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
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes

Definitions

• This invention relates to the production of antibodies specific for connective tissue proteins and, more particularly, to the production of monoclonal antibodies by fused cell hybrids against human collagens and enzymes involved in collagen degradation.
• Collagen is by far the most prevalent human protein, constituting almost half of the total body protein.
• the prolific research in recent years in the area of collagen biochemistry has demonstrated that there are at least six genetically distinct collagens and several related collagen-degrading enzymes.
• the collagen profile i.e., the types of distinct collagens and collagen-associated proteins present, their distribution in the tissue, and the concentration ratios among the distinct types, of any given tissue or body fluid sample varies with the tissue or fluid source.
• the collagen profile of a tissue or fluid sample also varies with the physiological or pathological state of its source.
• connective tissue disorders and other pathological conditions in which changes in the collagen profile occur, eventually resulting in such large scale tissue alterations as to cause organ impairment.
• a specific and reliable means for ⁇ detecting and/or quantitatively measuring changes in collagen types and distribution in tissue and body fluids is extremely useful for diagnostic evaluations of the stage of and specific organ involvement in certain diseases.
• the detection and/or quantitative measurement of different types of collagens and collagen-associated enzymes in body fluids provides a means for monitoring therapies that result in a release of collagens and
• OMPI collagen-associated enzymes into body fluids upon the eradication of cells, such as tumor cells, against which the drug is targeted.
• the use of monoclonal antibodies against connective tissue proteins to establish the collagen profile of histological, cytological and biological fluid samples is a novel and advantageous approach to disease diagnosis and therapy monitoring. Because of the high specificity and sensitivity of monoclonal antibodies, early detection of certain collagen-related pathological conditions " is possible as is early assessment of the efficacy of certain therapeutic programs. To achieve these goals, the invention provides: (1) a method for repeatedly producing large quantities of monospecific antibodies against distinct connective tissue proteins and (2) procedures for using the monoclonal antibodies individually or in combination as clinical probes for diagnosis and therapy monitoring. The potential prognostic importance of early and accurate disease diagnosis and determination of the usefulness of certain therapies using the methods of this invention is highly significant.
• the monoclonal antibodies produced by hybridomas are highly specific i munoglobulins of a single type.
• the single type of immunoglobulin secreted by a hybridoma is specific to one
• OMPI OMPI and only one antigenic determinant on an antigen, a complex molecule having a multiplicity of antigenic determinants.
• monoclonal antibodies raised against a single antigen may be distinct from each other depending on the determinant that induced their formation; but for any given clone, all of the antibodies it produces are identical.
• Monoclonal methods are generally applicable and have been used to produce antibodies to antigens other than the sheep red blood cells used by Kohler and Milstein. For instance, it has been reported that monoclonal antibodies have been raised against' umor cells [U.S. Pat. No. 4,172,124] and viruses [U.S. Pat. No. 4,196,265]. The production of monoclonal antibodies against certain collagens, procollagens (natural precursors of collagens) and a collagen-associated glycoprotein has also been reported. Linsenmayer et al. reported using the cell hybridization technique to produce monoclonal antibodies against chick Type I collagen [Proc. Natl. Acad. Sci. U.S.A.
• Each type of collagen has as its biosynthetic precursor a procollagen molecule which differs from the mature collagen molecule insofar as it has additional a ino acid sequences at the amino and carboxy termini of each chain that are eventually cleaved by specific processing enzymes.
• the interstitial collagen molecules comprise the majority of all the connective tissue proteins and account for nearly all fibrillar tissue components.
• This class of collagens represents four distinct molecular species: (1) the Type I collagen molecule which exhibits the chain composition [ ⁇ l(I)]2 ⁇ 2(I). Fibers derived from Type I collagen are found throughout the entire organism primarily in supporting tissues which normally exhibit very little distensibility under physical stress; (2) the Type I-trimer collagen molecule which is comprised of three identical ⁇ l(I) chains. This molecule has been described in certain chondrocyte cultures and other experimental systems, but its existence in normal tissue has not been firmly established; (3) the Type II collagen molecule which contains three ⁇ l(II) chains.
• this species forms relatively thin fibrils and displays a tissue distribution restricted predominantly to cartilaginous structures such as articular cartilage and nucleus pulposus and to certain parts of the embryonic eye; and (4) the Type III collagen molecule which is composed of three l(III) chains.
