EP0664001A1

EP0664001A1 - Method for screening hiv-1 positive individuals using antibodies against sulfatide

Info

Publication number: EP0664001A1
Application number: EP93923317A
Authority: EP
Inventors: Edwin H. Kolodny; Sirnavasa Raghavan; Miguel Angel Gama Sosa; Rita De Gasperi
Original assignee: New York University NYU
Current assignee: New York University NYU
Priority date: 1992-10-08
Filing date: 1993-10-08
Publication date: 1995-07-26
Also published as: AU5324994A; WO1994009369A1; EP0664001A4

Abstract

A method for determining neurodegeneration of nephrodegeneration in a subject, based on the detection of anti-sulfatide antibodies in a tissue or body fluid sample of the subject, wherein the degeneration is associated with at least one selected from the group consisting of an HIV infection, a neurodegenerative disease and a nephrodegenerative disease.

Description

METHOD FOR SCREENING HIV-1 POSITIVE INDIVIDUALS USING ANTIBODIES AGAINST SULFATIDE

Field of the Invention

The present invention relates to a meuhod for screening potential HIV-positive individuals by detecting the presence of antibodies against sulfatide in body fluids of such individuals.

Background of the Invention Sulfatide, (I³) -sulfogalactosylceramide, is a glycosphingolipid which occurs in high concentrations in the myelin of the nervous system and in the kidney. Although antibodies against this glycolipid have been found in some patients with a few specific autoimmune chronic liver diseases (see Toda et al., Hepatology 12:664-670 (1990)) and in patients with certain forms of polyneuropathy (see Pestronk et al. ,

Neurology 41:357-362 (1991)), the relationship between anti- sulfatide antibodies and the disease process has not yet been established.

There is evidence, however, that demyelination can also occur in patients with neuropathy who have antibodies against the sulfoglucuronyl epitope found in the glycolipid sulfoglucuronyl paragloboside and in the myelin associated glycoprotein. See, e.g., Pestronk, supra, and Chou et al. , J. B±ol . Chem. 261:11717-11725 (1986)). While neurological abnormalities are commonly found in individuals infected with human immunodeficiency virus (HIV) , a method for detecting the presence of such neurological abnormalities, which are specific for HIV-infected individuals and which would distinguish HIV-infected individuals from healthy individuals, has heretofore not been discovered.

Accordingly, there is a need to provide a diagnostic assay which may be commercially used by physicians or diagnosticians, and/or which may be provided in the form of a diagnostic kit, to identify potential neurological abnormalities in an HIV-infected individual and to distinguish between potential HIV-1 infected individuals and non-HIV infected individuals, taking into account alternative causes of neurological degenerations and/or nephrological degeneration autoimmune disorders.

Summary of the Invention

It is an object of the present invention to overcome the deficiencies of the related art.

It is also an object of the present invention to provide a method for detecting neurological and/or nephrological degeneration, as indicated by the presence of anti-sulfatide antibodies. It is also an object of the present invention to distinguish potentially HIV-infected humans or animals from otherwise healthy non-HIV-infected individuals, where neurological and/or nephrological degeneration, as indicated by the presence of anti-sulfatide antibodies, is due to HIV- infection, which detection made be made prior to the detection of HIV antibodies and/or HIV proteins in an animal or human.

It is another object of the present invention to provide methods whereby a sample of a body fluid or tissue of an individual may be tested for potential HIV infection, based on a determination of the presence of anti-sulfatide antibodies.

It is still another object of the present invention to provide a method for detecting neurological or degeneration in a subject by detecting the presence of anti- sulfatide antibodies in a body fluid or tissue of the subject. It is a further object of the present invention to provide kits which contain at least one reagent suitable for use in a diagnostic assay used to determine the presence of anti- sulfatide antibodies in HIV-infected individuals.

According to one aspect of the present invention, a method is provided for screening a subject suspected of being infected by HIV, comprising detecting the presence of anti- sulfatide antibodies in a tissue or body fluid sample from. the subject, wherein the presence of anti-sulfatide antibodies in the subject may be indicative of the presence of an HIV infection. Brief Description of the Figures

Figure 1 is a photographic representation of an antibody stained high performance thin layer chromatography (e.g., as an HPTLC-immunostaining assay) showing the presence of anti-sulfatide antibodies in HIV-1 positive individuals and the lack of anti-sulfatide antibodies in HIV-negative individuals with no history of neurological degeneration or autoimmune disease.

