WO1988006620A1

WO1988006620A1 - Monoclonal antibodies produced against glomerular proteoglycans

Info

Publication number: WO1988006620A1
Application number: PCT/US1988/000578
Authority: WO
Inventors: Eva Kemeny; Howard Fillit; John B. Zabriskie
Original assignee: The Rockefeller University
Priority date: 1987-03-04
Filing date: 1988-02-23
Publication date: 1988-09-07
Also published as: EP0304481A4; EP0304481A1; JPH01500798A; AU1625888A

Abstract

Monoclonal antibodies 4F2 and 7E12 and their method of production from hybridoma cell lines. The antibodies are useful for the detection of heparin sulfate proteoglycan in both normal and diseased tissue.

Description

MONOCLONAL ANTIBODIES PRODUCED AGAINST GLOMERULAR PROTEOGLYCANS

This invention is concerned with monoclonal antibodies identified herein as 4F2 and 7E12. These antibodies are produced against purified proteoglycans ( PG) isolated from bovine glomeruli (BG) containing heparan sulfate proteoglycan (HSPG). It is concerned also with methods of producing and utilizing such antibodies, compositions containing them, hybridoma cell lines useful for producing them and diagnostic kits containing them.

The techniques for producing hybridoma cell lines and monoclonal antibodies utilizing mouse myeloma cells and spleen cells from immunized mice were first described by Kholer and Milstein in Nature 256, 495 (1975). Subsequently considerable effort has been expended in the production of new cell lines and monoclonal antibodies. The general techniques applicable to such production are well known and understood. However, knowledge of the procedures is not a guarantee of success. There are many difficulties and unexpected impediments. In fact, there is no assurance, prior to attempting to prepare a given hybridoma, that it will produce antibody if obtained, or that the antibody produced will have the desired specificity. The degree of success depends on the type of antigen employed, the fusion technolog applied, and the selection techniques used for identifying and isolating the hybridoma with the desired specificity which subsequently must be maintained by long term culture technology.

HSPG is an integral component of the glomerular basement membrane (GBM) . It is also present in the basement membranes ( BM ) of other tissues. It has been demonstrated that the sera of patients with poststreptococcal glomerulonephritis contain antibodies to HSPG, suggesting that autoimmunity to HSPG .may be important in human glomerulonephri is ( GN ) . Monoclonal antibodies 4F2 and 7E12 to purified HSPG from BG tissue have been found useful for a number of purposes to be discussed hereinafter.

There follows a discussion of the various procedures utilized in connection with the practice of this invention including the preparation of purified HSPG, the production of hybridoma cell lines useful for the production of the monoclonal antibodies 4F2 and 7E12 from the purified HSPG, as well as the various chemical and immunological procedures used to test and characterize the antibodies.

Reagents Employed

DEAE-Sepharose CL-6B and Sepharose CL-4B were obtained from Pharmacia Fine Chemicals (Piscataway, New Jersey). Chondroitinase ABC and heparitinase were purchased from Miles Laboratories Inc. (Elkhart, Indiana). Pepstatin A,, benzamidine- HCl, 6-aminohexanoic acid, guanidine-CHl (grade I), phenylmethanesulfonyl fluoride (PMSF), N-ethylmaleimide (NEM), hyaluronic acid (grade I), chondroitin sulfate (CS) type A (C4- S), CS type C (C6-S), CS type B (DS; dermatan sulfate), and 3,3- diaminobenzidine tetrahydrochloride (grade II) were obtained from Sigma Chemical Co. (St. Louis, Missouri). Heparan sulfate (HS ) was purchased from Sei agaku Kogyo Co. (Tokyo, Japan). Papain was from Worthington Biochemical Corp. (Freehold, New Jersey). Polyethylene glycol (PEG; Mw:3-3700) and dimethylsulfoxide (DMSO) were purchased from American Type Culture Collection (Rockville, Maryland) . Dulbecco's modified Eagle medium (DMEM) with 4.5 g glucose and Penicillin/Streptomycin mixture were from Whittaker M.A. Bioproducts ( alkersville, Maryland) . Horse serum, L-glutamine, hypoxanthine-thymidme solution (100X) and aminopterine (100X) were obtained from Hazleton Dutchland, Inc. (Denver Colorado). Affinity purified, fluorescem or peroxidase or alkaline phosphatase conjugated F(ab)2 fragments of goat anti-mouse immunoglobulins ( IgA+IgG+IgM, heavy and light chain specific) and affinity purified, alkaline phosphatase conjugated F(ab)2 fragments of goat anti-mouse IgG (heavy chain specific) were purchased from Cappel Worthington Biochem. (Malvern, Pennsylvania ) .