• the fibrils formed by these molecules are usually found in a reticular network.
• the latter meshwork apparently contains in addition to Type III molecules certain quantities of a- form of Type III procollagen indicating that the conversion of Type III procollagen to Type III collagen is incomplete.
• These procollagen molecules participate in the formation of fine non-striated filaments which are associated with the Type III fibrils.
• the basement membrane collagens include at least two distinct collagen chains, the ⁇ l(IV) and 2(IV), which exhibit unique compositional features. The configuration of these chains within native collagen molecules is presently unknown. These collagens appear to be universally distributed as components of the morphologically distinct epithelial and endothelial basement membranes.
• Type V collagen The pericellular collagens commonly referred to as Type V collagen contain three distinct chains, ⁇ l(V), ⁇ 2(V) and ⁇ 3(V) , which combine to form a variety of molecular species. Histologically, they are more predominant in cells derived from the vascular system as ' compared to other tissues and appear to form a pericellular exocytoskeleton.
• Procollagens and cross-linked collagen molecules are susceptible to attack by specific collagen-degrading enzymes collectively called collagenases; cleavage by such enzymes yields procollagen peptides and collagen peptides.
• collagenases collagen-degrading enzymes
• elastase is a very distinct collagen-degrading enzyme which selectively cleaves Type III collagen, but not Type I collagen, and releases a distinct trimer peptide, ⁇ l(III) .
• the acquired connective tissue disorders are pathophysiological conditions in which large scale tissue alterations occur as the result of an apparent lack of coordination between collagen synthesis and degradation.
• the disorders include atherosclerosis, liver cirrhosis, lung fibrosis, bone marrow fibrosis, systemic progressive sclerosis, sleroder a, psoriasis, rheumatoid arthritis, osteoarthrosis and certain benign and malignant tumors.
• these conditions arise through fibroproliferative responses leading to an excessive accumulation of collagen in affected tissues though some disorders involve degenerative changes within previously normal connective tissues.
• the patterns of collagen deposition in three different fibroproliferative disorders,- atherosclerosis, liver fibrosis (or cirrhosis) and scleroder a of skin are quite similar and are illustrative of the types of changes in collagen profile that occur in the acquired connective tissue diseases.
• an increase in basement membrane collagen synthesis is first observed.
• the deposition of basement membrane matrix containing Type IV collagen is followed by a Type III collagen neosynthesis.
• the Type III collagen thereby forms the reticular network of granulation tissue.
• the dense collagen fiber forms the scar tissue which is almost completely comprised of Type -L collagen molecules [S. Gay, Ital. J. Gastroenterol. 12_:30-32 (1980)].
• a number of tumors such as'the various kinds of fibromatoses elaborate matrices containing copious quantities of fibrous collagen.
• Malignant tumors such as the osteosarcomas or chondrosarcomas may also produce large amounts of collagenous matrix.
• the collagen produced generally reflects the cellular origin of the tumor.
• osteosarcoma cells produce a matrix containing fibers derived from Type I molecules
• the fibrous elements of chondrosarcomas are c derived from Type II molecules, [K.. Remberger and S. Gay, Z. Krebsforsch. 9_ :95-106 (1977)].
• it is possible that less differentiated tumors may synthesize a number of different collagens.
• metastasized neoplastic mammary epithelial cells of breast carcinomas 10 retain the ability to synthesize Type IV (basement membrane) collagen [L.A. Liotta et al.. The Lancet, July 21, 1979: 146-147].
• Rheumatoid arthritis is an acquired disease 15 manifested by either fibroproliferative or degenerative changes in the connective tissue of diarthrodial joints.
• rheumatoid synovial tissue is characterized by the synthesis and deposition of additional Type I and III collagens.
• the blood vessels of the proliferating pannus tissue carry the bulk of vascular-derived Type V collagen.
• the endothelial basement membrane containing Type IV collagen often appears irregular, discontinuous, and sometimes multilamellated in the vessels of pannus tissue.
• the altered basement membrane barrier is reflected by the cellular synovial exudate.
• the exudate contains phagocytes that exhibit inclusions of various collagens.
• Osteoarthrosis is an example of a noninflammatory joint disorder that involves degenerative loss of the 5 articular cartilage.