Detailed Description of the Preferred Embodiments It has now been discovered that the body fluids of

HIV-1 positive individuals contain antibodies specific for sulfatide, while healthy HIV-1 negative individuals, with no neurological degeneration or autoimmune disorders which induces a humoral immune response to sufatide, lack such antibodies. Accordingly, assay methods have been discovered for diagnosing potential HIV-infected individuals which can be routinely accomplished using known method steps based on the teachings and guidance presented herein, without undue experimentation.

Body fluid or tissue samples, or extracts thereof, of individuals suspected of having anti- sulfatide antibodies in HIV infected or neurologically or nephrologically degenerated subjects, may include sera, CSF, urine, lymph blood cells (WBC's, RBC's, T-cells, B-cells, macrophages, monocytes, esinophils, basophils, and neutrophils) and saliva. According to methods of the present invention, anti- sulfatide antibodies can be detected in a sample of a body fluid of an individual, as a human or an animal, by showing reactivity towards sulfatide or towards anti-idiotipic antibodies thereto, including chimeric antibodies, made according to known method steps; see, e.g., Harlow and Lane, Antibodies : A Laboratory

Manual , Cold Spring Harbor Press, N.Y. (1988) ; and Ausubel et al., eds., Current Protocols in Molecular Biology, Wiley Interscience, N.Y. (1987; 1992); Sambrook et al. Molecular Cloning: A Laboratory Manual , Second Edition, Cυld Spring Harbor Press, N.Y. (1989) which are incorporated entirely herein by reference. The term "animal" in the context of the present invention refers to mammals and birds, including but not limited to, humans, cats, dogs, cattle, sheep, horses, rodents, primates, pigs, rabbits poultry, and the like. Thus humans are included in the term animal, but humans are also used separately to specify use in humans.

While methods of the present invention are not limited to any single theory as to why such antibodies are present in HIV infected individuals or individuals with neurological or nephrological degeneration, neurological and/or nephrological damage in such patients may be attributed to the presence of the anti-sulfatide antibodies which either can be a result in degeneration of the kidney or degeneration of myelin in the nervous system, which tissues contain sulfatide which is released or degrades such that the sulfatide induces a humoral response in an animal or human, resulting in the presence of anti-sulfatide antibodies in tissues or the circulation of such individuals. Anti-sulfatide antibodies could also be induced by the breakdown of the blood-brain barrier occurring in HIV- positive individuals wherein B cells enter the rain's blood supply and react with sulfatide, resulting in anti-sulfatide antibody products. For example, recent data have shown the presence of serum protein in the central nervous system parenchyma of a number of AIDS patients (see, e.g., Rhodes, J^". Exp. Neurpathol . Exp. Neurol . 50:171-183 (1991); Ilyas et al. , Ann . Neurol . 18:655-659 (1985); Tlyas et al. , Proc. Natl . Acad. Sci . USA 82:6697-6700 (1985); Tlyas et al. , Ann. Neurol . 23:440- 447 (1988) . Since sulfatide is also found in high concentration in the kidney. Thus, anti-sulfatide antibodies could also be responsible for, or a result of, the nephropathy frequently found in HIV-infected individuals (see, e.g., Rao, Annu . Rev. Med. 42:391-401 (1991)).

The term "sulfatide" in the context of the present invention includes any fragment or derivative of sulfatide, which can be prepared according to known methods, such as by enzymatic cleavage or chemical modification or degeneration, to provide at least one ceramide, sphingosine, sulfate or saccharide, which also corresponds to an epitope bending region of an anti-sulfatide antibody.

According to a method of the present invention, the presence of anti-sulfatide antibodies, as indicative of neurological or nephrological damage (e.g., due to HIV infection, neurodegenerative disease, or nephrodegenerative disease) may be detected by contacting a sample from a body fluid or tissue of an individual suspected to be HIV-1 positive, with sulfatide or an anti-idiotype antibody attached to a support.