Isolation of Proteoglycans and their Components

The PG ' s were purified from bovine glomeruli as described in Fillit et al. J. Exp. Med. , 161, 277 (1985). Briefly, glomeruli were isolated from fresh bovine kidneys by a sieving method. [Misra R.P.: Am J. Clin. Pathol 58:135, 1972]. After hypotonic lysis of the glomeruli in distilled water containing protease inhibitors (0.1M 6-aminohexanoic acid, 0.01M EDTA, 0.005M benzamidine-HCl ) , PGs were extracted with 4M guanidine-HCl, 0.05M sodium acetate, pH 5.8, in the presence of protease inhibitors. PGs were fractioned by stepwise elution from a column of DEAE- Sepharose CL-6B in 7M urea, 0.05M Tris-HCl, pH 7.0 by use of increasing concentrations of NaCl in the same buffer. The material eluted in 0.6M NaCl buffer was extensively dialyzed against distilled water, lyophilized, and purified further by chromatography on a Sepharose CL-4B column in 0.5M sodium acetate, pH 5.8. The fractions were analyzed for uronic acid by a variation of the borate/carbazole method. [T. Bitter and H.M. Muir, Anal. Biochem. , 4,330 (1962) ]. Hexosamines were quantitated on a Technicon TSM amino acid analyzer after hydrolysis in 4M HCl at 100°C for 8 h. [F.J. Kieras, J. Biol. C e.ii., 249, 7506 (1974) ]. Protein was measured by a modified Lowry procedure. [G.R. Schacterle and R.L.. Pollack, Anal. Biochem. , 51 , 654 (1973) ].

Glomerular PGs, were digested with 0.05 U/mg of chondroitinase ABC in 0.1M Tris, 0.1M sodium acetate, pH 7.3, containing 10 m.M N-ethylmaleimide, 1 M phenyl ethanesulfonyl fluoride, 10 M EDTA, and 0.035 mM Pepstatin A, for 17 hours at 37°C. The digest was chromatographed on a column (0.69 x 110 cm) of Sepharose CL-43 in 0.5M sodium acetate, pH 5.8. HSPG was located by uronic acid assay and glucosamine analysis, and the digestion products by uronic acid and galactosamine analyses. Chondroitinase ABC digestion of the glomerular PGs resulted in movement of a portion of the PGs to a higher Kav (first peak) on Sepharose CL-4B indicating the presence of a small amount of chondroitin sulfate proteoglycan. A second peak with Kav=0.6, contained the components of HSPG; uronic acid, protein, glucosamine and no detectable galactosamine, while a third pea emerging near the total volume contained galactosamine and uronic acid, representing fragments of CS. Fractions containing HSPG (peak B₁ ) were pooled, dialyzed, lyophilized, and then digested with 3 MlU/mg of heparin lyase (heparitinase) in 0.1M sodium acetate, 0.01M calcium acetate, pH 6.3, in the presence of protease inhibitors for 17 ^"hours at 37°C. The digested HSPG was chromatographed on a column (0.69 x 110 cm) of Sepharose CL-6B in 0.5M sodium acetate, pH 5.8. This specific treatment resulted in complete digestion of this material indicating that it was indeed HSPG. Biochemical analysis showed that a fourth peak with Kav=0.55 represented the core protein of HSPG, and a fifth peak contained glucosamine and uronic acid, representing fragments of HS. Hexosamine analysis of this HSPG core protein preparation revealed that 2.5% of the glucosamine of the original PG remained, probably representing the oligosaccharides that are part of the PG structure. Fractions containing the core protein of HSPG (fourth peak), the HS fragments (fifth peak) and the CS grafments (third peak), were used for ΞLISA inhibition studies and for lm unoblottmg as discussed hereinafter.

For the Western immunoblot described below, glomerular PGs were digested with heparitinase and with chondroitinase ABC using the orocedure described above. Immunization Schedules:

Female Balb/c mice were immunized with the glomerular PG isolated as described above. The antigen was mixed in equal parts with complete Freund's adjuvant and 100 ug/0.2 ml was injected I.P. in each mouse. This was- followed by two subsequent injections of the same amount of antigen in incomplete adjuvant at 3-4 week intervals. The final boosting injections of the antigen in PBS were given intraperitoneally four days before the actual fusion procedure.