• articular cartilage is characterized by a loss of proteoglycan aggregates, presumably due to the release of unusually large amounts of degradative enzymes, which results in demasking Type II collagen fibers on 0 fibrillated surfaces [S. Gay and R.K. Rhodes, Osteoarthritis Symposium, pp. 43-44, Grune & Stratton, Inc. (1981)].
• the fibrillated surface persists and the initial clefts eventually extend into the deeper layers of articular cartilage due to the 5 inefficient healing and repair capacity of cartilage tissue.
• Chondrocytes do proliferate and form clusters adjacent to the cartilage clefts. Although these chondrocytes apparently retain their capacity to form new proteoglycan aggregates, the capacity to synthesize new i cartilage specific Type II collagen molecules appears to be greatly diminished or lost. Instead, a small deposition of fibrocartilaginous material comprised of collagen fibers derived from Type I molecules occurs. The switch from Type II collagen synthesis to Type I collagen 5 synthesis appears to be an important step in the pathogenesis of osteoarthrosis and hence the presence of Type I collagen in biopsies can serve as an indicator of the progress of the disease.
• Type III collagen molecule contains about 30% more hydroxyproline than the Type I collagen molecule.
• the genetically distinct types of collagens are very similar to one another in their macromolecular structure, they are sufficiently different in their amino acid sequence to allow the production of specific antibodies.
• Antibodies can be raised against antigenic determinants located in five identifiable regions of collagen or procollagen molecules, specifically, the globular amino and carboxy termini of procollagen molecules, the non-helical termini of mature collagen molecules, the helical portion of collagen and procollagen molecules, and the central amino acid sequences of individual ⁇ -chains, obtained by denaturing collagen molecules.
• the antigenic regions in collagen consist both of sequential and conformational determinants.
• Enzyme-linked immunoadsorbent assays have been developed for types I, II, III, and IV collagen and for laminin and _ fibronectin by using antibodies prepared in rabbits and goats [S.I. Rennard e_t a_l., Anal. Biochem. 104:205-214(1980)] .
• the present invention provides a method for producing monoclonal antibodies against human collagens
• the monoclonal antibodies may be used in standard radioimmunoassays or enzyme-linked immunosorbent assays for the quantitative measurement of the spectrum of connective tissue proteins in a given sample of body fluid, thereby permitting non-invasive diagnosis of certain pathologocal states and the monitoring of therapies that result in release of connective tissue proteins into sera and other biological fluids.
• the monoclonal antibodies may be tagged with compounds which fluoresce at various wavelengths so that the distribution of collagens in tissue biopsies can be determined by immunohistological techniques.
• Radioimmunoassays and immunohistological methods employing the monoclonal antibodies of this invention can be used to detect and follow the pathogenesis of diseases, such as: genetic disorders affecting skeleton, skin and muscles; formation of excessive scar tissue; and deposition of pathological amounts of connective tissue in body organs, including kidney, intestines and heart, and in liver by liver cirrhosis, in skin by scleroderraa; in lung by lung fibrosis; in bone marrow by leukemia; in blood vessels by atherosclerosis; and in joints by rheumatic diseases.
• the methods involving monoclonal antibodies can also be used to detect changes in the neosynthesis of collagens that is indicative or suggestive of the malignant state of cells derived from such tumors as breast carcinomas.
• the present invention provides theoretically immortal cell lines capable of consistently producing high titers of single specific antibodies against the distinct connective tissue proteins. This is a distinct advantage over the traditional technique of raising antibodies in immunized animals where the resulting sera contain multiple antibodies of different specificities that vary in both type and titer with each animal, and, in individual animals, with each immunization.
• the invention contemplates the extension of the hybridoma technique to the production of monoclonal antibodies to other genetically distinct collagens and collagen-associated proteins and enzymes as they become known and their use in the _ijn vitro diagnosis of disorders and cancers involving connective tissue proteins.
• the invention further contemplates the use of monoclonal or polyclonal antibodies against connective tissue proteins for in_ vivo diagnostic and therapeutic
• Antibodies produced by either conventional methods or the monoclonal techniques of this invention can be labelled with radioactive compounds, for instance, radioactive iodine, and administered to the patient.
• the antibodies localize in areas of active collagen neosynthesis such as certain malignant tumors or other 0 :issues undergoing pathological changes involving collagen. The localization of the antibodies can then be detected by emission tomographical and radionuclear scanning techniques; such detection is of diagnostic value.
