diseases are accompanied by persistently high concentrations of blood-borne autoimmune complexes. Much of the damage caused by autoimmune disease can be traced to the efforts of the cell-mediated immune system to eliminate these autoimmune complexes, wherever they may be found. Control of these diseases has proven difficult in the past, partly because the detection methods that have been used inadequately discriminated between levels of immune complexes that reflected either normal or disease status.

Many different techniques have been attempted in research for accurate, rapid and inexpensive methods for immune complex detection of disease states characterized by immune complex formation including autoimmune diseases. Although often very ingenious, no method has proven to be completely adequate. Examples of the limitations of the methods from the state of the art regarding detection follow.

First, the so-called Raji cell assay relies on the presence of cell surface receptors for innum complexes, and these receptors only appear at precise times during the growth of the culture.

Secondly, complement fraction ions or antibodies directed against them are often used in radio-immuno- or enzyme-linked immunosorbant assays. Unfortunately, both complement and the antibody used to immobilize the complement are labile proteins. This instability leads to loss of sensitivity, specificity and reproducibility. Consequently, methods that depend on complement immobilization either by direct or antibody-mediated adherence are not sufficiently reliable for clinical testing due to this labile property.

Third, immunoglobulin class and size limit the utility of complement-based assays. Neither Raji cell nor complement-based assays can generally detect all classes and subclasses of antibodies in the immune complex; they are limited to IgM, IgCj, IgG2 and IgG^. Raji cell and other complement-based methods can also generally only detect complexes with a molecular weight greater than 1,000,000 daltons. These two criteria are the most limiting for Raji cell and complement-based assay systems.

Finally, liquid-physicochemical techniques where precipitation is used with substances such as polyethylene glycol, dextran or Staphylococcus aureus protein A are unsustainable because

the difficulties inherent in handling small and often flocculent precipitates. Moreover, accidental binding of immunologically unrelated immunoglobulins to the precipitate will further impair the results of such tests. These difficulties will collectively prevent the usefulness of these methods of immunocom-

spot measurement. To overcome these limitations, numerous tests must be performed, thereby increasing the costs for the patient.

As a consequence, the various tests are not normally performed with sufficient frequency to properly monitor the development of a patient's disease. Development of a reasonable, selective, effective aid to adhere the immune complex to a solid carrier is essential to overcome these problems.

It is therefore an object of the present invention

to provide compositions, methods and articles for the selective adsorption or fixation of immune complexes which will be effective in detecting a wide variety of classes and subclasses of immune complexes. It is a further object of the invention to overcome the aforementioned selectivity, stability and handling disadvantages which are inherent in all other methods of immune complex adsorption to solid surfaces for the purposes of assay, removal of immune complexes from serum and the like. It is further an object of the invention to provide new, highly adaptable and easily usable aids for the detection of components of said complexes after fixation to a carrier, and further to apply such techniques for the purposes of clinical detection, removal, concentration or whatever preferably another purpose associated with human or veterinary medical applications.

Brief summary of the invention

The present invention includes new preparations, new methods and articles for the direct selective absorption, adsorption or attachment, by any mechanism, of immune complexes from serum or other body fluids, for the purpose of identification, quantification or removal. The present invention uses, in its broadest sense, immunologically non-specific peptide-bound amino acids, which have particular affinity for connecting immune complexes. As described herein, the immunologically non-specific peptide-bound amino acids and similarly modified peptide-bound amino acids are used to directly and selectively fix immune complexes from serum or other body fluids.

The immunologically non-specific peptide-bound amino acids which can be advantageously used in carrying out the present invention include oligopeptides, polypeptides and proteins as well as modified or substituted oligopeptides, polypeptides and proteins. Preferably, polypeptides and proteins that are glycosylated or modified with functionally equivalent substituents, e.g. thiosugars, hydroxy or thioamino acids, hydroxy or thiolipids, or chemically related or similar substances, shown to be capable of direct binding with immune complexes. More preferably, glycosylated proteins are used (hereinafter called glycoproteins) and most preferred are certain globulin fractions, e.g. immunologically non-specific gamma globulins, which are particularly effective in carrying out the present invention.

Whether the immune complexes comprise a single class or subclass of antibody or whether the fluid containing the immune complexes has corporeal or extracorporeal origin does not seem to be limiting for the usefulness of the present invention, as physicochemical changes of the antibodies, after binding to antigens, make them selectively exposed for immobilization.

The source of the glycoproteins chosen for use here is a special purified gamma globulin fraction (Cohn, E.J.

(1946), J. American Chemical Society 68, 459). These glycoproteins were derived from animals that were not immunized against human proteins or immune complexes derived from them; therefore, the agents are shown not to fix immune complexes via any reaction in a Fab binding site with an antigenic determinant in the analyte. Instead, they are proven to fix complexes via reaction between the carbohydrate portion found mainly in the "Fc" portion of the antibody molecule. In this way, the present invention is completely different from previously known methods that describe protein fixation of immune complexes.

While such glycoprotein preparations have long been used to prevent random adherence of free antibodies to solid supports, it is unique and very surprising to find that a coating of glycoproteins on a solid support selectively fixes antibodies that are immunologically bound to antigens. Thus, immune complexes will be adsorbed to a coating of gamma globulins, while free antibodies will not. The present invention now provides a way to substantially improve detection methods for immune complexes in serum and in blood and other body fluids. It also provides a particularly useful method for the selective removal of such complexes from the blood and the sufferer of autoimmune and other diseases characterized by the formation of immune complexes.

According to the preferred embodiment of the present invention, a film of glycoproteins selected from the group described herein is first fixed to a solid support medium to serve as a selective adsorbent for fixing any immune complex that may be present therein. fluid to be brought into contact with the film. The glycoprotein coating can be any which possesses the described ability to fix the immune complex, an immunologically non-specific bovine gamma globulin fraction obtained as described is preferred.

After fixation of the described coating to the solid support medium, the glycoprotein-coated support can then selectively adsorb immune complexes from any fluid containing immune complexes brought into contact with the coating. The fixed immune complexes can then either be: 1) analyzed with respect to their components, by means of any immunoassay method, the enzyme-linked immunosorption assay method being preferred; or 2) concentrated and purified; or 3) the fluid may be returned to the patient without the removed immune complexes, if desired, for a clinical therapeutic benefit.

In the following examples, the described film is formed by

exposing the selected gamma globulin fraction to pH-dependent denaturation; however, any form of fixation of the chosen glycoprotein is suitable as long as the ability of the film to specifically and selectively fix immune complexes is preserved. Such examples of fixation include, but are not limited to, heat aggregation, chaotropic unfolding, cross-linking by chemical means, e.g. glutaraldehyde, drying, freezing and similar methods.

Subsequent qualitative and quantitative detection of the complex is thereby made relatively easy, since enzyme-conjugated antibodies specific for any immunoglobulin class are readily available. The relevant visual or machine-readable quantification of the immune complexes sod: adheres to the support is carried out using the ELISA techniques first described by Engvall and Perlman (1971) Immunochemistry 8, 871-874 and (1972) J. Immunology 109, 129 - 235),

and The Enzyme Linked Immunosorbent Assay (ELISA) by Voller, A., Bidwell, D.E. and Bartlett, A., (1979) Dynatech Laboratories, Ind., Alexandria, Virginia, both of which are hereby incorporated by reference in their entirety. It is also possible to detect the presence of specific complex-bound antigens using enzyme-conjugated antibodies directed against the antigen.

Brief description of the drawings

FIG. 1 is a bar graph of absorbance values for specific immunoassays for normal and disease states performed according to the present invention. FIG. 2 is a bar graph of absorbance values for specific immunoassays for normal serum and serum containing immune complexes, performed on uncoated polystyrene plates. FIG. 3 is a bar graph of absorbance values for specific immunoassays using immunologically non-specific glycoproteins from the indicated sources. FIG. 4 is a bar graph of immunoassay results on

sera before and after adsorption of the immune complexes in a column that has been pre-treated with immunologically non-specific glycoproteins.