Conditions may then be provided to allow the anti- sulfatide antibody to bind to the bound sulfatide or anti- idiotype antibody, followed by labeling and detection of the anti-sulfatide antibodies with a detectable label, such as labeled anti-human antibodies. In a preferred embodiment, labeled goat anti-human IgG may be used. The above antibody generation and/or detection can be routinely accomplished according to known method steps. See e.g., Harlow, supra; Ausubel, supra; and Sambrook supra . Neurodegenerative diseases include, but are not limited to, AIDS dementia complex, demyelinating diseases, such as multiple sclerosis and acute transverse myelitis; extrapyramidal and cerebellar disorders, such as lesions of the corticospinal system; disorders of the basal ganglia or cerebellar disorders; hyperkinetic movement disorders such as Huntington's Chorea and senile chorea; drug-induced movement disorders, such as those induced by drugs which block CNS dopamine receptors; hypokinetic movement disorders, such as Parkinson's disease; progressive supra-nucleo palsy; structural lesions of the cerebellum; spinocerebellar degenerations, such as spinal ataxia, Friedreich's ataxia, cerebellar cortical degenerations, multiple systems degenerations (Mencel, Dejerine- Tho as, Shi-Drager, and Machado-Joseph) ; systemic disorders (Refsum's disease, abetalipoprotemia,ataxia, telangiectasia, and mitochondrial multi.system disorder); demyelinating core disorders, such as multiple sclerosis, acute transverse myelitis; and disorders of the motor unit, such as neurogenic muscular atrophies (anterior horn cell degeneration, such as amyotrophic lateral sclerosis, infantile spinal muscular atrophy and juvenile spinal muscular atrophy); Alzheimer's disease; Down's Syndrome in middle age; Diffuse Lewy body disease; Senile Dementia of Lewy body type; Wernicke-Korsakoff syndrome; chronic alcoholism; Creutzfeldt-Jakob disease; Subacute sclerosing panencephalitis Hallerrorden-Spatz disease; and Dementia pugilistica. See, e.g., Berkow et al, eds. , The Merck Manual , 15th edition, Merck and Co., Rahway, N.J., 1987, which reference, and references cited therein, are entirely incorporated herein by reference.

Nephrodegenerative diseases include, but are not limited to, AIDS-associated nephropathy, immunologically related mediated renal diseases, glomerular diseases, tubulointerstitial disease, nephrotoxic disorders and hereditary chronic nephropathies. See, e.g., Berkow et al, eds., The Merck Manual , 15th edition, Merck and Co., Rahway, N.J., ppl552-l633 and 1647- 1648, 1987, which reference, and references cited therein, are entirely incorpor.ated herein by reference. Labeled or unlabeled anti-idiotype antibodies may also be used according to the present invention to identify anti- sulfatide antibodies. An anti-idiotypic (anti-Id) antibody is an antibody which recognizes unique determinants generally associated with the antigen-binding site of an antibody. See, for example Kohler and Milstein, Nature 256:495-497 (1975); U.S. Patent No. 4,376,110; Ausubel et al, eds., Current Protocols in Molecular Biology, Wiley Interscience, N.Y. , (1987, 1992); and Harlow and Lane Antibodies : A Laboratory Manual Cold Spring Harbor Laboratory (1988) , the contents of which references are incorporated entirely herein by reference. Such antibodies may be of any immunoglobulin class including IgG, IyM, IgE, IgA, GILD and any subclass thereof.1