Hybridization:

Spleen cells of immunized mice were fused with the Balb/c mouse myeloma line NS-1 according to the basic outline of Kohler and Milstein, Nature, 256, 495 (1975). Briefly, the washed spleen cells were mixed with NS-1 myeloma cells in 3:1 ratio ( spleen:myeloma) and were pelleted. by centrif gation at 230 g for 10 minutes. The cell pellet ws resuspended quickly in 1 ml of fusion mixture [40% PEG containing 5% DMSO or 50% PEG] at roo temperature and the cells were gently stirred for 90 seconds by rolling the tube on a vial rotator [TechniLab Instr. Inc. (Pequannock, NJ) ]. The fusion mixture was then diluted in serum free DMEM 1 :1 and rotated for 1 minute, diluted 1 :3 with the same medium and rotated for another 2 minutes and finally followed by 3 minutes rotation each time for the 1 :7 and 1 :15 dilutions of the fusion mixture. The last two dilutions utilized HT medium [DMEM supplemented with hypoxanthine, thymidine, 10% horse serum and antibiotics]. Following the last dilution, the cells were pelleted by centrifugation at 230 -g for ten minutes and resuspended in 40 ml of the HT medium. The cell suspension was distributed in microtiter plates [Linbro, Flow Lab. , Inc. (McLean, VA ) ] in 100 ul volume per well. The next day 100 ul of HAT medium containing double concentration of aminopterin 100X was added to each well. From day 7 postfusion, all cells were replenished every 2-3 days with HT medium.

Ten days after the fusion, hybrid colonies were seen in 604 out of 1150 wells. Supernatants were tested by the indirect IF technique using frozen bovine kidney sections as target organ and by ELISA utilizing glomerular PGs as the binding substrate. It was seem that antibody secreting clones from 26 wells showed immunofluorescent binding to bovine kidney, and 13 of these wells displayed significant binding to glomerular PG in the ELISA test. Hybridomas from selected wells were cloned and subcloned by the limiting dilution technique using Balb/c mouse spleen cells as feeders and were grown in bulk cultures utilizing DMEM and 10% horse serum supplemented with antibiotics.

The antibodies are isolated and purified by precipitation with 40% aqueous ammonium sulfate,. centrifugation at 12,100 x g. The pellet is separated, resuspended in PBS at pH 7.4, and dialyzed against 20 liters PBS to remove the ammonium sulfate. The dialyzed material is then passed over a Protein-A/Sepharose column equilibrated with PBS. The Protein A trapps the antibody via its Fc receptor. The antibody is eluted in 0.1m citrate buffer at pH3. The antibody fractions are collected and redialyzed back to pH 7.4 in a PBS buffer.

Other affinity chro otography procedures can be employed for further purification.

Enzyme Linked Immunosorbent Assay (ELISA):

ELISA was carried out essentially according to the method of Engvall and Perlmann, J. Immunol., 109, 129 (1972). Nunc immunoplates [Intermed, (Denmark) ] were coated with 50 ul of antigen per well at 5 ug/ml concentrations in 0.1 M Tris buffer pH 9.8 containing 0.3 mM MgCl-, and kept overnight at 4°C. After rinsing the plate and blocking the binding sites with PBSB for 2 x 30 minutes, 100 ul of supernatants diluted in PBSB were added to each well and incubated overnight at 4 C, then for 30 minutes at 37°C. After washing the immunoplates in 3 changes of PBSB, 100 ul of an alkaline phosphatase labelled F(ab) fragments of goat anti-mouse IgG diluted to 1 :500 in PBSB were added to each well for 1 hour at 37 C. Following washing, the enzyme reaction was developed by addition of 150 ul of substrate solution per well (1 mg/ml nitrophenyl phosphate [Sigma (St. Louis, MO) ] in 0.1M diethanola ine buffer, pH 9.8). The optical density was measured at 405 nm in a Titertek ELISA plate reader at intervals up to 6 hours.

Antibody Subclass:

To determine the immunoglobulin subclass and isotype of these monoclonal antibodies, 100 ng of affinity purified rabbit antisera (anti-mouse captured antibodies) [Bionetics, (Charleston, VA) ] specific to the Fc chains of mouse IgG, IgG2a, IgG2b, IgG3, IgA, IgM and to lambda and kappa light chains were adsorbed overnight at 4 C in sodium carbonate binding buffer, 0.01M, Ph 9.6 to microtiter plates [ unc , Intermed. (Denmark) ]. After blocking in two changes of phosphate buffered saline 0.5% Brij (PBSB) for 30 minutes each, 100 ul of hybridoma supernatants were added to each panel of anti-mouse captured antibody and incubated at 37 C for 2 hours. After washing, 100 ul of pretitered affinity purified goat anti-mouse F(ab)2 [Biochemical,

(Malvern, PA) ] conjugated to alkaline phosphatase were added and iinnccuubbaatteedd ffoorr oonnee hhoouurr aatt 3 377°CC.. T Thhee rr<eaction was developed as described above at the ELISA Drocedure Western Immunoblot;