FIG. 5 is a bar graph of the results of another

embodiment of the present invention.

FIG. 6 is a bar graph of the results of applying

present invention on whole blood.

FIG. 7 is a bar graph showing the relative removal of relative immune complexes according to the present invention. FIG. 8 is a diagram showing gel filtration of human serum and resolution and detection of immune complexes therefrom. FIG. 9 is a diagram showing molecular weight-weight calibration

of the gel filtration shown in fig. 8.

FIG. 10 is a top plan view of a microtiter plate which

is adapted to form the article according to

invention and which are particularly useful in immunoassays thereof; and

FIG. 11 is a fragmented cross-section, taken along the line 11-11 in fig. 10 on an enlarged scale, showing the microtiter plate prepared for use.

Detailed description of the invention

The following definitions are provided for the purposes of clarifying aspects of this invention.

Immunologically non-specific peptide-linked amino acids: As used herein, this means oligopeptides, polypeptides and proteins, as well as modified oligopeptides, polypeptides and proteins, derived from organisms that have not been immunized against any antigenic determinant derived from any part of the animal species being tested or otherwise is associated with such species, or immune complexes derived therefrom and has the functional ability to attach to, adhere to, fix or otherwise immobilize in situ, immune complexes present in such species. In addition to the aforementioned naturally occurring substances, this definition also includes peptide-linked amino acids that are created synthetically. Such synthesis can be carried out in a peptide synthesizer or via similar chemical processes, or produced by genetic modification of existing or newly created viruses, bacteria, fungi, yeast or other recombinant, or hybrid cell vectors or hosts. It is therefore included that the defined oligopeptides, polypeptides and proteins, and modified oligopeptides, polypeptides and proteins, which originate from one species that has not been immunized against another species, or synthesized as described, should be useful in carrying out the present invention if they exhibit the ability to attach to, adhere to, fix or otherwise immobilize, in situ, immune complexes present in the tested species.

Glycoprotein: As used herein, it shall mean any combination of polysaccharide and protein or polypeptide from an immunologically naive or immunologically non-specific peptide-linked amino acid for which the two agents are linked via covalent bonds between the polysaccharide and the protein/polypeptide.

Gamma globulin: Any member of globular glycoproteins having electrophoretic mobility in the "gamma" region of serum proteins subjected to the method.

Immunoglobulin: Any member of the gamma globulin fraction that has the chemical ability to bind to another agent, such agents including proteins, carbohydrates, nucleic acids, complex lipids, simple organic compounds, or any other compound that interacts with the immunoglobulin via topographically determined binding at the "Fab "-region.

Antibody: A member of the immunoglobulin class of proteins. Mammary gland antibody molecules comprise at least two Fab and one Fc region in their structures.

Antigen: Any compound against which antibody molecules can be directed provided that binding occurs to the antibody at the binding point found in the Fab part of the antibody molecule.

Immunoglobulin classes: Immunoglobulins separated according to electrophoretic mobility. Recognized classes of immunoglobulins include, but are not limited to, immunoglobulins A, D, E, G, and M. These classes are abbreviated IgA, IgD, IgE, IgG, and IgM, respectively.

Immune complex: Any combination of antibodies and antigens in which binding occurs via interaction between Fab points on the antibody and topographical features of the antigen.

Immune response: Any response of a particular immune system during which specific antigens serve to cause the formation of antibodies directed against the antigen or to cause specific activation of cellular defense mechanism resulting in antigen engulfment, cytotoxic response or other action by cells or cell-mediated system.

Immunologically naive: The state of the immune system in a

animal or group of animals, including humans, which have never been exposed to an antigen in such a way that the product of an immune response, either humoral or cell-mediated, is produced.

Immunologically non-specific: For the purposes of this patent application, the term "immunologically non-specific" shall

mean antibodies or antigens that are not selective for or directed against components of the immune complex that exist

examination during the test. Such non-specific antibodies would be expected to be present in the serum of animals immunologically naive to the components of the immune complex, whereas immunologically specific antibodies would be found in animals that had been immunized against any component of the immune complex, including the whole complex or any part thereof .

Non-specific: Immunologically non-specific (see above).

Complement: A thermolabile substance, normally present

in serum, which is destructive to certain bacteria and other cells sensitized by a specific complement-fixing antibody.

Serum: Meant to mean the fluid component of any body fluid that remains after cells and coagulable proteins, e.g. fibrin, which may be present in such body fluidic components has been removed by appropriate physical, chemical or physicochemical means. Typically, this term refers to the remaining watery fluid that remains after the blood has clotted and the clotted blood has been removed, but in its broadest sense it is meant to include the fluidic component of cerebrospinal fluid, urine, disc fluid, cellular cytoplasm and the like .

Alkaline carbonate buffer: Unless otherwise specified, this shall mean a solution prepared to be the equivalent of 1/10 mol of sodium bicarbonate dissolved in 900 ml of demineralized water. The pH value was adjusted to 9.6 with sodium hydroxide, and the volume of the solution was adjusted to 1 liter by adding demineralized water.

Bovine gamma globulin solution: Purified bovine gamma globulins (Cohn, E.J. (1946) J. American Chemical Soc. 68, 459) were dissolved in alkaline carbonate buffer at a rate of 1.0 mg bovine gamma globulins/ml of solution.

Phosphate buffered saline: Sodium chloride was added to water to a final concentration of 9 g/l. To this solution was added 0.01 mol of potassium phosphate (monobasic). The pH value was adjusted to 7.4, and the volume of the solution was brought to exactly 1.00 liters by adding demineralized water.

Tween-20: A polyoxyethylene sorbitan monolaurate: In the description, this compound was added to phosphate-buffered saline in several steps. Where indicated, Tween-20 was used at a concentration of 0.05 vol/vol%. Non-ionic synthetic detergents such as Tween-20 is used to prevent further binding of proteins.

Conjugated antibodies: For the enzyme immunoassay part

of the determination, antibodies directed against human immunoglobulin fractions were obtained. These antibodies had been chemically conjugated with horseradish peroxidase to serve as a detection agent. Said antibody preparations were diluted between 500 and 2000 times in phosphate-buffered saline containing Tween before use.

Substrate solution: To quantify the horseradish peroxidase, a solution of orthophenylenediamine (400 µg/ml) was prepared together with 10 µM hydrogen peroxide in phosphate buffered saline. The solution was prepared just before use and stored in the dark for

to prevent photolytic decomposition.

Labeled antibodies: Any antibody substance that has been covalently or otherwise combined with a molecule or ion for the purpose of selectively identifying said group of antibodies. Such adduct molecules or ions include enzymes, fluorescent substances, radionuclides and the like.

Labeled antigens: Any antigenic substance that has been covalently or otherwise combined with a molecule or ion for the purpose of selectively identifying said group of antigens. Such adduct molecules or ions include enzymes, fluorescent substances, radionuclides and the like.

Optical density (OD) or absorbance: A number that refers to the color absorbance of a sample. Optical density is related to the percent light transmitted through the sample according to the following formula:

OD = 2 - log(% transmittance)

Proteins and polypeptides, including glycoproteins and glycopeptides, can be fixed on plastics or other solid supports including polymers, resins, glass and the like, by denaturation at a predetermined pH value or by covalent coupling, or by other physical or chemical methods, e.g. . heat aggregation, ultraviolet-mediated cross-linking with or without chemical cross-linking agents, or by other chemical or physical aids or any combination thereof. In addition, solid carriers can consist of the glycoproteins or glycopolypeptides, or similar proteinaceous materials either chemically or physically or both chemically or physically modified and it either spun into insoluble protein fibers with or without further supporting and constitutive materials such as e.g. polymers, resin, silicon dioxide or mineral fibres, or are used after treatments other than spinning, which results in a solid phase material containing the desired glycoprotein, glycopolypeptide or other similar material. As described later, such glycoprotein films or insoluble preparations possess the unusual and unexpected characteristics of selectively adhering immune complexes that may be present in a sample of fluids, including, but not limited to, fluids of biological origin that have been contacted with the film-coated solid carrier or is brought into contact with the glycoprotein preparation. What follows is a description of a preferred embodiment of the coatings according to the present invention, together with the preferred methods for adhering immune complexes, e.g. such as may be found in a sample of body fluid, including the method of detecting the complexes so bound to the solid support. Gamma globulins obtained from an animal population not previously challenged with human serum proteins are a preferred embodiment of this method.