An anti-Id antibody may be prepared by known method steps by conventional hybridoma technology or by recombinant methods to produce single or multiple species hybridomas or antibody producing cells, including, but no limited to urine- murine, murine-human, human-human hybridomas and alternative chimeras with other species, such as rat, rabbit, hamster, primate or any other mammalian cells for hybridoma production, e.g., by immunizing an animal of the same or related species and genetic type (e.g. mouse strain) as the source of the antibody with the antibody (or at least the idiotypic determinants thereof) to which an anti-Id is being prepared. The immunized animal will recognize and respond to the idiotypic determinants of the immunizing antibody by producing an antibody to these idiotypic determinants (the anti-Id antibody) . Alternatively, anti-idiotype antibodies specific for anti-sulfatide antibodies, or fragments thereof, according to the present invention, can be produced using conventional procedures via the expression of isolated DNA which codes for variables regions of such an Mab in host cells like E. coli, see Ward, et al., Nature 341:544-46 (1989) , or transfected murine myeloma cells, see Gillies, et al., Biotechnol . 7:799-804 (1989), and Νakatani, et al. , loc. ci t . , 805-10. In addition, Fab molecules can be expressed and assembled in a genetically transformed host like E. coli . A lambda vector system is available thus to express a population of Fab's with a potential diversity equal to or exceeding that of subject generating the predecessor antibody. See, e.g., Huse, et al., Science 246:1275-81 (1989).

Accordingly, antibodies, or fragments thereof, isolated or generated against sulfatide, may be used to induce anti-Id antibodies in suitable animals, such as in Balb/c mice. Spleen cells from such immunized mice may then be used to produce anti-Id hybridomas secreting anti-Id mAbs, according to known method steps. See, e.g. Harlow, supra; and Ausubel, supra . The anti-Id mAbs thus have their own idiotypic epitopes, or "idiotopes" structurally similar to the epitope being evaluated, such as an epitope of the anti-sulfatide antibody present in HIV infected subjects.

The term "antibody" is also meant to include both in- tact molecules as well as fragments thereof, such as, for example, Fab and F(ab')₂, which are capable of binding antigen. Fab and F(ab')₂ fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody (Wahl et al., J. Nucl . Med. 24:316-325 (1983)).

It will be appreciated that Fab and F(ab')₂ and other fragments of anti-idiotype antibodies useful in the present invention may be used for the detection and/or quantitation of anti-sulfatide antibodies according to methods of the present invention. Such fragments are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')₂ fragments). See, e.g., Harlow, supra .

The anti-idiotype antibodies, or fragments of such antibodies, according to the present invention, may be used to quantitatively or qualitatively detect the presence of anti- sulfatide antibodies in body fluids or tissues of HIV infected subjects. This can be accomplished by immunofluorescence tech¬ niques employing a fluorescently labeled antibody by known method steps coupled with light microscopic, flow cytometric, or fluorimetric detection. See, e.g., Ausubel, supra; Harlow, supra; and Sambrook, supra .

Sulfatide or a derivative thereof; anti-idiotype antibodies to anti-sulfatide antibodies from HIV infected subjects; or anti-human Ig antibodies, can be labeled according to the present invention and used diagnostically in a assay or histologically, as in immunofluorescence or immunoelectron microscopy, for in situ or in vitro detection of anti-sulfatide antibodies in a sample. In si tu or in vitro detection may be accomplished by known methods steps by removing a cell, tissue or body fluid sample from a subject and associating the labeled sulfatide or antibody with the bound or contained anti-sulfatide antibody in the sample by contacting the sample to the labeled sulfatide or derivative, or antibody, bound to a support, optionally followed by associating the labeled antibody with the bound anti-sulfatide antibody. The antibody (or fragment) is preferably provided by applying to or by overlaying on the biological sample. Through the use of such a procedure, it is possible to determine not only the presence of anti-sulfatide antibodies but also their distribution on the examined tissue. Using the present invention, those of ordinary jkill will readily perceive that any of a wide variety of histological methods can be modified in order to achieve such in situ or in vitro detection. See e.g., Ausubel, supra, Sambrook, supra, and Harlow, supra .

Accordingly, antibodies used to label anti-sulfatide antibodies can be used to detect the presence of anti-sulfatide antibodies in a biological sample or tissue which has contacted with a support having sulfatide, or an analog or derivative thereof, attached thereto.

The biological sample may be treated with a solid phase support or carrier (which terms are used interchangeably herein) such as nitrocellulose, or other solid support which is capable of immobilizing cells, cell particles or soluble pro¬ teins. The support may then be washed with suitable buffers followed by treatment with the detectably labeled Ig-specific or anti-idiotype antibody. The solid phase support may then be washed with the buffer a second time to remove unbound antibody. The amount of bound label on said solid support may then be detected by known method steps.