Immunoblot was performed essentially as described by M.S. Blake, K.H. Johnston, G.J. Russel-Jones and E.C. Gotschlich, Anal. Biochem., 136, 175 (1984). PGS or protein cores in SDS sample buffer (25 ug/100 ul) were first electrophoresed on a 7% SDS-polyacrylamide gel. [Laemmli, Nature, 27, 680. (1970)]. The material was electroblotted to nitrocellulose [Schleicher and Scheull] in ethanol buffer in a blotting transfer system. After washing the nitrocellulose in PBS-0.05% Tween 20 (PBST) four washes of 30 minutes each, monoclonal antibody was added (1:50, monoclonal supernatant:PBST) overnight with rotation at room temperature. After washing in PBST three times for five minutes each, an alkaline phosphatase conjugated goat anti-mouse IgG antibody at 1:1000 dilution [Sigma (St. Louis, MO)] was added for one hour. After several washes in PBST the blot was developed by addition of enzyme substrate solution prepared as follows: To 9ml of 500 mM Tris, pH 10.0 containing 3mM MgC12; 1ml of 0.1% w/v nitro blue tetrazolium [Sigma (St. Louis, MO)] in H-,0_; and 0.1 ml of 0.5% w/v 5-brorao-4chloro-3-indolyl phosphate was added in anhydrous dimethylformamide.

Tissue Processing:

Pieces of bovine, human organs and other tissues utilized were embedded in OCT medium (Miles Scientific, Naperville, IL) , snap frozen in liquid nitrogen and stored at -70°C. Kidney biopsies taken at a clinic were embedded in OCT medium, snap frozen, and stored at -70°C. Immunofluorescence (IF) Techniσue

The monoclonal antibody binding was determined by the indirect IF technique. 4 urn frozen sections were washed in PBS, pH 7.4, followed by preincubation with 10% normal goat serum [Hazelton, Dutchland Inc. (Denver, CO)] in PBS for 20 minutes. Then the sections were incubated with culture supernatants for at least 30 minutes at room temperature. After washing in PBS, sections were layered with fluorescein conjugated F(ab)2 fragment of goat anti-mouse IgG at 1 :40 dilution in PBS for 30 minutes. The sections were washed, mounted with 50% glycerol in PBS, and examined under a Nikon Optiphot microscope with appropriate dichroic filters.

Double immunolabeling; Frozen sections were preincubated with 10% goat serum, then with rhodamine conjugated anti-human IgG or C3 diluted 1 :60 in 7E12 monoclonal antibody supernatant. Following washing the sections were incubated with FITC conjugated sheep F(ab)2 fragments of anti-mouse IgG (non cross- reactive with human immunoglobulins) .

Immunoperoxidase (IP) Technique:

Frozen sections were washed in PBS, preincubated with 10% goat serum and incubated with supernatants in the same way as described for the indirect IF technique. The second antibody layer was formed by incubation with a peroxidase coupled F(ab)2 fragments of goat anti-mouse IgG at 1 :40 dilution for 30 minutes. ^'Incubation with the substrate 3 , 3 ' -diaminobenzidine tetrahydrochloride [0.5 mg/ l, 0.01% H₂0₂ 0.05M Tris-DCl buffer, pH 7.6] was performed for 5-10 minutes according to the method of Graham and Karnovsky , J. Histochem. Cytochem. , 14, 291 (1966) . The sections were counterstained with Mayer's he atoxylin [Sigma (St. Louis, Mo) ], dehvdrated and mounted. There follows a section of this disclosure describing the results observed and the conclusions reached from completing and analyzing the procedures described above.

Isolation and Characterization of Glomerular Proteoglycans:

Chromatography of the 0.6M NaCl eluate using Sepharose CL-4B as described above resulted in three protein peaks of which peak 1 and peak 2 were PGs. The monoclonal antibodies described and claimed herein were produced by immunization with peak 2, referred to as glomerular PGs. This material, with Kav=0.48 was shown to contain protein, glucosamine and galactosamine in the ratio of 9:1, and uronic acid in an approximate 1 :1 ratio of hexosamine, which are characteristics of PGs. Enzymatic susceptibility of the glomerular PGs to specific enzymatic treatment as described in the section on the isolation of proteoglycans and their components was used to further characterize the purified material'. Galactosamine containing PG susceptible to chondroitinase ABC was found in a small amount. Approximately 90% of the purified glomerular PGs represented heparitinase susceptible HSPG.

HSPG reactivity of 4Ξ2 and 7E1 Monoclonal Antibodies:

Monoclonal antibodies 4F2 and 7E12 are both IgG1 immunoglobulins with kappa light chains. In order to determine the specific reactivity of the monoclonals, 4F2 and 7E12 were tested first m an ELISA system using glomerular PG as the binding substrate. Each monoclonal showed significant binding to the glomerular PG. Addition of high concentrations of salt (1M NaCl) to the test system did not inhibit the proteoglycan- antibody reaction, indicating that the binding of the monoclonal antibodies to the highly anionic proteoglycans was not due to non-specific charge interactions. A clear titration effect was seen with both 4F2 and 7E12 when the amount of glomerular PG was varied and the antibody concentration was held constant. In addition, in these tests which were carried out by direct ELISA, the antibodies showed no cross-reactivity with laminin, type IV collagen, and fibronectin.