Step 1: FIXATION OF THE GLYCOPROTEIN COATING

Purified bovine gamma globulins are contacted with a buffer comprising 0.1 molar sodium bicarbonate, pH 9.6. The final concentration of gamma globulins in the resulting solution is 1.0 mg/ml (w/v). That solution is used for the production of the glycoprotein coating.

To fix a thin film of the bovine gamma globulin to a receiving surface, 200 µl of the previously prepared solution of bovine gamma globulin is added to each well of a 96-well titer plate P, e.g. Dynatech Immulon II plate with a series of wells 20. As seen in fig. 10 and 11, the wells 20 in plate P are divided into both horizontal and vertical rows, with appropriate index systems to indicate each specific well. In the system shown in fig. 10, the wells in the vertical rows can be identified by numbers, such as

1 - 12, while the wells in the horizontal rows can be identified by letters, such as e.g. A-H, to provide an accurate correlation of the results with the samples to be tested. It will clearly appear that there are 96 wells shown on the plate in fig. 10, although the specific number of wells can be varied as desired. The plate is placed in a humidified chamber maintained at 37°C, and is removed from the chamber after 4 hours. Although the embodiment described below is described for 96-well microtiter plates, the assay method works equally well in plastic tubes, including tubes molded with internal fins to increase surface area. The coating time and bovine gammaglobulin concentration differ only slightly from what is required for plate sensitization, and the results are comparable when the method is performed in tubes, with or without fins, or in plates.

Unbound bovine gamma globulin proteins are removed by shaking the solution from the plate, then washing the plate three times with a 0.9% (w/v) saline solution containing 0.01 molar potassium phosphate, pH 7.4, leaving a thin coating 21 of bound gamma-globulin proteins. Preferably, but not necessarily, a protective layer or film 22 formed of a suitable polymeric material may be fixed over open wells 2D as shown in fig. 11.

Step 2: ADHESION OF IMMUNE COMPLEXES TO THE PLATE PREPARED ACCORDING TO STEP 1

Before the body fluid is brought into contact with the prepared plate, the fluid is properly diluted by adding aliquots of serum to phosphate-buffered saline. Dilution is necessary to give appropriate amounts of color in step 3 (the enzyme-linked assay). For most of the following examples, the optimally preferred dilution for this assay is 1:15 (volume serum:volume diluent), although wide dilution ranges may be used, depending on the nature of the body fluid subjected to the method and the assay detection techniques used are used. Preparation of the fluid may also include adding immunologically non-specific peptide-linked amino acids (as defined herein) to the fluid.

The diluted sample of body fluid is then placed

in appropriate wells in the titer plate. Adherence of the immune complexes contained in the samples to the solid carrier was enhanced

ket by incubated at 3 7°C for not less than 10 min. In the following assay examples, the adherence incubation described in this section was continued for 30 min.

After incubation to achieve complex adherence, the samples are shaken from the wells together with such non-complexed antibodies as are present in the sample. This is done since there are many more free antibodies than there are antibodies bound in complexes, and remaining free antibodies would increase the background absorbance values. The plates are then washed three times with phosphate-buffered saline, as described under step 1 above. To this buffered medium, 0.05% (volume/volume) Tween-20 is also added to further reduce further binding of the free antibodies.

Step 3: ASSAY FOR IMMUNE COMPLEX FIXED TO THE PLATE

Standard enzyme-linked assay techniques, described previously, are used for the assay of immune complexes, although any suitable detection aids, e.g. radioactive labelling, fluorescence or the like, can be used. For the examples described later, anti-human IgG induced in goats was used to clarify whether the complex contained human IgG. These antisera were coupled with horseradish peroxidase, an enzyme that gives a colored product whenever one of its substrates is present together with hydrogen peroxide. The substrate should be chosen to be in accordance with the enzyme activity added to the antibody. For the examples described below, the substrate was ortho-phenylenediamine. The product of the reaction catalyzed by horseradish peroxidase under these circumstances is nitroaniline, which has a chestnut-brown color in acidic solution. Many other substrates exist for this enzyme, and for other enzymes as well. For example, peroxidase catalyzes the oxidation of tetramethylbenzidine to an intense blue product that can be easily detected with the naked eye. Alkaline phosphatase catalyzes the removal of phosphate groups from

a large selection of substrates, including p-nitrophenyl phosphate. The product of the p-nitrophenyl phosphate reaction is p-nitrophenol,

which itself is intensely yellow in colour. Other substrates, e.g. thymol phosphate, gives different colors when exposed to phosphatase action. The description of this embodiment does not limit the assay method to any single means of detection,

either by direct attachment of enzymes such as e.g. horseradish peroxidase, alkaline phosphatase, beta-galactosidase or the like, or to chemical reagents, rather the assay method will work equally well regardless of the detection core chosen. Such detection schemes may be, but are not limited to, enzyme-linked immunoassay methods, whereby the enzyme used to detect the presence of the complex may be linked either to an antibody or to an antigen or other component selective for the presence of the immune complex which fixed to the coating, fluorescent antibody methods whereby any fluorescent substance of the antibody or antigen or any other substance is used to detect the presence of the immune complexes fixed to the coating by a chemical, physical, physicochemical or other kind of aid, or any another direct method for detecting the presence of immune complexes that can be fixed by plating. In addition, indirect means of detection are also equally useful for the detection of immune complexes by this assay method. Such indirect aids include, but are not limited to, second-antibody techniques, biotin-avidin or biotin-streptavidin or similar adjunctive amplification schemes, or any other indirect means of detecting the presence of immune complexes in the coating. Furthermore, the assay method is equally useful if biological aids to detect the presence of the immune complex are fixed to the coating. Such aids include the use of living or killed cells obtained either from cell culture or from living or dead organisms, whether prokaryotic or eukaryotic,

and without regard to colonial or solitary organization. Any component derived from such biological preparations, including but not limited to nucleic acids, nuclear components, cytoplasmic or membrane components, lectins, receptors, opsonins and similar materials, may be used to detect the presence of immune complexes fused to the coating, provided that such lectins, receptors, opsonins, or the like have the ability to interact with immune complexes or any component thereof. Finally, detection of immune complexes fixed to the coating by any aid either chemical or physical is also equally applicable to the assay protocol ranging from the embodiment

as stated here. Such aids may include, but are not limited to, changes in the pH value of the solution, polarity of light, changes in refractive thicknesses, changes in electronic characteristics such as conductivity or piezoelectric behavior or dielectric constants of capacitors, changes _l. the specific heat content of the surface, or any other physical means used to demonstrate the presence of the materials in close proximity to other materials.

The enzyme-conjugated goat antibody solution, prepared

as described, is then added to each of the wells in the titer plate. Binding of these probe antibodies to the human IgG fraction of the complex is allowed for at least 15 min. at 37°C. The plate is removed from the incubation chamber, emptied of its contents and washed as in step 2. The last washing is best carried out with TVeen-

free buffer to avoid inhibition of attachment of the horseradish peroxidase enzyme.

The amount of enzyme marker, as described earlier, is determined by incubating the plates with a solution of phosphate-buffered saline containing ortho-phenylamine-diamine (400 µg/ml) and 3-10 micromolar hydrogen peroxide at room temperature in the dark. The reaction is allowed to continue for 10 min., or until sufficient color can be read on the spectrophotometric device used. The reaction is then stopped via the addition of the same volume of 2.5 molar sulfuric acid, and the color intensity (the optical density, or OD or absorbance) is read with a spectrophotometric instrument, e.g. Dynatech MR600 or similar.