A support for the sulfatide, derivative or anti- idiotype antibody can include a solid phase support or carrier which is intended to be any support capable of binding antigen or anti-idiotype antibodies. Well-known supports, or carriers, include glass, polystyrene, silica, C18, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite. The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. The support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody. Thus, the support configuration may be spherical., as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, etc. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same without undue experimentation.

The binding activity of a given lot of anti-human Ig antibody may be determined according to well-known method steps. Anti-human Ig antibodies include IgG, IgM and IgA. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.

Other such steps as washing, stirring, shaking, fil- tering and the like may be added to the assays as is customary or necessary for the particular situation.

One of the ways in which the anti-idiotype antibody for anti-sulfatide antibodies can be detectably labeled is by linking the same to an enzyme and use in an enzyme immunoassay (EIA) . This enzyme, in turn, when later exposed to an appropriate substrate, will react with the substrate in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorimetric or by visual means. Enzymes which can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6- phosphate dehydrogenase, glucoamylase and acetylcholinesterase. The detection can be accomplished by calorimetric methods which employ a chromogenic substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzy- matic reaction of a substrate in comparison with similarly pre¬ pared standards. See, e.g., Ausubel, supra; Sambrook, supra; and Harlow, supra .

Detection may be accomplished using any of a variety of other immunoassays. For example, by radioactively labeling the anti-human Ig antibodies or antibody fragments, it is possible to detect the presence of anti-sulfatide antibodies through the use of a radioimmunoassay (RIA) . A good description of RIA may be found in Laboratory Techniques and Biochemistry in Molecular Biology, by Work, T.S., et al. , North Holland Publishing Company, New York (1978) with particular reference to the chapter entitled "An Introduction to Radioimmune Assay and Related Techniques" by T. Chard, incorporated by reference here¬ in. The radioactive isotope can be detected by such means as the use of a gamma counter or a liquid scintillation counter or by autoradiography.

It is also possible to label the anti-idio type antibody with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wave length, its presence can then be detected due to fluorescence. Among the most commonly used fluorescent labelling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine. Such compounds are readily available from commercial sources, such as Molecular Probes, Inc. (Eugene, Ore.).

The anti-idiotype antibody can also be detectably labeled using fluorescence emitting metals such as ¹⁵²Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA) , according to known method steps. The anti-idiotype antibody also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.

Likewise, a bioluminescent compound may be used to label an anti-idiotype antibody of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin. The above labeling compounds method steps are well known and available from commercial sources. See, e.g., Ausubel, supra; Harlow, supra; and Sambrook, supra .

The anti-Id antibody molecules of the present invention may be adapted for utilization in an .immunometric assay, also known as a "two-site" or "sandwich" assay. In a typical immunometric assay, a quantity of unlabeled antibody (or fragment of antibody) is bound to a solid support and a quantity of detectably labeled soluble antibody is added to permit detection and/or quantitation of the ternary complex formed between solid-phase antibody, antigen, and labeled antibody. Typical, and preferred, immunometric assays include

"forward" assays in which the antibody bound to the solid phase is first contacted with the sample being tested to "extract" the antigen from the sample by formation of a binary solid phase antibody-antigen complex. After a suitable incubation period, the solid support is washed to remove the residue of the fluid sample, including unreacted antigen, if any, and then contacted with the solution containing an unknown quantity of labeled antibody (which functions as a "reporter molecule") . After a second incubation period to permit the labeled antibody to complex with the antigen bound to the solid support through the unlabeled antibody, the solid support is washed a second time to remove the unreacted labeled antibody.

In another type of "sandwich" assay, which may also be useful with the antigens of the present invention, the so-called "simultaneous" and "reverse" assays are used. A simultaneous assay involves a single incubation step as the antibody bound to the solid support and labeled antibody are both added to the sample being tested at the same time. After the incubation is completed, the solid support is washed to remove the residue of fluid sample and uncomplexed labeled antibody. The presence of labeled antibody associated with the solid support is then determined as it would be in a conventional "forward" sandwich assay.