The specific reactivity of these antibodies to the glomerular PGs was confirmed by competitive ELISA inhibition experiments. The reaction of each antibody was significantly inhibited by glomerular PG while cartilage PG (50ug/ml) from bovine nasal septa failed to inhibit the antibody binding. To determine whether or not the antibody reactivity was against the HS chains of the molecule, 4F2 and 7E12 mAbs were mixed with different concentrations of HS and were tested for binding to the glomerular PG in the ELISA. The results of this competitive ELISA experiments showed lack of inhibition of the antibody reactivity demonstrating that the antigenic determinants recognized by 4F2 and 7E12 do not reside on the HS chains of the molecule.

The nature of the antibody binding to the immunizing glomerular PGs, and the site of the antigenic determinant recognized by 4F2 and 7E12 monoclonal antibodies was studied by Western immunoblot using the procedures described above. Intact, heparan lyase and chondroitinase ABC digested PGs were separated on PAGE and were electroblotted onto nitrocellulose membranes. When the monoclonal antibodies were blotted against the intact PG, each antibody stained a high molecular weight smear characteristic for the electrophoretic pattern of PGs. Heparan lyase treatment of the glomerular PG resulted in a significant movement of the high molecular weight material stained by the antibodies to a lower position in the gel indicating that 4F2 and and 7E12 are reactive with the core protein of the heparan lyase susceptible HSPG. As was expected from these results, digestion of the PG with chondroitinase ABC had no effect on the blotting pattern of the PG stained by the monoclonal antibodies.

These immunoblot and ELISA ^'results demonstrate that both 4F2 and 7E12 are specific for HSPG of glomerular origin and strongly suggests that the antigenic determinant recognized by them is located in the core protein of the molecule.

Immunolocalization of HSPG in normal tissues

The immunolocalization of HSPG in the bovine kidney cortex was studied by the indirect IF and IP techniques described above, and both gave essentially similar results. 4F2 and 7E12 evidenced identical staining patterns. They labeled exclusively the BMs in a linear fashion. The most intense sta-ining was noted along the GBM. Binding was also seen along the extraglomerular BMs including the BMs of the tubular epithelium, Bowman's capsule and the BM material of the vessels. In the arterioles the staining was stronger in the media layer and weak in the subendothelial region. The matrix of the mesangium and the juxtaglomerular apparatus (JGA) exhibited no or only trace labeling. Inhibition of the tissue binding of 4F2 (1 :50 in PBS) by glomerular PG (20 ug/ml), core protein of HSPG intact HS was also tested by indirect IF. In correlation with the previous immunochemical experiments, glomerular PG and the HSPG core protein inhibited the binding of the monoclonal antibody to kidney tissue while HS had no effect on the tissue labeling.

The binding of 4F2 and 7Ξ 2 monoclonals to sections of normal kidneys of different species was also tested. 4F2 bound strongly to sections of rabbit and dog kidney tissues, and showed no reactivity with human kidney tissue. 7E12 exhibited strong cross-reactivity to human kidney. Neither of the antibodies exhibited cross-reactivity to rat and mouse kidneys.

To determine whether or not there was binding of these monoclonal antibodies to other than renal BMs, immunolocalization studies were performed with frozen sections of the bovine brain, testis, heart and lymph node. In the cerebral cortex and white matter of the brain the staining was localized to the small blood vessels with each antibody. No staining was detected in^' the brain parenchyma itself. The binding was also restricted to the vascular BM in the heart and in the lymph node. In the testes, 4F2 and 7E12 both labeled the wall of arterioles, and the BM of the ductuli deferentes.

Immunolocalization of HSPG in human kidney biopsies.

The distribution of HSPG core protein in human kidney biopsies obtained from patients with various forms of glomerulonephritis was investigated. Monoclonal antibody 7E12, which exhibited strong cross-reactivity to human kidney tissue was utilized in these studies. Intact staining for HSPG was found in biopsies obtained from patients with minimal change, postreptococcal and mesangioproloferative G . Loss of HSPG was detected only in one obsolent glo erulus seen in a case of minimal change GN. Negative staining was restricted to the area of sclerosis. Although in postreptococcal GN the GBM itself in general showed intact staining for HSPG, the expansion of the mesangium in a rather clubbed shaped appearance seen in the severe cases, was dramatically demonstrated by the wide negatively stained mesangial areas in the center of the glomeruli, which were filled with C3 according to the double i munolabeling study described above. In the two biopsies showing histological characteristics of membranous nephropathy 7E12 detected a diffuse increase in the width of the GBM associated with spike formation. In addition to the spikes, in the HSPG positive thickened GBM numerous "0" shaped HSPG negative foci were seen, which provided a so-called bubbling appearance of the GBM where the membrane was cut en face. When IgG or C3 was detected together with the HSPG in the same histological section utilizing the double immunolabeling technique it was clearly demonstrated that the immunodeposits were sitting in between the epimembranous spikes or in the center of the "0" shaped HSPG positive GBM extensions. In diffuse proliferative lupus nephritis various types of GBM abnormalities were detected with 7E12 such as double contour "moth eaten" membrane areas, focal spike formation and "0" shaped GBM projections. Double immunolabeling studies showed that the noted GBM changes were associated with im unoglobulin or C3 deposition. For instance intramembranous wormlike deposits of C3 were observed in the area of double contour, intra and epimembranous C3 granules in the "moth eaten" membrane areas. The intra and epimembranous im unodeposition was associated with different forms ^"of irregular GBM thickening. In one biopsy with severe form of proliferative lupus nephritis besides the above mentioned GBM changes loss of HSPG was seen in the areas of focal segmental necrosis.