• monoclonal or polyclonal antibodies 5 against connective tissue proteins can be conjugated to certain cytotoxic compounds (radioactive compounds or other therapeutic agents) and can be used for therapeutic purposes, for instance, cancer therapy.
• the antibodies, targeted for malignant cells expressing the appropriate 0 . . . . collagen antigen localize on or in the vicinity of the individual cells or tumor at which point the conjugated cytotoxic compound takes effect to eradicate the malignant cells. 5
• the genetically distinct types of collagens and 0 other connective tissue proteins can be derived from a variety of tissue sources throughout the human body.
• any one of these distinct connective tissue proteins is a suitable antigen with which to immunize animals, such as mice or _ rabbits, to obtain antibody-producing somatic cells for fusion.
• the choice of animal can influence , the type of antibody obtained vis a vis the determinant on the antigen against which the antibody is directed. For example, if antibodies directed toward amino or carboxy terminal determinants are desired, rabbits should be immunized. When rats or mice are immunized, antibodies produced against determinants in the more stable helical portion of the various collagen molecules are usually the result.
• Somatic cells with the potential for producing antibody and, in particular, B cells are suitable for fusion with a B-cell myeloma line. Those antibody- producing cells that are in the dividing plasmablast stage fuse preferentially. Somatic cells may be derived from the lymph nodes and spleens of primed animals. Once-primed or hyperimmunized animals can be used as a source of antibody-producing lymphocytes. Mouse lymphocytes g-ive a higher percentage of stable fusions with the mouse myeloma lines described in Section 4.3. However, the use of rabbit, human and frog cells is also possible.
• myeloma cell lines have been developed from lymphocyte tumors for use in hybridoma-producing fusion procedures [G. Kohler and C. Milstein, Europ. J. Immunol. :511-519 (1976); M. Shul an et al., Nature 276:269-270 (1978)].
• myeloma cell lines may be used for the production of fused cell hybrids, including X63-Ag8,
• mice including
• MOPC-21 mice 210.RCY3.Agl.2.3 derived from rats and U-226AR, and GM1500GTGAL 2 , derived from rats and U-226AR, and GM1500GTGAL 2 , derived from humans.
• G.J. Hammerling, U. Ha merling and J.F. Kearney (editors) Monoclonal antibodies and T-cell hybridomas in: J.L. Turk (editor) Research Monographs in Immunology, Vol. 3, Elsevier/North Holland Bio edical Press, New York (1981)].
• Methods for generating hybrids of antibody- producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2:1 proportion (though the proportion may vary from about 20:1 to about 1:1), respectively, in the presence of 0 an agent or agents that promote the fusion of cell membranes. It is preferred that the same species of animal serve as the source of the somatic and myeloma cells used in the fusion procedure. Fusion methods have been described by Kohler and Milstein [Nature 256:495-497
• the fusion-promoting agent used by those investigators were Sendai virus and polyethylene glycol (PEG) , respectively.
• the fusion procedure of the example of the 0 present invention is a modification of the method of
• PEG is added to the mixture of mouse spleen and myeloma cells to promote the formation of fused cell hybrids.
• Dimethyl sulfoxide (DMSO) another agent affecting cell membranes, may also be included, in 5 addition to PEG, in the fusion mixture.
• Fusion procedures usually produce viable hybrids _ at very low frequency.
• the frequency of heterokaryon formation using state-of-the art techniques with PEG as fusing agent is generally 1x10 -2.
• Ensuing nuclear fusion and formation of synkaryons has a frequency of
• the selection of fused cell hybrids is accomplished by culturing the cells in media that support the growth of hybridomas but prevent the growth of the myeloma cells which normally would go on dividing indefinitely.
• myeloma cells lacking hypoxanthine phosphoribosyl transferase HPRT
• HAT hypoxanthine/aminopterin/ thymidine
• HPRT-positive genotype of the spleen cells HPRT-positive genotype of the spleen cells.
• myeloma cells with different genetic deficiencies e.g., other enzyme deficiencies, drug sensitivities, etc.
• myeloma cells with different genetic deficiencies e.g., other enzyme deficiencies, drug sensitivities, etc.
• OMPI Generally, around 3% of the hybrids obtained produce the desired antibody, although a range of from 1 to 30% is not uncommon.