In the "reverse" assay, stepwise addition first of a solution of labeled antibody to the fluid sample followed by the addition of unlabeled antibody bound to a solid support after a suitable incubation period is utilized. After a second incuba¬ tion, the solid phase is washed in conventional fashion to free it of the residue of the sample being tested and the solution of unreacted labeled antibody. The determination of labeled antibody associated with a solid support is then determined as in the "simultaneous" and "forward" assays.

The above described assay methods step are well known in the art. See Harlow, supra; and Ausubel, su^ra.

According to the present invention, it is thus possible to diagnose circulating or non-circulating antibodies in a subject, which antibodies are specific for sulfatide. This is accomplished by means of an immunoassay, as described above, using sulfatide, a derivative thereof, or an anti-idiotype antibody to anti-sulfatide antibodies, according to known method steps, as described or referenced herein, without undue experimentation.

Pharmaceutical Preparations of Anti-Id mAbs of the Present Invention.

According to the present invention, it is also possible to treat the neurological and/or nephrological degenerative effects caused or associated with the presence of anti-sulfatide antibodies in an animal or human by inhibiting circulating or ron-circulating antibodies in a jubject, which antibodies are specific for sulfatide. This is accomplished by the use a pharmaceutical composition comprising an anti-Id Ab, or a fragment or peptide thereof, which is capable or specifically binding the epitope binding region of a circulating anti-sulfatide Ab in an animal or human, as described herein, according to known method steps, as described or referenced herein, without undue experimentation.

Preparations of anti-Id antibodies, or fragments or peptides thereof, for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions, which may contain auxiliary agents or excipients which are known in the art.

By the term "protection" from infection or disease as used herein is intended "prevention, " "suppression" or

"treatment." "Prevention" involves administration of an anti-Id mAbs, which include fragments and derivatives thereof, prior to the induction of the disease.

"Suppression" involves administration of the composition prior to the clinical appearance of the disease.

"Treatment" involves administration of the protective composition after the appearance of the disease. It will be understood that in human and veterinary medicine, it is not always possible to distinguish between "preventing" and "suppressing" since the ultimate inductive event or events may be unknown, latent, or the patient is not ascertained until well after the occurrence of the event or events. Therefore, it is common to use the term "prophylaxis" as distinct from "treatment" to encompass both "preventing" and suppressing" as defined herein. The term "protection, " as used herein, is meant to include "prophylaxis. "

At least one anti-Id mAb of the present invention may be administered by any means that achieve their intended purpose, for example, to treat sulfatide antibody related pathologies, such as neurodegenerative or nephrodegenerative diseases, as described herein and as would be clear to one of ordinary skill in the art, using an anti-Id mAbt* corresponding to at least one epitope recognized by a circulating anti- sulfatide antibody in an animal or human, the anti-Id Ab in the form of a pharmaceutical composition.

For example, administration of such a composition may be by various parenteral routes such as subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, tran-dermal, or buccal routes. Par<-.ιteral administration can be by bolus injection or by gradual perfusion over time. A preferred mode of using an anti-Id Ab containing pharmaceutical composition of the present invention is by intravenous, intramuscular or intraperitoneal application. A typical regimen for preventing, suppressing, or treating sufatide antibody-related pathologies, comprises administration of an effective amount of an anti-Id mAbs or fragments or derivatives thereof, administered over a period of one or several days, up to and including between one week and about 24 months. It is understood that the dosage of an anti-Id mAbs of the present invention, administered in vivo or in vitro, will be dependent upon the age, sex, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired. The ranges of effective doses provided below are not intended to limit the invention and represent preferred dose ranges. However, the most preferred dosage will be tailored to the individual subject, as is understood and determinable by one of skill in the art, without undue experimentation. See, e.g., See, e.g., Berkow et al, eds., The Merck Manual , 15th edition, Merck and Co., Rahway, N.J. , 1987; Goodman et al., eds., Goodman and Gilman ' s The Pharmacological Basis of Therapeutics, 8th edition, Pergamon Press, Inc., Elmsford, N.Y., (1990); Avery' s Drug Treatment: Principles and Practice of Clinical Pharmacology and Therapeutics, 3rd edition, ADIS Press, LTD., Williams and