As with any enzyme-linked immunoassay, the resulting color of the reaction product is proportional to the number of conjugated antibodies that have bound to the immune complex. For most cases, the number of bound conjugated antibodies is linearly related to the number of human IgG molecules present in the immune complex. Therefore, as the amount of immune complex fixed on the film increases, so does the optical density, or absorbance of the enzyme reaction.

EXAMPLES

EXAMPLE 1

Selectivity of glycoprotein films for absorption of immune complexes from serum

A polystyrene plate was coated with a film of bovine gamma globulins by the following method: 1) Purified bovine gamma globulins from a Cohn preparation fraction II were dissolved in an alkaline buffer (0.1 M sodium carbonate, pH 9.6). 2) The alkaline solution containing the bovine gamma globulins was contacted with the plate for 4 hours at 37°C. 3) The coating solution was shaken from the plate, after incubation. The plate was washed three times with a solution of sodium chloride (0.15 M), buffered with potassium phosphate (0.01 M, pH 7.4)

for the removal of unbound gamma globulin proteins.

The bovine gammaglobulin-coated plate was then used

to determine the presence of immune complexes in human serum samples taken from people with:

1) no visible pathology; (NORMAL)

2) systemic lupus erythematosus in remission (REM SLE); 3) moderate systemic lupus erythematosus or advanced systemic lupus erythematosus, (SVR SLE);

and

4) moderate rheumatoid arthritis (MOD RA).

These sera had previously been shown to contain immune complexes appropriate for their pathological status by C1q ELISA tests and radial immunodiffusion methods.

A representative immune complex assay, to be described later, using the bovine gamma globulin coated plates, prepared as previously described, is shown in Table 1 and graphically in Fig. 1. The methods used to derive these data are summarized below: 1) Sera were properly diluted with a solution of 0.15 M NaCl in 0.01 M sodium phosphate buffer (pH 7.4) before the test (in which the following denoted PBS). In this example, a volume of serum was diluted with

15 volumes of PBS.

2) 0.1 ml aliquots of the diluted sera were placed in appropriately labeled wells. For this example, 7 replicate determinations were performed on each sample and averaged. 3) The plate containing the samples was placed in a humidified incubator at 37°C for 30 min. After the incubation, the plate was removed from the incubator, the liquid shaken out and the plate washed three times with PBS containing 0.5 ml polyoxyethylene sorbitan monolaurate (Tween-20) per liter of solution. 4) The plate was exposed to a mixture containing horseradish peroxidase-conjugated antibodies specific for the gamma chain of human immunoglobulin C. The plates were incubated at 37°C for 15 min. to attach the conjugated anti-IgG. After the incubation, the plates were washed three times with PBS for removal

of the unbound enzyme-conjugated antibodies.

5) Each well of the plate was examined for horseradish peroxidase activity by addition of PBS containing o-phenylenediamine and hydrogen peroxide. The presence of complexes was thus demonstrated by the intense chestnut-brown color that appeared after the reaction was terminated with sulfuric acid. The color was quantified at 490 nm using a Dynatech MR 500 plate reading spectrophotometer. Reagent blanks for spectrophotometric calibration were wells in the plate that had not been brought into contact with human serum.

Explanations:

The numbers given under AVERAGE OPTICAL DENSITY (AVERAGE ADSORBANCE) refer to the optical density read at 490 nm obtained during the assay described above.

The figures given are averages of the results

from 7 assays per try.

The number under the heading Standard error refers to the standard error of the mean.

Abbreviations:

SLE: Systemic Lupus Erythematosus

RA: Rheumatoid Arthritis

The ability of bovine gamma globule-treated support media to adsorb immune complexes is demonstrated by the increased absorbance shown in wells exposed to serum taken from individuals diagnosed with immune complex disease. Furthermore, good agreement was observed between the severity of the disease and the amount of color found by the method. Normal human serum did not extract much color on examination, providing further evidence of the specificity of the coated plates for immune complexes. The same method has been shown to work equally well for whole blood and anticoagulated plasma.

EXAMPLE 2

Adsorption of immune complexes from serum by uncoated polystyrene plates

To demonstrate the beneficial effect of glycoprotein coating, a polystyrene plate was left uncoated, but was exposed to the sera described in Example 1 under identical incubation conditions. The plate was then exposed to peroxide-conjugated anti-IgG and probed for peroxidase as before. As expected, only weak selectivity is shown by untreated plates. In addition, very high background values were also observed, unlike the selectivity and the background values found for plates that are pre-coated with glycoproteins such as e.g. bovine gamma globulins. The results obtained by this manipulation are shown in table 2 and graphically in fig.

2. Each post height shown in fig. 2 represents the mean adsorbate for 7 replicate assays.

Explanations:

As in Table 1, the numbers given under the heading Optical Density refer to the AVERAGE OPTICAL DENSITY (AVERAGE ADSORBANCE) at 490 nm obtained during the assay described above.

The figures given are averages of the results

from 7 assays per try.

The number under the heading Standard error refers

to standard error of the mean.

Spectrophotometric blanks were performed using uncoated plastic wells that had not been exposed to human serum. Such blanks were processed

with all the following steps, including exposing them to conjugated antibodies and peroxidase assay. The blank test vein showed no visible color.

Abbreviations:

SLE: Systemic Lupus Erythematosus

RA: Rheumatoid Arthritis

EXAMPLE 3

Purified gamma globulins from a selection of species were used for plate processing as described in Example 1. The plates were then exposed to immune complex-rich or immune complex-poor sera as described in Example 1. After exposure and washing, the plates were examined for the presence of bound immune complexes using horseradish-peroxidase-conjugated anti-IgG as described in example 1. The results of these treatments are shown in table 3 and graphically in fig. 3.

Explanations:

The figures given in Table 3 refer to the AVERAGE OPTICAL DENSITY (AVERAGE ADSORBANCE) at 490 nm obtained following the treatment mentioned above for plates coated with gamma globulin fractions recovered from the species listed in the left column of the table.

Serum sources are indicated along the upper edge of the table. Abbreviations:

SLE: Systemic Lupus Erythematosus

RA: Rheumatoid Arthritis

The fact that gamma globulin mediates the ability of coated carriers to specifically fix immune complexes is illustrated by comparing Table 3 with Table 1. Each of the coating groups was able to fix immune complexes from certified human sera appropriate for the pathological status of the the human source. Therefore, all gamma globulin coatings tested resulted in selectivity for immune complex adsorption, although different qualities may be observed for different coatings. The same method should work equally well for whole blood or anticoagulated plasma.

EXAMPLE 4

Removal of immune complexes by coated polystyrene beads

An alkaline preparation of bovine gamma globulins (1 mg bovine gamma globulins/ml in pH 9.6 carbonate buffer, as in Example 1) was used to treat polystyrene beads (BioBeads, SM4,

20 - 50 mesh, BioRad Laboratories, Richmond, California) for the purpose of adhering the immune complex to the beads. Serum-containing immune complexes, previously examined for their immune-complex level as in Example 1, were passed through a column of said beads maintained at 37°C, and the amount of immune complexes was determined both before and after column filtration. The results from this method are shown in table 4 and graphically in fig. 4.

Explanations:

Separately, serum samples from unaffected and from known cases of systemic lupus erythematosus were passed over columns treated with alkaline bovine gamma globulins as described previously.

Immune complex assays were performed before and after treatment (pre- and post-column).

The optical densities shown by the samples before and

after treatment is listed in the table.