• the detection of antibody-producing hybrids can be achieved by any one of several standard assay methods, including enzyme-linked immunoassay and radioimmunoassay techniques which have been described in the literature [R. Kennet, T. McKearn and K. Bechtol (editors) , Monoclonal antibodies, hybridomas: a new dimension in biological analyses, pp. 376-384, Plenum Press, New York (1980)].
• the detection method used in the example of the present invention was an enzyme-linked immunoassay employing an alkaline phosphatase-conjugated anti-mouse immunoglobin.
• each cell line may be propagated in either of two standard ways.
• a sample of the hybridoma can be injected into a histoco patible animal.
• the injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid.
• the body fluids of the animal such as serum or ascites fluid, can be tapped to provide monoclonal antibodies in high concentration.
• the individual cell lines may be propagated _in vitro in laboratory culture vessels.
• Rheumatoid arthritis and osteoarthrosis synovial fluid may be withdrawn from the knee which can be subjected to radioimmunoassays (and immunofluorescent assays for any cell which may be present in the fluid) in which monoclonal antibodies against the various types of collagen are used. If Type II collagen, for instance, is detected in the synovial fluid, this may indicate the destruction of articular cartilage which is characteristic of osterarthrosis, but which also occurs in other erosive joint disorders. On the other hand, if Types I and III collagens are detected, this may be more indicative of the inflammatory-proliferative phase of rheumatoid arthritis.
• the ability to diagnose early lesions of articular cartilage can effect the choice of the appropriate therapy with which to treat the patient.
• wavelengths to various tissue sections represents a sensitive means of detecting changes in the collagen distribution within biopsied tissue samples. For instance, if liver cirrhosis is suspected, part of the affected tissue can be i munohistologically stained with monoclonal antibodies against Types I, III and IV collagens. Normally, liver contains very little collagen; thus a lack of significant fluorescent staining would indicate a healthy liver. On the other hand, if Type IV collagen was detected in the sample, this would suggest
• fibrotic diseases affecting the liver and the connective tissue of the organs such as skin and bone can also be detected by serological (i.e., non-biopsy) means utilizing monoclonal antibodies on serum samples.
• Type IV collagen collagen.
• the production of this collagen continues as the cell metastasizes to other locations, such as the lymph nodes surrounding the breast area.
• monoclonal antibodies against Type IV collagen the presence of a single metastasized cell can be detected immuhohistologically in a lymph node biopsy.
• An early diagnosis of infiltrating cells and lymph node metastasis as judged on the basis of as little as one basement membrane collagen-synthesizing tumor cell is of significant progostic importance.
• malignancies may also be diagnosed by detecting the neosynthesis of collagens.
• monoclonal antibodies against collagens may also prove useful for locating malignant cells in cytological samples such as in Pap smears taken to diagnose cervical and/or uterine cancers.
• Monoclonal antibodies may also be used in immunohistological differential diagnoses to distinguish, for example, malignant melanomas from benign naevi. 4.8. THERAPY MONITORING USING
• Monoclonal antibodies against connective tissue proteins may be used to monitor the effectiveness of antifibrotic drug therapies. They provide the immunoserological, immunohistological and immunocytological means to detect an inhibition or suppression of collagenous connective tissue neosynthesis, the resulting diminution in the accumulation of the collagenous matrix, and hence, the antifibrotic effect of the drug.
• monoclonal antibodies may be used to monitor the effectiveness of certain chemotherapies aimed at eradicating malignant tumor cells.
• tumor cells present in bone marrow malignancies produce the enzyme elastase which selectively cleaves one fourth of the Type III collagen molecule to yield a peptide fragment. If such cells are successfully destroyed by cheraotherapeutic means, both elastase and the Type III peptide fragment are released and enter the serum.
• a radioactively labeled connective tissue protein is mixed with monoclonal antibodies specific for that particular protein as antigen and with a serological sample containing an unknown amount of unlabeled connective tissue protein.
• the labeled and unlabeled antigen compete for binding with the monoclonal antibody.
• the more unlabeled connective tissue protein there is in the serological sample the less labeled antigen binds with antibody to form an insoluble complex.
• EIA enzyme-linked immunosorbent assay
• RIA radioimmunoassay
• the slide is then layered with another coating of a second type of monoclonal antibody.
• the localization of the connective tissue proteins within the sample is then determined by fluorescent light microscopy and optionally photographically recorded.