Wilkins, Baltimore, MD. (1987), Katzung, ed. Basic and Clinical Pharmacology, Fifth Edition, Appleton and Lange, Norwalk, Conn. (1992) , which references and references cited therein, are entirely incorporated herein by reference. The total dose required for each treatment may be administered by multiple doses or in a single dose. An anti-Id mAbs may be administered alone or in conjunction with other therapeutics directed to sulfatide antibody related pathologies, as described herein. Effective amounts of the an anti-Id antibodies (Abs) or an anti-Id antibody containing composition, which may also include pharmaceutically acceptable carriers or diluents, are from about 0.01 μg to about 100 mg/kg body weight, and preferably from about 10 μg to about 50 mg/kg body weight, such 0.05, 0.07, 0.09, 0.1, 0.5, 0.7, 0.9, 1, 2, 5, 10, 20, 25, 30, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 mg/kg. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions, which may contain auxiliary agents or excipients which are known in the art. Pharmaceutical compositions of the present invention can be prepared according to routine methods. See, e.g., Berkow, supra, Goodman, supra, Avery, supra and Ebadi, supra, which are entirely incorporated herein by reference, included all references cited therein.

Pharmaceutical compositions comprising at least one anti-Id Ab or ταl n , such as 1-10 or 1, 2, 3, 4, J, 6, 7, 8, 9 or 10 anti-Id Abs or MAbs, of the present invention may include all compositions wherein the anti-Id Abs are contained in an amount effective to achieve its intended purpose. In addition to at least one anti-Id mAb, a pharmaceutical composition may contain suitable pharmaceutically acceptable carriers, such as excipients, carriers and/or auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.

Pharmaceutical compositions comprising at least one anti-Id antibody or fragment thereof may also include suitable solutions for administration intravenously, subcutaneously, der ally, orally, mucosally, rectally or may by injection or orally, and contain from about 0.01 to 99 percent, preferably from about 20 to 75 percent of active component (i.e. the antibody) together with the excipient. Compositions which can be administered rectally include suppositories. See, e.g.,

Berkow, supra, Goodman, supra, Avery, supra, Katzung, supra and Ebadi, supra, which are entirely incorporated herein by reference, including all references cited therein.

Having now generally described the present invention, the following example is provided by way of illustration, and not of limitation, to more particularly describe an embodiment of the present invention. EXAMPLE 1

METHOD FOR DETECTING THE PRESENCE OF ANTI - SULFIDE ANTIBODIES AS AN INDICATOR OF HIV INFECTION IN HUMANS AND

ANIMALS Sulfatide (Sigma, MO) (5 μg/lane) was applied to aluminum-backed Silica Gel 60 HPTLC plates (Merck, Darmstadt) and the plates were developed in chloroform/methanol/water (65:25:4, v/v/v) . The plates were subsequently mixed in 0.15% polyisobutylmetachrylate in hexane for 30 seconds, dried, and blocked for l h in phosphate buffered saline (PBS) containing 2% bovine serum albumin (BSA) and 0.05% Tween-20. The plates were incubated with a 1:100 dilution of the respective sera samples from human patients known to be HIV positive or HIV negative according to a Western blot or DNA assay in PBS/BSA for 1 h at room temperature and washed extensively with PBS/tween-20. The plates were then extensively washed as above, and incubated with peroxidase-labelled goat anti-human IgG (Boehringer Mannheim, IN) (1:400 dilution in PBS/BSA) for 1 h. After washing with PBS/Tween-20, reactivity towards sulfatide was visualized by incubating the plates in 0.01 M sodium citrate, pH 6.0, containing 0.2 mg/ml 4-chloronaphthol and 0.03% H₂0₂.

As presented in Figure 1, lanes a-c are control HIV-l negative healthy individuals; lanes d and e are HIV-l positive individuals, without fully developed AIDS; and lane f is an HIV- 1 positive patient with fully developed AIDS and demonstrated neurological involvement; lane g is a sulfatide standard, chemically stained with the orcinol/H₂S0₄ spray reagent. The doublet is due to the presence of two sulfatide species containing non-hydroxy fatty acids (upper band) and hydroxy fatty acids (lower band) respectively. A method of the present invention was thus found to routinely and correctly diagnose human patients having no history of neurological or nephrological disease as HIV positive or negative.

