WO1991017257A1 - MONOCLONAL ANTI-IgM ANTIBODIES, THEIR PRODUCTION AND USE, AND HYBRIDOMAS FOR PRODUCING THE SAME - Google Patents
MONOCLONAL ANTI-IgM ANTIBODIES, THEIR PRODUCTION AND USE, AND HYBRIDOMAS FOR PRODUCING THE SAME Download PDFInfo
- Publication number
- WO1991017257A1 WO1991017257A1 PCT/US1991/002822 US9102822W WO9117257A1 WO 1991017257 A1 WO1991017257 A1 WO 1991017257A1 US 9102822 W US9102822 W US 9102822W WO 9117257 A1 WO9117257 A1 WO 9117257A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- antibody
- igm
- cells
- antibodies
- igg
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
- C07K16/4283—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an allotypic or isotypic determinant on Ig
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
- A61K47/6873—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting an immunoglobulin; the antibody being an anti-idiotypic antibody
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- This invention is in the fields of immunology and monoclonal antibody production. More particularly it concerns monoclonal anti-IgM antibodies, hybridomas that produce these antibodies, immunochemicals made from those antibodies, and the use of those immunochemicals.
- Antibodies are endogenous proteins produced by the immune system in response to antigenic stimuli. These proteins specifically bind to antigen molecules at defined sites (epitopes). Polyclonal antibodies are derived from immunization of animals with antigens and they bind to these antigens at multiple sites (epitopes). Monoclonal
- antibodies are a specific, defined set of antibodies which are derived from a single clone
- the IgM sub-type antibodies are generally of low affinity, are difficult to purify and often comprise the bulk of antibodies produced by hybridomas.
- IgM and IgG secreting are generally of low affinity, are difficult to purify and often comprise the bulk of antibodies produced by hybridomas.
- IgM secreting cells often overgrow the IgG secreting hybrid cells.
- the IgG producing hybridoma cells are then further analyzed to determine the antigen specificity of the antibodies produced.
- immunotoxins has been disclosed by the applicants and others. Recent interest has centered on immunotoxins of monoclonal antibodies conjugated to the enzymatically active portions (A chains) of toxins of bacterial or plant origin via hetero-bifunctional agents. Nevelle, D.M. and Youle, R. J., Immunol Rev (1982) 62: 75-91; Ross, W.C.J., et al., European J Biochem (1980) 104; Vitteta, E.S., et al.,
- a principal aspect of the invention concerns rat monoclonal antibodies that:
- the preferred embodiment of these antibodies is one designated 2G10, and functional equivalents thereof.
- the invention also includes a method of preparing a hybridoma as defined above comprising fusing rat tumor cells with rat splenocytes obtained from a rat immunized with murine IgM sub-type immunogen and selecting for hybridomas producing antibody as defined above.
- a further aspect of the invention is a method of producing antibody as defined above comprising culturing a hybridoma having the ability to produce such antibody, or optionally a hybridoma which has been prepared by effecting a method as claimed above.
- Another aspect of the invention relates to immunotoxins and their preparation by conjugating
- derivatives of the above described monoclonal antibodies that are labeled with a detectable label that permits the derivatives to be used in targeting, specific selection or sorting.
- Another aspect of the invention concerns a method of killing IgM producing hybridoma or B cells by contacting the cells with a cytocidally effective amount of one or more of the above described immunotoxins.
- kits for determining whether a cell is producing IgM antibodies or to determine whether an antibody is of the IgM isotype involve incubating the cells with the monoclonal antibodies or labeled derivatives thereof. When the labeled derivatives are used, the presence of labeled binary immune complexes on the cells as read directly. When unlabeled antibody is used the cells are further incubated with a labeled antibody against the monoclonal antibody and the presence of labeled ternary immune complexes on the cells is read.
- Figure 1 demonstrates screening of hybridoma
- Figure 2 demonstrates the dose-dependent specific binding of antibody 2G10.
- Figure 3 demonstrates the effect of increasing absorbed murine immunoglobulin content on 2G10 binding.
- Figure 4 shows selective recognition of murine IgM by 2G10 in an indirect ELISA.
- Figure 5 illustrates the purity of 2G10 antibody by SDS-PAGE.
- Figure 6 characterizes the subclass of rat 2G10
- Figure 7 demonstrates utilization of 2G10 for binding to cells expressing IgM subclass antibodies.
- Figure 8 illustrates the elution profile of immunotoxin (composed of 2G10 coupled to gelonin) on a gel filtration matrice.
- Figure 9 demonstrates the purity of 2G10-gelonin immunotoxin by SDS-PAGE.
- monoclonal antibody means an antibody composition having a homogeneous antibody
- the term "functional equivalent” means a monoclonal antibody that: (a)
- crossblocks an exemplified monoclonal antibody (b) binds selectively to murine IgM antibody; (c) has a G isotype; and (d) does not bind to IgG 1 or lgG 2 isotype.
- the present invention may be utilized to produce antibodies that will bind to IgM antibodies of any species. It is only necessary to utilize the teaching of the present invention to obtain a hybridoma cell line which is stable and continues to produce the anti-IgM antibody directed to the immunizing specie.