• the cell hybridization techniques of this invention are adopted from the protocol of Drs. J. Kearney, A. Anderson and P. Burrows, of the Cellular Immunobiology Unit, 224 Tumor Institute, University of Alabama in Birmingham. [G.J. Hammerling, U. Hammerling and J.F. Kearney (editors) , Monoclonal antibodies and T-cell hybridomas in: J.L. Turk (editor) , Research Monographs in Immunology, Vol. 3, Elsevier/North Holland Biomedical Press, New York (1981)].
• mice At 5 to 6 weeks of age, e_.c., BALB/c female mice
• mice are immunized with 200 ug of a purified connective tissue protein as antigen.
• the antigen is delivered in 0.5 ml of complete Freund's adjuvant by subcutaneous inoculation.
• An immunization schedule is followed wherein the mice are boosted intraperitoneally with a similar amount of antigen 21 days after the initial priming. Only a single boost is administered, though other immunization schedules with multiple boosts may be used with similar success.
• the spleens and lymph nodes are removed 4 days after the booster inoculation following standard techniques
• Spleens of immunized BALB/c mice are removed 5 under sterile conditions and washed in serum-free RPMI 1640 medium (Sero ed, Munchen, F.R.G.). The spleens are- macerated through cheesecloth and are then resuspended in serum-free RPMI 1640 medium and centrifuged; this washing procedure is performed three times at 4°C. After the final washing, the cells are resuspended in the same
• the number of cells in the preparation is determined microscopically before mixing with myeloma cells for fusion (see Section 5.4).
• F.R.G. 100 units/ml penicillin, 100 ug/ml streptomycin, and 0.25 ug/ml Fungizone (Flow Laboratories, Bonn,
• F.R.G. F.R.G.
• complete medium X630-Ag8.653
• HAT thymidine-(HAT) -sensitive cell line.
• X63-Ag8.653 has lost immunoglobulin expression entirely and does not synthesize 1 or K chains of X63 origin upon fusion with antibody-forming cells.
• the 0 myeloma cells are cultured in complete medium and harvested during the exponential phase of growth.
• the cells are counted microscopically prior to O fusion with spleen cells.
• Spleen cells and X63-Ag8.653 myeloma cells are combined in a ratio of 2:1 or 1:1 (spleen cells:myeloma cells) and washed once in serum-free RPMI 1640 medium at 37°C.
• the cell mixture is centrifuged at room temperature at 1,000 rpm for 7 minutes.
• the pellet fraction is carefully aspirated to leave it as dry as possible.
• the pellet is loosened by gentle tapping and is "*resuspended with gentle agitation in 1.0 to 1.5 ml of PEG-4000 (polyethylene glycol) solution at 37°C.
• PEG-4000 polyethylene glycol
• the PEG-400 solution is prepared by autoclaving 20 gm PEG 4000 in a 100 ml bottle, cooling and adding 28 ml of sterile phosphate buffered saline. After approximately 30 seconds, the cell mixture is slowly diluted dropwise to a volume of roughly 20 ml with serum-free RPMI 1640 medium at 37°C; the tube is then filled to 50 ml with the same medium. The cells are centrifuged at room temperature and resuspended at 37°C in HAT medium, which is selective for fused cells.
• HAT medium is prepared by adding 1 ml of a stock solution (100X) of hypoxanthine (H) and thymidine (t) and 1 ml of a stock solution (100X) of aminopterin (A) to 100 ml of complete medium.
• the HT stock contains 272.2 mg hypoxanthine and 7.75 mg thymidine in 200 ml distilled water. Because the hypoxanthine does not dissolve well, the pH of the solution is adjusted to pH 8.1-8.5 with 1-2 drops of in NaOH. The solution is sterilized through a 0.45 u Millipore filter and stored at 4°C.
• the A stock contains 3.52 mg aminopterin in 200 ml distilled water.
• the cell mixture is resuspended in HAT medium at a concentration of about 2 to 5 x 10 spleen cells/ml.
• Peritoneal exudate feeder cells are then added (roughly, the peritoneal washout of one normal, non-immunized mouse per 100 ml of HAT/fused-cell suspension) and 1 ml of the cell _ suspension is added per well of a 24 well macrotiter plate (Costar, Cambridge, Massachusetts) .
• the cells are observed with an inverted microscope to check for myeloma cell death.