All of the above establishes the utility of 7E12 for recognizing normal and abnormal kidney tissue.

The foregoing disclosure describes the distrubution of HSPG in normal bovine and human kidney tissues as well as in renal biopsies obtained from patients with different types of glomerulonephritis utilizing the monoclonal antibodies of this invention which were produced against HSPG of glomerular origin. By application of the various procedures described above, it has been observed that both 4F2 and 7E12 are specific for the core protein of HSPG isolated from bovine glomeruli. It has been further observed that 7E12 antibody binds to the BM of human kidney tissue and other human organs such as testes and lung.

The two antibodies differ in their cross reactivity to human tissue. The antibody 7E12 binds to human renal and other BM. Therefore, it can be applied to several human clinical studies. The antibody 4F2 does not cross react with human kidney. It has a much higher activity against HSPG in both ELISA and Western immunoblot. These facts clearly indicate that the two monoclonals are different and are specific for different antigenic determinants on the core portion of HSPG-

The novel monoclonal antibodies of this invention may be employed for a number of useful purposes. These include:

1. The anatomic detection of HSPG in various tissues in order to define normal anatomic distribution of such molecules.

2. To detect pathologic anatomic distribution of HSPG in various diseases including diseases of various organs which contain it such as the kidney, heart brain and testes.

3. The isolation of HSPG from tissue specimens and other biological fluids using affinity chromatography in which the monoclonal antibody is linked to a solid phase such as a Sepharose to which the HSPG is first preferentially absorbed and then desorbed. The HSPG, thus isolated, can be used diagnostically to recognize antibodies in human fluids such as urine or urum. 4. To detect HSPG in biological fluids such as cerebrospinal fluid, urine and serum for the detection of certain diseases. For example, the detection of HSPG in pathological urine may be useful in the diagnosis of certain types of renal disease.

5. The 4F2 can be employed for standardization of immunologic tests such as ELISA and imunoblotting for the detection of antibodies to HSPG in human fluids.

The hybridoma cell lines of this invention and the novel antibodies 4F2 and 7D12 can be produced by the techniques, described above. The antibodies can be isolated and purified by standard procedures. Typically useful procedures include, for example, precipitation, dialysis, chromatography, membrane filtration and electrophoresis. For most of the utilities described above it is not necessary to isolate and purify the antibody. Most of the tests can be applied utilizing antibody containing compositions of various concentrations.

The processes of this invention permits the preparation of hybridomas which secrete a monoclonal antibody into a culture medium.^" The supernatants of the culture medium will contain the monoclonal antibody in sufficient- concentration so that the supernatant can be used for almost any purpose as though it were a monoclonal antibody. The hybridoma culture can be employed in accordance with standard procedures with any of a number of known and readily available culture mediums to expand or propogate the hybridomas and thus produce additional antibodies.

While BALB/c mice are the presently preferred subjects for immunization, it is recognized that other mouse strains may be employed, and that other rodents, particularly rats, may also be used. The presently used mouse myeloma NS-1 cells are available from a number of sources such as public culture deposits were obtained. However, many other mouse myeloma cell lines are known and available, for example from deposit banks. These can be utilized in the practice of this invention. The cell line should preferably be of the drug resistant type illustrated above so that unfused myeloma cells will not survive. The most common class are the 8-azaquanine resistant cell lines which lack the enzyme HGPRT and therefore will not multiply in HAT medium. The myeloma cell lines are also preferred to be of the immunoglobulin non-secreting kind, such as the line described herein, so as to avoid contamination of the monoclonal antibody by antibodies produced by the cell. A suitable ratio of spleen cells to myeloma cells for the fusion process is from about, 5:1 to 1 :05. The preferred ratio is 1:1.

While the presently preferred fusion promotor is polyethylene glycol with an average- molecular weight of from about 1000 to 4000, other fusion promotors are known and can be used.