Accordingly, the methods of the present invention which detect the presence of anti-sulfatide antibodies in body fluids or tissues of a subject can be used to screen or diagnose human or animal subjects which are HIV positive, or, alternatively or additionally, subjects with neurodegenerative and/or nephrodegenerative diseases.

All references cited herein, including journal articles or abstracts, published or corresponding U.S. or foreign patent applications, issued U.S. or foreign patents, or any other references, are entirely incorporated by reference herein, including all data, tables, figures, and text presented in the cited references. Additionally, the contents of the references cited within the references cited herein are also entirely incorporated by reference.

Reference to known method steps, conventional methods steps, known methods or conventional methods is not in any way an admission that any aspect, description or embodiment of the present invention is disclosed, taught or suggested in the relevant art.

The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the sk ll of the art (including the contents of the references cited herein) , readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the generic concept of the present invention. Therefore, such adaptations and modifications are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein.

Claims

What is Claimed is:

1. A method for screening or diagnosing a subject potentially infected by a human immunodeficiency virus, comprising assaying for the presence of anti-sulfatide antibodies in a tissue or body fluid sample from said subject, wherein the presence of anti-sulfatide antibodies in said subject is indicative of the presence of human immunodeficiency virus infection.

2. A method according to claim 1, wherein said assay step comprises:

(A) contacting said tissue or body fluid sample with a sulfatide bound to a support;

(B) labeling said anti-sulfatide antibody bound to said sulfatide bound to a support; and (C) qualitatively or quantitatively detecting the labeled anti-sulfatide antibody.

3. A method according to claim 2, wherein said support comprises silica on a TLC plate.

4. A method according to claim 1, wherein said labeling comprises binding an anti-human Ig antibody which is labeled with a detectable marker.

5. A method according to claim 4, wherein said anti- human Ig antibody is IgG selected from anti-mouse, anti-goat or anti-rabbit.

6. A method according to Claim 2, wherein said sulfatide is replaced with an anti-idiotype antibody which binds an epitope of said anti-sulfatide antibodies,

7. A method for screening or diagnosing a subject suspected of having a neurodegenerative or nephrodegenerative disease, comprising assaying for the presence of anti-sulfatide antibodies in a tissue or body fluid sample from said subject, wherein the presence of anti-sulfatide antibodies in said subject is indicative of the presence of an HIV infection.

8. A method according to claim 7, wherein said assaying step comprises: (a) contacting said tissue or body fluid sample with a sulfatide bound to a support;

9. A method according to claim 8 , wherein said support comprises silica on a TLC plate.

10. A method according to claim 7, wherein said labeling comprises binding an anti-human Ig antibody which is labeled with a detectable marker.

11. A marker according to claim 10, wherein said anti-human Ig antibody is IgG selected from anti-mouse, anti- goat or anti-rabbit.

12. A method according to Claim 8, wherein said sulfatide is replaced with anti-idiotype antibodies which bind an epitope of said anti-sulfatide antibodies.

13. An anti-idiotype antibody, fragment or derivative thereof, comprising an anti-idiotype antibody having an epitope binding region that specifically binds an anti-sulfatide antibody.

14. An anti-idiotype antibody according to claim 13, wherein said anti-idiotype antibody is a monoclonal antibody.

15. A pharmaceutical composition, comprising at least one anti-Id antibody, or a fragment or derivative thereof, having an epitope specific for an anti-sulfatide antibody; and a pharmaceutically acceptable carrier or diluent.

16. A method for treating an animal subject suffering from a pathology related to the presence of circulating anti- sulfatide antibodies, comprising administering an anti-sulfatide antibody inhibiting amount of a pharmaceutical composition according to claim 13, wherein said binding of said anti-Id antibody to said antisulfatide antibody inhibits the binding the anti-sulfatide antibody to sulfatide or a portion thereof.

17. A method according to claim 16, wherein said pathology is an HIV infection.

18. A method according to claim 16, wherein said pathology is a neurodegenerative disease.

19. A method according to claim 16, wherein said pathology is a nephrodegenerative disease.