• the X63-Ag8.653 cell line is HAT-sensitive and thus, unfused myeloma cells cannot survive in this medium; unfused spleen cells naturally die out of the culture.) Contamination of the wells is also
• the fused cells are allowed to incubate in HAT medium for two weeks at which time 0.5 ml of the 0 supernatant of each well is discarded and replaced with
• non-HAT complete (non-HAT) medium. This medium replenishment is repeated daily for another week. Two to three days after the last medium replenishment, which is enough time to allow for sufficient production of 5 antibodies for testing, the macrotiter plates are scored for hybrid growth and assayed for antibody activity by the
• the wells of a 96-well polyvinyl microtiter plate are coated with 100 ul/well of 1 mg/ml solution of . collagen antigen in borate saline. The plate is incubated for four hours at 25°C or overnight and 4°C. The plates are then blocked with 1% bovine serum albumin (BSA) in borate buffered saline (BS-BSA) and incubated for one hour at 25°C. The wells of the microtiter plate are washed twice with saline, after which the supernatants (containing monoclonal antibodies) from the wells of the macrotiter plates used for outgrowth and selection of fused hybrids are added to the microtiter wells.
• BSA bovine serum albumin
• BS-BSA borate buffered saline
• the plate is incubated for four hours at 25°C (or overnight at 4°C) and washed two to three times with saline.
• 100 ul of alkaline phosphatase-labeled antibodies (goat antimouse-immunoglobulin) diluted 1:500 in BS-BSA is added.
• the wells are washed 4-5 times with saline and 200 ul of substrate (p-nitrophenylphosphate) is added per well.
• the reaction is stopped by the addition of 50 ul 3 N NaOH to each well.
• the absorbance of the fluid in the wells is then determined spectrophotometrically.
• the alkaline phosphatase converts colorless p-nitrophenylphosphate into yellow p-nitrophenol.
• the colorometric reaction permitts the easy identification of those culture supernatants containing collagen-specific antibodies and hence the identification of the desired fused hybrids. This step is performed to exclude from 31
• the extent of hybrid cell growth in the wells of the macrotiter plates (see Section 5.1.6.) is determined 3-4 weeks after the initial plating in HAT medium.
• 15 is diluted further by delivering 10 ml in 20 or 30 ml of medium containing feeder cells and 200 ul aliquots are added to each well of -another microtiter plate. Further dilutions of the cell suspension can be performed if necessary. This method is used to insure that the wells
• 20 of at least one plate contain clones derived from a single cell. Samples of cells from the original hybridoma-containing macrotiter wells are frozen for safekeeping.
• the supernatants of the microtiter wells are rescreened for monoclonal antibody production using the ELISA assay described in Section r - n 5-1.7. 30
• the culture media from hybrids that survived two successive clonings and that continued to exhibit a stable phenotype are screened for cross-reactivity against the other types and individual molecular forms of collagen as well as other connective tissue proteins using the .ELISA assay of Section 5.1.7.
• the other individual collagens and connective tissue proteins are used in the ELISA assay. Only those monoclonal antibodies exhibiting no cross-reactivity are used in the procedures for detecting connective tissue proteins in body fluids and tissue samples described in Sections 5.2, 5.3, and 5.4 below.
• Hybrids which synthesized antibodies of the desired specificity are amplified in cell culture and stored in liquid nitrogen so that an adequate supply of cells producing identically monospecific antibodies are available.
• samples of the fused cells are injected intraperitoneally into BALB/c mice (10 cells/mouse) resulting in the subsequent induction of palpable tumors within a few weeks.
• the tumors generally produce ascites fluid (approximately 2 ml per mouse) containing antibody amounts significantly greater 33
• mice (as high as 60 mg per mouse) than those obtained by in vitro cell culture techniques.
• Sera samples from the inoculated mice contain antibody titers comparable to that - of ascites fluid.
• the mouse hybridoma-produced monoclonal antibodies are purified by subjecting samples of ascites fluid, sera, or media to immunoadsorption chromatography.
• Iodinated connective tissue protein antigens are prepared as described by Rohde et al. and are used in a modification of the radioimmunoassay described by the same authors [J. Immunol. Meth. 11:135-145 (1976)].
• Antibody titrations are carried out by diluting the monoclonal antibody preparation with PBS.