The monoclonal antibodies of this invention can be produced from the hybridoma cell lines which have been deposited at the American Type Culture Collection and asigned the accession numbers HB9336 and HB933? The former is employed to produce 7E12, the latter to produce 4F2. The cell lines can be utilized to produce additional antibody in either of two ways.

The purest moloclonal antibody is produced by _i_n vitro cultures of the selected hybridoma in a suitable medium for a suitable period of time. Suitable procedures are well known, or may be readily determined. Typical of the several mediums which can be employed are Dulbecco' s modified Eagle's medium and RPMI 1640, available from MA Bioproducts, Walkersville, Md. This _irι vitro technique produces essentially monospecific monoclonal antibody, essentially free from other antibodies. However, this _in_ vitro method may not produce a sufficient quantity of concentration of antibody for some purposes, since the concentration of monoclonal antibody produced is only about 1-20 ug/ml.

To produce a much greater concentration of slightly less pure monoclonal antibody, the selected hybridoma may be injected intraperitoneally into mice, preferably syngeneic or semi- syngeneic mice. The hybridoma will cause formation of antibody producing tumors in the mice after a suitable incubation time, which will result in high concentration of the desired antibody (about 5-20 mg/ml) in the blood stream and peritoneal exudate (ascites) of the host mouse. Although the host mice also have normal antibodies in their blood and ascites, the concentration of these normal antibodies is only about 5% of the monoclonal antibody concentration.

^'The monoclonal antibody compositions of this invention are distinguished from antibody compositions normally available, such as human antisera. These latter compositions, even in highly purified form, contain significant amounts of contaminating antibodies. In contrast, the compositions of this invention are essentially free of contaminating antibodies. They therefore lend themselves readily to a number of medical utilities, as will be readily apparent to those skilled in the art. The compositions comprise the antibodies in a medically inert medium, that is a medium which is not toxic and does not adversely affect the physiological activity of the antibody in the selected use. The composition of choice for many uses will be the supernatant of the culture medium in which the hybridoma grows and produces the antibody. It may also be an aqueous medium, e.g., isotonic saline or glucose solution or an oil such as peanut or sesame oil. One of the important medical uses anticipated for the products of this invention is to detect individuals with renal dysfunction or a malfunction in another organ. For this diagnostic purpose, the selected antibody will react with HSPG from the individual under test to produce, in the case of positive individuals, a detectable product. The HSPG of positive individuals will be characterized by an antigen reactive with the monoclonal antibody. An antibody containing composition used in any test must contain sufficient antibody to react with the antigen to produce a detectable product. Such diagnostically effective amounts of antibody will vary appreciably with a number of factors well known to those skilled in the art. These include, for example, the sensitivity of the test employed, the instrumentation available and the amount of sample under test, and the identity of the sample, e.g. serum or urine.

Any of a large number of clinical tests may be employed utilizing the hybridoma and antibodies of this invention. Typical tests include radioimmunoassay, enzyme linked im unoassay, precipitation, agglutination, direct and indirect immunofluorescence, and complement fixation. These tests may employ competitive and sandwich type assays. The tests may employ detectable labels. The antigen, the monoclonal antibody, or antiantibody such as rabbit anti-mouse serum may be labeled.

Useful labels include fluorescent labels such as fluorescein, rrhhcod _Aami ■ne or aurami ■ne. R_Dad _Ai-osot.opes suchu as 14C-, 131 I_τ, 125I_τ andj 25 'S. may be employed. Enzyme labels which may be utilized include, for example ,

, _ -0- galactosidase, urease,' glucose oxidase plus peroxidase, and acid or alkaline phosphatase. Methods for labeling biological products such as cells, antibodies, antigens and antisera are well known and need not be described. There are several currently available procedures for . detecting these labels including, for example colorimetric, spectrophotometric, fluorospectrophotometric, photometric and gasometric techniques, as well as various instrumental methods of detecting isotopes.

All of the tests which may be usefully employed in accordance with this invention involve the formation of a detectable reaction product which includes a monoclonal antibody of the invention and HSPG. Of course there may be other components such as an antiantibody in the detectable reaction product.