- the anti-IgM monoclonal antibody of the present invention is directed to a murine or human IgM.
- the antibody-producing fusion partners that are used to make the hybridomas of this invention are generated by immunizing rats with murine IgM antibody.
- the rats are inoculated subcutaneously and intraperitoneally with an immunogenic amount of the murine IgM antibody in Feund's adjuvant and then boosted with similar amounts of the
- Spleens are collected from the immunized rats a few days after the final boost and a cell suspension is prepared therefrom for use in the fusion.
- Hybridomas are prepared from the splenocytes and a rat tumor partner using the general somatic cell hybridization technique of Kohler, B. and Milstein, C., Nature (1975) 256: 495-497 [as modified by Buck, D. W. , et al, In Vitro (1982) 18: 377-381].
- Available rat myeloma lines such as YB2/0 and Y3-Ag 1.2.3, may be used in the hybridization.
- the technique involves fusing the tumor cells and splenocytes using a fusogen such as polyethylene glycol.
- the cells are separated from the fusion medium and grown in a selective growth medium, such as HAT medium, to eliminate unhybridized parent cells.
- a selective growth medium such as HAT medium
- hybridomas are expanded, if desired, and supernatants are assayed for anti-murine IgM activity by conventional
- immunoassay procedures e.g., radioimmunoassay, enzyme immunoassay, or fluorescence immunoassay
- immunizing agent IgG 1 , IgG 2 and IgM murine IgM antibody
- Positive clones are characterized further to determine whether they meet the criteria of the invention antibodies.
- Hybridomas that produce such antibodies may be grown in vitro or in vivo using known procedures.
- the monoclonal antibodies may be isolated from the culture media or body fluids, as the case may be, the conventional immunoglobulin purification procedures such as ammonium sulfate
- antibodies are (1) their immunoglobulin class, (2) their selectivity for murine IgM antibody, and (3) their
- the selectivity and range of a given antibody is determined by testing it against panels of (1) IgG 1 , IgG 2 and IgM producing hybridoma cells and (2) IgG 1 , IgG 2 and IgM antibodies. In selecting the claimed antibodies
- N-succinimidyl-3-(2-pyridyldithio) propionate SPDP
- iminothiolane IT
- immunotoxins conjugates of the antibody and a cytotoxic moiety and labeled (e.g., radiolabeled, enzyme-labeled, magnetic-labeled or fluorochrome-labeled) derivatives in which the label provides a means for identifying and/or sorting immune complexes that include the labeled antibody.
- labeled e.g., radiolabeled, enzyme-labeled, magnetic-labeled or fluorochrome-labeled
- the cytotoxic moiety of the immunotoxin may be a cytotoxic drug or an enzymatically active toxin of bacterial or plant origin, or an enzymatically active fragment ("A chain") of such a toxin.
- Enzymatically active toxins and fragments thereof are preferred and are exemplified by gelonin, diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas
- ricin A chain abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytoiacca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, saponaria officinalis inhibitor, mitogellin, restrictocin, phenomycin, and enomycin. Gelonin is most preferred.
- Conjugates of the monoclonal antibody and such cytotoxic moieties may be made using a variety of bifunctional protein coupling agents. Examples of such reagents are SPDP, IT, bifunctional
- imidoesters such as dimethyl adipimidate - HCl
- active esters such as disuccinimidyl suberate
- aldehydes such as glutaraldehyde
- bis-azido compounds such as bis(p-azidopenzoyl) hexanediamine
- 2,6-diisocyanate 2,6-diisocyanate, and bis-active fluorine compounds such a 1,5-difluoro-2,4-dinitrobenzene.
- the conjugates will typically be added to the cell culture medium at a concentration of at least about 10 nM.
- concentration of at least about 10 nM.
- the formulation and mode of administration for in vitro use are not critical. Aqueous formulations that are compatible with the culture or perfusion medium will normally be used. Cytotoxicity may be read by conventional techniques to determine the presence or degree of IgM producing hybridoma cells.
- the immunotoxins When used in vivo for suppression of IgM producing cells, the immunotoxins are administered to the immunized animals in therapeutically effective amounts (i.e., amounts that eliminate or reduce the IgM producing splenocytes). They will normally be administered parenterally, preferably intravenously.
- the dose and dosage regimen will depend upon the nature of the IgM producing cell to be suppressed, the characteristics of the particular immunotoxin, e.g., its therapeutic index, and onset of action.
- the amount of immunotoxin administered will typically be in the range of about 0.1 to about 10 mg/kg of body weight.
- the immunotoxins will be formulated in a unit dosage injectable form (solution, suspension, emulsion) in association with a pharmaceutically acceptable parenteral vehicle.
- a pharmaceutically acceptable parenteral vehicle Such vehicles are inherently nontoxic and nontherapeutic. Examples of such vehicles are water, saline. Ringer's solution, dextrose solution, and 5% human serum albumin. Nonaqueous vehicles such as fixed oils and ethyl oleate may also be used. Liposomes may be used as carriers.