Abbreviations:

SLE: Systemic Lupus Erythematosus

Polystyrene beads coated with bovine gamma globulins in this way selectively and rapidly adsorbed immune complexes. Approximately 90% of the complexes were removed in one pass through the column. Alkaline gamma globulin treatment of polystyrene beads is thus effective in relatively quickly removing these agents from serum samples. Therefore, a sufficiently large column separated by similar glycoproteins should remove significant amounts of immune complexes when whole blood is passed through the column at flow rates that may enable the present invention to be used in therapeutic treatment methods. Serum treatment will in this way facilitate the selective removal of circulating immune complexes that are present. Furthermore, column isolation of immune complexes from large amounts of blood will provide concentration of said complexes, as it became possible to detect small components such as e.g. immunoglobulin idiotypes, rare subclasses of antibodies and rare or sequestered antigens that may be present in said complexes. The same method should work equally well for anticoagulated plasma.

EXAMPLE 5

The purpose of this example is to illustrate the beneficial effect of liquid phase bovine gamma globulin on immune complex assays performed on immobilized glycoprotein coatings. This supplement proved highly effective in reducing baseline values for this normal serum sample containing low levels of immune complexes. For this example, two serum samples were tested.

The first of these samples originated from an individual who showed no visible clinical pathology, but nevertheless reacted positively in immune complex assays as described earlier. The second serum sample was obtained from an individual who had a severe case of systemic lupus erythematosus. These two serum samples were diluted with 0.15 M NaCl containing 0.01 M potassium phosphate pH 7.4 (phosphate-buffered saline), or with phosphate-buffered saline containing bovine gamma globulin at a concentration of 1 mg of bovine gamma globulin per ml puffer.

After dilution of the samples, each of the four preparations was tested for immune complexes according to solid-phase assays as described previously, but especially in examples 1-3. This maroon color resulting from the enzyme-linked portion of the method was read at 490 nm on a Dynatech MR600 plate reader, and the results are both reported in the accompanying table and plotted on a bar graph.

Explanations:

Serum from stored blood was used for the tests in the table above. The term "normal" is used herein to denote serum derived from an individual showing no clinically apparent manifestation of disease at the time of phlebotomy. The term "SLE" is used in this example to denote serum derived from an individual clinically diagnosed as suffering from systemic lupus erythematosus. Before this assay, 1 part serum was diluted with 15 parts diluent. For the "BGG Absent" column, the diluent used in this example was 0.15 M NaCl buffered to pH 7.4 with 0.01 M potassium phosphate. For the "BGG Present" column, bovine gammaglobulin was dissolved in phosphate-buffered saline (defined earlier) to the extent of 0.5 mg bovine gammaglobulin per

ml of phosphate-buffered saline.

The diluted sera were subjected to the immune complex assay described in Examples 1-3. The numbers presented in the table represent the average absorbance for four replicate determinations.

In fig. 5 shows results from serum samples diluted either with phosphate-buffered saline or with phosphate-buffered saline containing 1 mg of bovine gamma globulin per ml. After dilution, the samples were assayed for immune complexes using the solid phase assay described in Examples 1, 2 and 3. The height of the bar in FIG. 5 represents the mean absorbance found after 4 replicate assays. In fig. 5 normally refers to serum obtained from an individual showing no apparent clinical pathology. SLE refers to serum obtained from an individual suffering from severe systemic lupus erythematosus. -BGG refers to serum diluted in phosphate-buffered saline alone, while +BGG refers to serum diluted in phosphate-buffered saline containing 1 mg of bovine gamma globulin per ml.

Addition of bovine gamma globulin to the serum diluent reduced the absorbance found in the normal serum sample approximately 40-fold. In fact, very little color was observed when this assay was performed on bovine gammaglobulin-diluted normal serum. Conversely, dilution of serum obtained from an individual with an authentic autoimmune disorder with bovine gamma globulin-containing buffer produced very little change in color, either visually or as detected by the spectrophotometric effect. Accordingly, a 16-fold difference between serum in normal and autoimmune conditions was noted after dilution with bovine gamma globulin-containing buffer, while no differentiation existed without such addition. In further similar assays, this method actually eliminates false positives for individuals without visible pathology while individuals with significant disease remain elevated. These results clearly indicate the beneficial effect of incorporating proteins similar to bovine gamma globulin into diluents, such an effect being effective in eliminating the false positive rate without significantly altering the same positive rate.

EXAMPLE 6

The foregoing examples illustrate the utility of the immune complex filtration method described herein. The data presented in this example demonstrate that the fixation techniques described earlier can also be applied to plasma and whole blood, regardless of which common anticoagulant is used.

For this example, blood was taken from a healthy donor and from a subject with rheumatoid arthritis. Four glasses were taken from each donor. The first glass contained no anticoagulants. The three remaining glasses contained anticoagulants. The second glass contained sufficient ethylenediamine tetraacetate (EDTA) to make the final concentration 1.5 mg EDTA/ml blood; the third vial contained sufficient heparin to make the final concentration 28 USP units of heparin/ml of blood; the fourth glass contained sufficient sodium citrate to make the final concentration 3.5 mg sodium citrate/ml blood.

The samples were prepared and analyzed as follows:

The first pair of samples (one each from normal and CIC-elevated subjects) was allowed to grow normally, and the sera were collected. These sera were subjected to the immune complex assay methods described in Examples 1 and 5.

An aliquot of whole blood was taken from each of the other pair of samples anticoagulated with EDTA and subjected to the immune complex assay as described in Examples 1 and 5. The plasma components from each sample were separated by centrifugation and the plasma samples analyzed for immune complexes as described in examples 1 and 5.

An aliquot of whole blood was taken from each of the third pairs of specimens, anticoagulated with heparin and analyzed as above. The plasma from each pair of samples was separated by centrifugation and analyzed as above.

An aliquot of whole blood was taken from each of the fourth pairs of specimens, anticoagulated with sodium citrate and analyzed as above. The plasma components from each sample were separated by centrifugation and analyzed as above.

The results of these investigations are shown in table 6, and illustrated graphically in fig. 6.

Referring to Table 6, it is clear that the addition of anticoagulant to whole blood for the purpose of preventing clot formation has no detrimental effect on the method's ability to discriminate between samples containing normal and elevated amounts of immune complexes. In general, the addition of anticoagulants appeared to improve assay performance slightly, as evidenced by the double-deficient values observed for serum as opposed to plasma or whole blood samples. This finding fundamentally excludes mechanisms of immune complex adherence in this method based on components in serum such as e.g. complement or conglutinin. Such agents require calcium ions for activity, and the addition of either EDTA or citrate removes free calcium from blood. As a result, it can be said that this example both illustrates the usefulness of the thin-glycoprotein film method for immune complex fixation and highlights the surprising properties of such thin-glycoprotein films as used in the method.

Serum samples from people showing immune complex disorders are significantly elevated compared to those from normal individuals, regardless of whether the blood from these individuals has been inhibited against clotting or not, and regardless of the anticoagulant used. Because of this behavior, the method described for the fixation of immune complexes can be used with plasma or whole blood samples for analysis or for the removal of such complexes that could be present in them according to the described processes. It is obvious from the foregoing tests that there is no reason to believe that the method of removing immune complexes described here is limited to those found in serum, plasma or blood. It is obvious that immune complexes which are present in various sources such as e.g. urine, ascites fluid, cerebro-spinal fluid or from in vitro mixtures that have been made in the laboratory, will have the ability to be fixed to the glycoprotein coating described here and thereby greatly enhance the usefulness of the invention described here. As shown in Example 7, the principle of immune complex removal from anticoagulated whole blood and/or plasma by passage through a column made of glycoprotein coated beads also applies

on the therapeutic apheritic removal of immune complexes from patients affected by auto-immune disorders.

Explanations: Samples were obtained either from an individual free of clinically visible pathology (Normal) or from individuals with rheumatoid arthritis (RA) or vasculitis. Serum refers to serum samples obtained after centrifugation of clotted blood. Plasma refers to the supernatant obtained after centrifugation of whole blood containing an anticoagulant. Anticoagulants are abbreviated as follows: hep, heparin; EDTA, ethylenediaminetetraacetate; one, citrate. When indicated in the table, anticoagulated whole blood was tested. Factors refer to volume corrections that are necessary due to added volume (citrate) or cell volume in whole blood. Citrate solutions made up 10% of the final volume. Hematocrits were taken as 34% of blood volume for normal values and 44% for CIC-elevated samples. Times elevation refers to the adsorbance in any given CIC elevated sample divided by that found for

the respective normal value.