• 0.1 ml monoclonal antibody preparation (ascites fluid or tissue culture fluid), 0.1 ml labeled antigen, and 0.2 ml
• Non-specific precipitation of labeled antigen is determined by replacing the monoclonal antibody preparation by non-immune Ig.
• Antigen binding capacity of the monoclonal antibody preparation is calculated according to Minden and Farr [D.M. Weir,
• the monoclonal antibody preparation is first incubated with a sample containing unlabeled connective tissue protein at 4°C for 24 hours and then the labeled antigen is added to the reaction, followed by incubation and finally addition of and incubation with anti-mouse Ig as above. Precipitable counts are measured, also as above in a Beck an Gamma 300 counter.
• Monoclonal antibodies directed against connective tissue proteins are conjugated to alkaline phosphatase by the method of Hammerling et al. [Monoclonal antibodies and
• Dialysis tubing is boiled for 20 minutes in deionized water. Alkaline phosphatase ⁇ 1.5 mg as an ammonium sulfate-precipitated slurry) is centrifuged at 4°C for 2-3 minutes at 12,000xg and the supernatant is discarded. The pelleted enzyme is dissolved in buffer (Dulbecco's PBS with magnesium and calcium cations, DPBS) containing an appropriate amount of monoclonal antibody, in a volume of approximately 0.2 ml.
• buffer Dulbecco's PBS with magnesium and calcium cations, DPBS
• the antibody-enzyme mixture is dialyzed against 100 ml of
• Vortex mixer and is incubated for 2 hours at room temperature. After dialyzing overnight against DPBS at
• the enzyme-coupled antibody is diluted to 10 ml with
• Enzyme-linked monoclonal antibodies thus prepared are mixed with serological samples containing unknown amounts of the specific connective tissue protein being _ assayed.
• the mixtures are transferred to the wells of • microtiter plates pre-coated with the appropriate antigen and the enzyme activity of the conjugated alkaline phosphase is measured as described in Section 5.1.7.
• Sections of tissues 4-6 um thick are prepared from frozen, unfixed biopsy samples by cryostat sectioning. The air-dried sections are incubated with a particular monoclonal antibody. For controls, sections 20 are incubated with immunoglobulin (Ig) from pre-i mune serum. After 30 minutes of incubation in a humidified chamber at room temperature, the sections are rinsed three times with phosphate-buffered saline (PBS, pH 7.4) and, in a second step, layered with a 1:30 dilution of 25 fluorescein-isothiocyanate conjugated (FITC) rabbit anti-mouse Ig for 30 minutes. Finally, the slides are washed exhaustively to remove nonspecifically associated reagents and are sealed with a solution of 90%
• Tissues for instance kidney, are fixed in phosphate buffered 4% paraformaldehyde at 4°C for 2 hours with one change . Tissues are then washed for 36 hours in
• Frozen sections 8 um thick are cut and placed in albumin coated slides and air-dried for at least 5 minutes.
• Tissue sections are reacted with the appropriate monoclonal antibody in a moist chamber overnight at 4°C or at room temperature for 2 hours. Slides are washed thoroughly with PBS and then incubated an additional 2 hours with secondary antibody (goat or rabbit anti-mouse Ig) . This is followed by washing with cold PBS and a
• Fab-PAP Fab-peroxidase- anti-peroxidase
• Anchorage-dependent cells which have grown to confluent monolayers on solid supports are detached by exposure to trypsin and are replated in the Dulbecco-Vogt modification of Eagle's medium containing 10% fetal calf serum in 35 x
• the dishes are rinsed three times with 0.15 M NaCl, 0.02 M sodium phosphate, pH 7.4, and are layered with 1 ml of a 1:32 dilution of fluorescein-isothiocyanate-conjugated rabbit antimouse Ig.
• the dishes are first exposed to one type of monoclonal antibody against a connective tissue protein and then to the fluorescein-isothiocyanate- conjugated rabbit .antimouse Ig.
• the dishes are exposed to monoclonal antibodies against a different type of connective tissue protein, washed, and then reacted with rhodamine-conjugated rabbit antimouse Ig. Finally, all dishes are washed extensively to remove adventitiously associated reagents and sealed from the air with a solution of 90% glycerol/10% saline under a cover slip. The localization of fluorescent stains on the dishes is observed in a Zeiss Universal fluorescence microscope and recorded photographically.

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• 1983
  • 1983-05-05 CA CA000427504A patent/CA1251729A/en not_active Expired
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