The 4F2 monoclonal antibody may be used a standard reagent in an ELISA for the detection of antibodies to HSPG in human serum or other bodily fluids. The assay was as follows: an ELISA employing the HSPG was performed by incubating Nunc (Intermed) Immunoplates with 5 ug/ml HSPG in 0.1M carbonate buffer, pH=9.8 for one hour at room temperature and overnight at 4°C. After washing the plates in PBS-0.5% Brij (PBSB) five times, the plates were incubated with human sera at various dilutions in triplicate. In triplicate control wells, 100 ul of 4F2 monoclonal antibody (1:80 dilution in PBSB) was added. The plates were incubated for one hour at room temperature, washed five times in PBSB, and an alkaline phosphatase conjugated goat anti-human IgG antibody was added to wells which contained human sera, while an alkaline phosphatase conjugated goat anti-mouse IgG antisera (1 :1000 in PBSB) was added to the wells which contain 4F2. The plates were again incubated for one hour at room temperature, and washed five times in PBSB. Finally, a developing reagent (nitrophenvl phosphate in diethanolamme) was added and the plates were incubated at 37 C for one hour. The optical density (0D) at 405 nm of the color reaction which develops in positive wells was determined in an ELISA reader apparatus such as the Titertek (Flow). .In wells which contained the 4F2, a reading of 1.000 +/- 0.1 OD was obtained as a standard reading. When this standard reading was not obtained, the plate was discarded and the assay repeated. In this way, population data for the measurement of antibody to HSPG in human sera may be obtained, and the test used for diagnostic and prognostic purposes in a clinical laboratory in which strict quality control is necessary. Thus, variations in the assay due to any factor may be determined.

The 7E12 is useful for the assessment of the integrity of the basement membrane, and specifically of the HSPG component of the basement membrane, in diseases which affect the basement membrane such as glomerulonephritis. For this procedure, human kidney biopsy material obtained fresh from a patient with the clinical diagnosis of glomerulonephritis is used. An immunoflourescence technique was employed. This technique is described in detail hereinabove. I munoperoxidase and other similar labelling techniques are also suitable. The biopsy material was snap frozen on OCT medium in liquid nitrogen. The biopsy material was then sectioned in a cryostat to 4 urn and placed on a microscope slide. The sections were washed in PBS, pH 7.4, and incubated with 10% normal goat serum (Hazelton) for 20 minutes. The sections were then washed three times for five minutes each in PBS, and incubated with the 7E12 monoclonal antibody in PBS for 30 minutes. After washing three times in PBS, the sections were incubated with a fluorescein conjugated F(ab)2 fragment of goat anti-mouse IgG at 1 :40 dilution in PBS for thirty minutes. Finally, the sections were washed three times in PBS, and layered with 50% glycerol in PBS. A coverslip was added and the slides examined under epifluorescence in an appropriate microscope (eg. Nikon Optiphot with appropriate filters). In normal human kidney, a fine linear pattern of staining of the glomerular and tubular basement membranes was seen. In diseases which affect the basement membrane, such as various forms of glomerulonephritis, alterations of the basement membrane were clearly seen. The test may be used diagnostically, since certain pattersns of staining are generally typical of specific forms of glomerulonephritis. A clinico-pathologic judgement regarding the general integrity of the basement membrane filtration surface may be made, which may have clinical significance in terms of prognosis, in conjunction with clinical^' evaluation of renal function. Similar pathologic studies of biopsies from other organs may be employed using the 7E12 monoclonal antibody as an indicator of the integrity and pathology of the basement membrane.

Claims

WHAT IS CLAIMED IS

1. A monoclonal antibody 4F2 derived from hybridoma cell line HB 9337 or a subclone thereof which reacts specifically with the core protein of HSPG and does not react with human kidney tissue.

2. A composition comprising hybridoma cell line HB 9337 or a subclone thereof and a culture medium therefore.

3. A process for preparing a monoclonal antibody 4F2 which reacts specifically with the core protein. of HSPG and does not react with human kidney tissue which comprises culturing hybridoma cell line HB 9337 in a culture medium therefore and recovering the antibody from the supernatant of the culture medium.

4. A method for detecting the presence of HSPG in a sample containing HSPG which comprises contacting the sample with a composition containing a sufficient quantity of monoclonal antibody 4F2 to react detectably with the core protein of HSPG, and detecting the presence of a reaction product.

5. A monoclonal antibody composition containing the monoclonal antibody 4F2 in sufficient quantity to react detectably with the core protein of HSPG.

6. A monoclonal antibody 7E12 derived from hybridoma cell line HB 9336 or a subclone thereof which reacts specifically with the core protein of HSPG and reacts specifically with human renal tissues .

7. A composition comprising hybridoma cell line HB 9336 or a subclone thereof and a culture medium therefore. 24

8. A process for preparing a monoclonal antibody 7E12 which reacts specifically with the core protein of HSPG and reacts specifically with human renal tissue which comprises culturing hybridoma cell line HB 9336 in a culture medium therefore and recovering the antibody from the supernatant of the culture medium.

9. A method for detecting the presence of HSPG in a sample containing HSPG which comprises contacting the sample with a composition containing a sufficient quantity of monoclonal antibody 7E12 to react detectably with the core protein of HSPG, and detecting the presence of a reaction product.

10. A monoclonal antibody composition containing the monoclonal antibody 7E1.2 in sufficient quantity to react detectably with the core protein of HSPG.