Four blood samples were taken from each subject, one in a glass that did not contain anticoagulants and three separately that contained different anticoagulants. The following abbreviations refer to the anticoagulant contained in thus obtained samples: 1) hep - heparin, 28 IU/ML; 2) EDTA - ethylenediaminetetraacetate, 1.5 mg/ml; 3) eit - sodium citrate, 3.5 mg/ml. Whole blood, plasma or serum (SER) taken from the samples were subjected to the assay described fully in Examples 1 and 5.

In the figure, the height of the bar represents the absorbance which

was measured by following the assay method. The data from this table is shown graphically in fig. 6.

EXAMPLE 7

As indicated in Example 3, columns prepared from glycoprotein-coated solid supports exhibit the capacity to extract immune complexes from serum samples. Further use of such columns is provided by the data presented in this example. Samples of body fluids such as blood, is often prevented from clotting by the addition of anticoagulants, e.g. heparin, ethylenediaminetetraacetate (EDTA) or salts of citric acid. Such compounds inhibit clotting either by interfering with the proteolytic steps leading to fibrin activation (heparin), or by chelating calcium (EDTA or citrate). In this example, it is shown that such agents as heparin, EDTA or citrate are without effect on the ability of glycoprotein-coated solid supports to adsorb or fix immune complexes regardless of whether the sample passed through the column was whole blood or plasma derived therefrom. Furthermore, immune complex fixation in the presence of these anticoagulants was at least equal to that of serum containing no anticoagulants. The concentrations of anticoagulants tested for this example were higher than those normally used for apheretic purposes in humans. Because these concentrations of anticoagulants did not show any harmful effects on immune complex adsorption, it is obvious that the intermediate anticoagulant concentrations used for apheretic purposes will likewise be without harmful effects on immunoadsorption of complexes.

For the purposes of this example, blood samples were taken from a subject afflicted with rheumatoid arthritis in vials containing either heparin, EDTA or sodium citrate. A control sample was also held in a glass containing no anticoagulant. The control sample was allowed to be delivered and the serum reserved. Immune complex assay was performed as described in Example 1, with the exception that the incubation for immune complex detection (second incubation) was performed with horseradish peroxidase-conjugated goat antiserum, directed against immune classes IgG, -M and -A was used instead of the IgG-specific antibodies that were used in example 1. The use of such multivalent antibodies as these (IgG, -M and -A-specific) further enhances the chemical sensitivity of the test and thereby improves the general applicability of the adsorption method presented here.

After performing the immune complex assay, 0.25 ml aliquots of undiluted samples were added to columns prepared with BGG-coated polystyrene beads as described for example 3. The columns were incubated at 37° for 30 minutes, as before.

Then the sample was eluted as a bolus, and was therefore not very diluted. Additional phosphate-buffered saline was passed through the column to make the final dilution 1:16 and to chase any loosely bound immune complexes off the column. The resulting diluted eluate was then assayed for immune complexes without further dilution. A further control sample was also prepared in which serum was incubated in contact with untreated polystyrene beads. Eluates from this column were also examined as indicated above.

Percentage of immune complexes removed by the method,

was calculated as follows:

The results from this method are shown in table 7 and illustrated graphically in fig. 7.

With reference both to table 7 and fig. 7, it is clearly stated that the presence of anticoagulants has no detrimental effect on the ability of glycoprotein-treated carriers to remove immune complexes from either plasma or whole blood. This is expected, as calcium is not required for the glycoprotein-immune complex interaction that fixes the complexes to the carrier as taught in the previous examples. Uncoated polystyrene support materials were unable to fix significant amounts of immune complex, again consistent with the principles taught in Examples 1-3.

As demonstrated by the teachings of this example, glycoprotein films applied to suitable support materials will be very useful for the purpose of removing immune complexes from whole blood or from plasma, regardless of the anticoagulant that may be present. This allows immune complexes to be adsorbed from the blood for the therapeutic treatment of any immune complex-associated disease or disorder. It also adds to the utility of the method since the column can serve to concentrate certain rare antibody or antigen types present in the immune complexes. After elution of the concentrated complexes from such columns, detection of rare antigens or antibodies can be performed with great ease using the assay method taught in Examples 1 and 5.

Explanations: Removal of immune complexes from serum, plasma and whole blood using columns of glycoprotein-coated polystyrene beads.

Blood was drawn into separate "vacutainer" glasses, each containing a different anticoagulant. The glasses were labeled

as follows:

Heparin, final blood concentration 28USP units/ml. Citrate, final blood concentration of sodium citrate on

3.5 mg/ml.

EDTA, final blood concentration of sodium EDTA of 1.5 mg/ml.

After collection, an aliquot of each blood sample was subjected to centrifugation and the plasma drained. These plasma samples were used in each case indicated in the table. Whole blood refers to the anticoagulated sample before centrifugation. Serum refers to a blood sample that was drawn into a glass

and allowed to settle before separation of the fluid component.

For column extraction of immune complexes, a 0.25 ml aliquot of each sample listed in the table was added to each of 8 columns and allowed to incubate for 30 min. at 37°C. The remaining serum components were eluted from the columns with 3.76 ml of PBS and the eluate analyzed for immune complexes. Percent complexes removed were calculated as indicated in the accompanying text.

For comparison purposes, serum control samples were also passed through columns prepared with thin glycoprotein films on polystyrene supports. Serum refers to a blood sample that has been drawn into a glass and allowed to settle before separating the fluid component. Such serum control samples were free of anticoagulants. The purpose of such serum control samples was to demonstrate that, regardless of how high or low the anticoagulant concentration was, there was no deleterious effect of said anticoagulants on the ability of glycoprotein-coated substrates to remove immune complexes from fluids for either purpose of assay or immune complex immunoadsorption from blood or plasma for the purpose of therapeutic apheretic applications.

For column extraction of immune complexes, 0.25 ml aliquots of each sample as indicated in the table were added to each of 8 columns and incubated for 30 min. at 37°C. The remaining serum components were eluted from the columns with 3.75 ml PBS and the eluate examined for immune complexes. The percentage of complexes removed was calculated as indicated in the accompanying text.

In fig. 7 is the sample that was added to the column indicated below the bar. The height of the bar represents the percent immune complexes removed from the sample. The abbreviations used are as follows:

SER: Serum

N/C: Serum passed over a column of uncoated

polystyrene

H/WB: Heparin-treated whole blood.

H/P: Plasma derived from heparin-treated

whole blood.

C/WB: Citrate-treated whole blood.

C/P: Plasma derived from citrated whole

blood.

E/WB: EDTA-treated whole blood.

E/P: Plasma derived from EDTA-treated whole blood.

EXAMPLE 8

Films of glycosylated polypeptides have the ability to fix immune complexes that include wide variations in molecular size and composition. To illustrate this capability, plasma derived from a subject suffering from systemic lupus erythematosus was subjected to column chromatography over a cylindrical column of agarose A-15M (BioRad) measuring 1.6 x 35 cm and capable of separating molecular substances in according to size over a range of 40,000 - 15,000,000 daltons. The column was eluted with phosphate buffered saline. The protein content was monitored in the eluate using an Isco UV monitor, set at 280 nm. The eluate was then fractionated into 2.8 ml aliquots as it flowed from the column, and each fraction was examined for immune complex content as described in the examples for IgG-, IgM- and IgA-containing complexes using peroxidase-labeled anti-human IgG, IgM or IgA as applicable for the method.

With reference to fig. 8, it appears that the peak immune complex binding, which appears by the immune complex assay method, was significantly shifted from the protein peaks (revealed by the OD280 monitor). Furthermore, complexes containing a predominant class of immunoglobulin behaved distinctly as calibration of the column (Fig. 9) revealed that the immune complexes present in this patient had molecular weights ranging from 200,000 to more than 5,000,000 daltons. The utility of the assay method described here can best be understood in the light of such characterization studies. Very few assay methods have such wide areas of application for the measurement of immune complexes. Explanation: Fig. 8 Agarose A15M gel filtration of human immune complexes in serum.

