Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans

Definitions

• 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.
• PG proteoglycans
• BG bovine glomeruli
• HSPG heparan sulfate proteoglycan
• 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.
• 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.
• 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).
• DMEM Dulbecco's modified Eagle medium
• 100X hypoxanthine-thymidme solution
• aminopterine 100X
• 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
• affinity purified, alkaline phosphatase conjugated F(ab)2 fragments of goat anti-mouse IgG were purchased from Cappel Worthington Biochem. (Malvern, Pennsylvania ) .
• 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.
• protease inhibitors 0.1M 6-aminohexanoic acid, 0.01M EDTA, 0.005M benzamidine-HCl
• 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 B 1 ) 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.
• mice 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.
• 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.
• 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].
• 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.
• 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.
• 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.
• affinity purified rabbit antisera anti-mouse captured antibodies
• 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) ].
• 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.
• 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.
• 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) .
• Monoclonal antibodies 4F2 and 7E12 are both IgG1 immunoglobulins with kappa light chains.
• 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.
• the antibodies showed no cross-reactivity with laminin, type IV collagen, and fibronectin.
• 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.
• digestion of the PG with chondroitinase ABC had no effect on the blotting pattern of the PG stained by the monoclonal antibodies.
• HSPG core protein 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.
• 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.
• 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.
• novel monoclonal antibodies of this invention may be employed for a number of useful purposes. These include:
• HSPG pathologic anatomic distribution of HSPG in various diseases including diseases of various organs which contain it such as the kidney, heart brain and testes.
• HSPG monoclonal antibody
• 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.
• HSPG can be used diagnostically to recognize antibodies in human fluids such as urine or urum.
• biological fluids such as cerebrospinal fluid, urine and serum for the detection of certain diseases.
• the detection of HSPG in pathological urine may be useful in the diagnosis of certain types of renal disease.
• 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.
• mice 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.
• 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 _ir ⁇ 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.
• 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.
• 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.
• aqueous medium e.g., isotonic saline or glucose solution or an oil such as peanut or sesame oil.
• aqueous medium e.g., isotonic saline or glucose solution or an oil such as peanut or sesame oil.
• 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.
• 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 A ami ⁇ ne or aurami ⁇ ne.
• R D ad A i-osot.opes suchu as 14C-, 131 I ⁇ , 125I ⁇ 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.
• 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 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.
• 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.
• 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.
• an appropriate microscope eg. Nikon Optiphot with appropriate filters

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