Immune complexes were separated by size on a column of Agarose A15M gauge with a diameter of 1.6 cm and a length of 35 cm. The abscissa and coordinate indicate elution fraction number and adsorbance, respectively. One column volume corresponds to approximately 25 fractions. A 1.0 ml sample of human serum obtained from an individual suffering from systemic lupus erythematosus was applied to the top of the column at fraction 0. Separation and elution was performed with phosphate-buffered saline as described previously (0.15 M NaCl with 0.01 M potassium phosphate, pH 7.4.

In fig. 8, P represents the absorbance monitored at 280 nm with an Isco column monitor. The void volume in the column is marked by the arrow Vq. The absorbances resulting from immune complex assays specific for IgG, IgA and IgM

is indicated by the appropriate line (marked G, A or M). The absorbance for IgM-containing complexes (curve M9) is the result of enhanced sensitivity of the IgM probe that was used for the analysis. The sensitivity of G- and A-specific antibodies turns out to be approximately the same under controlled conditions in the laboratory.

Explanation: Fig. 9: Calibration of Agarose A15M column:

The chromatography column described in the legend to fig. 9, was subjected to calibration using a selection of proteins to determine the molecular weight range of immune complexes detected by the immune complex assay. Five different column elutions were performed, one each with purified aldolase (M.V. 158,000), IgG (M:V. 170,000) catalase (M.V. 232,000), ferritin (M.V. 440,000) and thyroglobulin (M.V. 670,000). The void volume was determined in a sixth experiment using both aggregated protein (M.V. >20,000) and blue dextran (M.V. range between 1,000,000 and 50,000,000). The column volume was determined by ribonuclease chromatography (M.V. 13,000). The elution K^ value indicated on the ordinate was calculated with the following formula:

The log of the molecular weight is plotted on the abscissa in fig. 9. The resulting line was extrapolated to the Y-intercepts, giving an exclusion limit of 10,000,000 daltons, well within the manufacturer's estimate of 15,000,000. Molecular weights of protein fractions subjected to chromatography on this medium can be determined by first calculating Kav , then refer to fig. 9 for the molecular weight.

EXAMPLE 9

The purpose of this example is to further illustrate the utility of the assay method for immune complexes as described previously. Many auto-immune disorders, e.g. systemic lupus erythematosus, it is difficult for the doctor to diagnose at an early stage, but still they give complexes that include an antigen or a small family of antigens. If these immune complexes could be tested for their antigenic components, it would greatly simplify disease recognition and diagnosis.

If one brings the coated plates described in example 1 into contact with blood serum or plasma or whole blood, then the immune complexes, including the antigenic components and exposed Fab binding points thereof, will adhere to the coating and thus to the solid support. Addition of either enzyme-labeled antibodies specific for antigens or families of antigens unique to the given autoimmune disease, or enzyme-labeled antigens that may be present in the complex, which can bind to exposed Fab portions of the immune complex, will then demonstrate presence of the complexed, disease-specific, antigen that is in question, by development of the characteristic color that comes after the addition of substrate. On the other hand, the immune complexes should contain neither such antigens nor Fab regions which may be specific or selective for the disease, because then the addition of enzyme-labeled antibodies or antigens will fail with regard to forming color after substrate addition. As a result, antigen specificity for immune complex testing will greatly simplify the work of auto-immune disease diagnosis and monitoring of the disease's development, which is necessary for good medical care and practice. The utility of this method can be readily realized by one skilled in the art of healing.

The foregoing examples serve to illustrate the effectiveness and utility of thin glycoprotein films for fixing immune complexes to solid supports. While the illustrative examples have shown that the antibody molecules present in the complexes can be readily determined, there is no reason to limit the detection method to the antibody classes or subclasses contained therein. A further benefit of the method is provided by the use of antigen-specific detection means by which the disease state can be determined. The following descriptions illustrate ways of performing such detection.

Antigen and antibody components of immune complexes have unoccupied binding sites that can be detected via specific interaction with labeled or conjugated materials. As a result, immune complex fixation as described in Examples 1, 3 and 4, followed by incubation with an enzyme-conjugated antigen, will lead to subsequent fixation of the conjugated antigen. This property will lead to an antigen-specific enzyme-catalyzed reaction if the antibodies directed against the antigen are present in the complex. As a result, the enzyme-immunoassay method will be positive if the complex contains antibodies directed against the antigenic substance, while the enzyme-immunoassay method will be negative if these components are absent. This method offers the additional benefit of allowing disease-specific query regarding the exact antibody composition of complexes. Conjugation need not be limited to enzymes. Addition of fluorescent chemicals, e.g. fluorescein or similar to the antigen will cause fluorescence to the complex if said antibody substances are present; likewise, conjugation of the antigen with a radionuclide will give radioactivity to the complex, provided that said antibody substances are present in the previously fixed complex. Many other detection methods also exist and each of these methods will give positive indications provided that the antibody component exists in the complex fixed according to the methods described here.

Likewise, the presence of precise antigens in complexes, as described herein, will also allow detection of such antigens using conjugated antibodies directed against this antigen. As a result, if one incubates enzyme-conjugated antibodies with an immobilized immune complex, prepared by the methods described here, the enzyme-catalyzed reaction will indicate the presence of said antigens in the complex. Conjugation, again, need not be limited to enzymes. Addition of fluorescent chemicals such as fluorescein or similar to the antibody will cause fluorescence to the complex if said antigenic substances are present. Likewise, conjugation of the antibody with a radionuclide will result in radioactivity to the complex, provided that said antigenic substances are present in the previously fixed complex. As with the detection of antibodies described, there are also many other detection methods for the antigens, and each of these methods will give positive indications provided that the antigenic component exists in the complex fixed by the methods described here.

The described technology for coating carriers with immunologically non-specific peptide-linked amino acids including oligopeptides, modified oligopeptides, polypeptides, modified polypeptides, proteins and modified proteins, which have the ability to directly and selectively fix immune complexes by absorption, adsorption or other mechanisms of attachment, has wide useful either in analyzing body fluids for the presence of circulating immune complexes and in removing circulating immune complexes. In particular, glycosylation of any of the previously described immunologically non-specific peptide-linked amino acid-based classifications should produce the most useful materials for the isolation and identification of immune complexes. The ease of use and the widespread applicability of this technology will be readily realized by the person skilled in the art, after he has thoroughly familiarized himself with the method. The treatment of solid carriers in this way provides many important and useful solutions for the detection and treatment of autoimmune diseases, as a large selection of new methods is suggested. Such methods may include, but are not limited to, the detection of the antibody class and subclass composition of the immune complex, the determination of the antigenic nature of the immune complex, and the removal of such complexes that may be present in serum, other body fluid, or fluid from any source, whether it is corporeal or extracorporeal. The techniques described here therefore have wide applicability and broad utility for directly detecting circulating immune complexes in serum and for directly removing circulating immune complexes from serum.

Without being bound by any specific theory, it is contemplated that varying degrees of glycosylation or other similar, functionally equivalent substituents for immunologically naïve species of the disclosed oligopeptides, polypeptides and proteins will produce a family of materials capable of directly creating association with immune complexes present in serum, for the purpose of detection and removal.

It is included that the inventive ideas described here can be varied in other ways, and it is intended that the accompanying claims should be understood to include alternative embodiments of the invention with the exception of what limits it by the state of the art.

Claims (12)

1. Method for performing an immunoassay of a body fluid sample to determine the composition and/or concentration of immune complexes present in said sample, characterized by: introducing the sample to be examined and an immunologically non-specific peptide-linked amino acid onto a recipient, said peptide-linked amino acid adhering to said recipient and having the ability to fix the immune complexes that may be present in said sample to said recipient; allowing said peptide-linked amino acids to fix immune complexes present in said sample to said recipient; and processing said fixed immune complexes in a manner that will produce an indication of the composition and/or concentration of the immune complexes and antibody and antigen components present in said sample. 2. Method as set forth in claim 1, characterized in that it includes, in steps in sequence, treating said recipient with a solution containing immunologically non-specific peptide-bound amino acid to form a layer of said peptide-bound amino acid on said recipient, said recipient preferably comprises a solid phase base having aids formed thereon for receiving a sample to be examined, and then placing said sample to be examined on said receiver. 3. Method as stated in claim 1 or 2, characterized in that the immunologically non-specific peptide-bound amino acid is selected from molecules consisting of oligopeptides, modified oligopeptides, polypeptides, modified polypeptides, proteins and modified proteins, preferably glycosylated polypeptides and glycosylated proteins , which molecules are further characterized by their affinity for immune complexes; wherein the glycosylated protein is preferably selected from a globulin fraction, preferably originating from a vertebrate, more preferably a mammalian species, most preferably bovine gammaglobulin, equine gammaglobulin, lagomorph gammaglobulin, canine gammaglobulin, human gammaglobulin, ovine gammaglobulin, caprine gammaglobulin, murine gammaglobulin, procine gammaglobulin, feline gammaglobulin, or the Cohn fraction from any of the aforementioned gammaglobulins, especially the Cohn fraction from bovine gammaglobulin; or where said glycosylated peptide that is selected is chemically synthesized, or in organisms with altered genetic structure resulting from recombinant or hybrid cell techniques or other techniques that modify or alter cells to cause the modified cells to produce the desired protein. 4. Method for removing immune complexes from body fluid, characterized by the following steps: 1) fixing a material containing molecules of immunologically non-specific peptide-linked amino acid to a substrate under conditions preselected to facilitate the fixing of such material to said substrate to form a coating thereon; 2) bringing said coating into contact with specific body fluid containing immune complexes, in a manner that will facilitate adherence of the immune complexes contained in the body fluid, to said coating; whereby immune complexes present in said body fluid are fixed to said coating on said substrate. 5. Method as stated in claim 4, characterized in that the material containing immunologically non-specific peptide-linked amino acid contains peptide-linked amino acid molecules selected from molecules consisting of oligopeptides, modified oligopeptides, polypeptides, modified polypeptides, proteins and modified proteins, preferably glycosylated peptides and glycosylated proteins, which molecules are further characterized by their affinity for immune complexes; wherein the glycosylated protein is preferably selected from a globulin fraction, preferably derived from a vertebrate, more preferably a mammalian species, most preferably bovine gammaglobulin, equine gammaglobulin, lagomorph gammaglobulin, canine gammaglobulin, human gammaglobulin, ovine gammaglobulin, caprine gammaglobulin, murine gammaglobulin, porcine gammaglobulin, feline gammaglobulin or the Cohn fraction from any of the aforementioned gammaglobulins, especially the Cohn fraction from bovine gammaglobulin; or where said glycosylated peptide that is selected is chemically synthesized, or in organisms with altered genetic structure resulting from recombinant or hybrid cell techniques or other techniques that modify or alter cells to cause the modified cells to produce the desired protein. 6. Method for removing immune complexes from body fluid, characterized by the following steps: 1) fixing a material containing an immunologically non-specific peptide-linked amino acid molecule to a substrate by incubation at a preselected temperature for a sufficient period of time to complete the fixing of a coating of such material on said substrate; 2) contacting said fixed coating on said substrate with a solution such as immune complex and phosphate buffered saline; and 3) inducing adherence of the immune complexes contained in the solution to said coating by incubation at a preselected temperature and for a sufficient period of time to adhere sufficient immune complexes to said coating; whereby immune complexes present in said body fluids will be able to be identified. 7. Method as stated in claim 6, characterized in that said material containing immunologically non-specific peptide-linked amino acids is alkaline and contains peptide-linked amino acid molecules selected from the group of molecules consisting of oligopeptides, modified oligopeptides, polypeptides, modified polypeptides, proteins and modified proteins , preferably glycosylated peptides and glycosylated proteins, which molecules are further characterized by their affinity for immune complexes; wherein said glycosylated protein is preferably selected from a globulin fraction which preferably originates from a vertebrate, more preferably a mammalian species, most preferably bovine gammaglobulin, equine gammaglobulin, lagomorph gammaglobulin, canine gammaglobulin, human gammaglobulin, ovine gammaglobulin, caprine gammaglobulin, murine gammaglobulin, procine gammaglobulin, feline gammaglobulin, or the Cohn fraction from any of the aforementioned gammaglobulins, especially from the Cohn fraction from bovine gammaglobulin; or where said glycosylated peptide that is selected is chemically synthesized, or in organisms with altered genetic structure resulting from recombinant or hybrid cell techniques or other techniques that modify or change cells to ensure that the modified cells produce the desired protein. 8. Object for selectively removing immune complexes from body fluid that has been brought into contact with such an object, characterized by support agents (P) adapted to be brought into contact with solutions containing immune complexes; and coating agents (21) comprising a material containing immunologically non-specific peptide-linked amino acid-containing compounds which are fixed to said support agents, such a coating or material having the ability to adhere to immune complexes that are present in specific body fluid when such body fluid is brought into contact with said coating or material, whereby immune complexes that are present in such body fluid can be selectively linked to said coating agent. 9. Item as stated in claim 8, characterized in that said material containing immunologically non-specific peptide-linked amino acids contains peptide-linked amino acid molecules selected from oligopeptides, modified oligopeptides, polypeptides, modified polypeptides, proteins and modified proteins, preferably glycosylated peptides and glycosylated proteins, which molecules are characterized by their affinity for immune complexes, the glycosylated protein being preferably selected from a globulin fraction, which preferably originates from a vertebrate, more preferably a mammalian species, most preferably bovine gammaglobulin, equine gammaglobulin, lagomorph gammaglobulin, canine -gammaglobulin, human gammaglobulin, ovine gammaglobulin, caprine gammaglobulin, murine gammaglobulin, procine gammaglobulin, feline gammaglobulin, or the Cohn fraction from which any of the aforementioned gamma globulins, especially the Cohn fraction from bovine gamma globulin; or where said glycosylated peptide that is selected is chemically synthesized, or in organisms with altered genetic structure resulting from recombinant or hybrid cell techniques or other techniques that modify or alter cells to cause the modified cells to produce the desired protein. 10. Procedure for the production of an object which has . ability to directly and selectively fix immune complexes to such a prepared substrate, characterized by the following steps: providing a substrate having a receiving surface; bringing said substrate into contact with a material comprising a moiety selected from oligopeptides, modified oligopeptides, polypeptides, modified polypeptides, proteins and modified proteins, which has the ability to bind to said substrate to form a coating and which has ability to adhere immune complexes present in the material brought into contact with said coating in a manner that allows subsequent identification of the presence of said immune complexes. 11. Method as stated in claim 10, characterized in that said contact-creating material is in a solution that has an alkaline pH value, and that said material is incubated in contact with said substrate at a preselected temperature for a sufficient period of time to achieve attachment of said parts to said substrate. 12. Method as stated in claim 10 or 11, characterized in that said part is selected from a globulin fraction, which preferably originates from a vertebrate, more preferably a mammalian species, most preferably bovine gammaglobulin, equine gammaglobulin, lagomorph gammaglobulin, canine -gammaglobulin, human gammaglobulin, ovine gammaglobulin, caprine gammaglobulin, murine gammaglobulin, procine gammaglobulin, feline gammaglobulin, or the Cohn fraction from any of the aforementioned gammaglobulins, especially the Cohn fraction from bovine gammaglobulin; or where said glycosylated peptide that is selected is chemically synthesized, or in organisms with altered genetic structure resulting from recombinant or hybrid cell techniques or other techniques that modify or alter cells to cause the modified cells to produce the desired protein.