- the vehicle may contain minor amounts of
- the immunotoxin will typically be formulated in such vehicles at concentrations of about 1 mg/ml to 10 mg/ml.
- Cytotoxic radiopharmaceuticals for eliminating IgM producing hybridoma cells may be made by conjugating high linear energy transfer (LET) emitting isotopes (e.g., Y, Pt) to the antibodies.
- LET linear energy transfer
- cytotoxic moiety as used herein is intended to include such isotopes.
- Examples of such labels are 3 2 P, 125 I, 3 H, 14 C, fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferia,
- 2,3-dihydrophthalazinediones horseradish peroxidase, alkaline phosphatase, lysozyme, and glucose-6-phosphate dehydrogenase.
- the antibodies may be tagged with such labels by known methods. For instance, coupling agents such as aldehydes, carbodiimides, dimaleimide, imidates,
- succinimides bis-diazotized benzadine and the like may be used to tag the antibodies with the above-described
- the antibodies may also be labeled with magnetic beads for use in magnetic sorting regimens.
- the antibodies and labeled antibodies may be used in a variety of cell sorting procedures to separate the IgM producing hybridoma cells from IgG producing hybridoma cells or to eliminate the IgM producing hybridoma cells from cultures containing such cells.
- Common assay techniques that may be used include direct and indirect assays. Direct assays involve incubating a hybridoma or antibody of unknown isotype with a labeled antibody of the present invention. If the sample includes IgM producing cells, the labeled antibody will bind to those cells. After washing the cells to remove unbound labeled antibody, the sample is read for presence of labeled immune complexes. In indirect assays the cell sample is incubated with unlabeled monoclonal antibody. The sample is then treated with a labeled antibody against the monoclonal antibody (e.g., a labeled anti-rat antibody), washed, and read for the presence of labeled ternary complexes.
- kits will typically comprise: the antibody in labeled or unlabeled form in suitable
- kits for the incubations and washings, labeled anti-rat antibody if the kit is for an indirect assay, and substrates or derivatizing agents depending on the nature of the label.
- a rat hybridoma designated 58.6 was obtained from Dr.
- 58.6 cells were cultured in Iscove's medium for 3 days at 37°C in a humidified atmosphere of 5 CO 2 in air. When the expanded cell culture had reached 50% confluency, cells were harvested by centrifugation and counted using a
- Fluid was collected into tubes containing 5 ml of PBS with 20 itiM EDTA. After centrifugation at 2000 x g for 10 min, the supernatant was saved, made 0.1% in sodium azide, and stored at 4°C or frozen at -20°C in small aliquots. This fluid provided a rich source of monoclonal antibody (approximately 5-10 mg/ml).
- Hybridoma colonies which grew to a density of
- Hybridoma culture medium from these colonies was assayed for the presence of rat anti-mouse IgM by the
- Hybridomas IC2 and 2G10 both bound equally well to IgM-kappa and IgM-lambda coated wells, indicating that the binding specificity of the anti-IgM antibodies was on the heavy (mu) chain of the IgM antibody. That the other 7 antibodies tested also bound to murine IgM is indicated by the results shown on Figure 1. As can be seen in Figure 1A and IB, nine different hybridomas were tested against either IgM-kappa or IgM-lambda coated plates. A standard ELISA assay was performed to measure rat
- the specificity of the rat monoclonal 2G10 antibody was tested by ELISA under a variety of conditions.
- 100ng of mouse IgM-lambda protein or IgG 3 -lambda protein was absorbed onto microtiter plates (100 ul) and incubated with
- IgM-coated plates at all coating concentrations. Ten-fold greater reactivity was measureable by ELISA on IgM-versus IgG-coated plates at a coating dose of 10000 ng of IgM or IgG (compare IgG3-kappa to IgM-kappa).
- the 2G10 antibody again demonstrates selective IgM binding.
- Antibody 2G10 was also tested for its ability to selectively recognize IgM in an indirect ELISA assay.
- Antigens were coated to microtiter plates and were incubated with hybridoma culture media which contained antibody which recognizes these antigens. Antibodies of the IgM and IgG1 subclass were used in this assay. After incubation of antigen-coated plates with their interacting antibody, increasing concentrations of rat 2G10 antibody were added to the wells and the binding of 2G10 was evaluted by ELISA. As shown in Figure 4, 2G10 sensitively and selectively
- Rat Anti-Mouse IgM Antibodies Mouse immunoglobulin of various subtypes (IgM-kappa and lambda, IgG 1 , lgG 2 , and IgG 3 ) were coated on microtiter plates as in Example 2 and the binding of the rat anti-mouse IgM antibodies produced by the hybridoma cells which were positive in Example 2 were further characterized. All plates were read in a Bio-Tek ELISA plate reader at a wavelength of 405 nm. Absorbance (compared to controls) was used an an indication of the presence of antibody against mouse IgM.
- Example 3 the 2G10 antibody was positive for IgM kappa and lambda.
- antibody was purified by centrifugation and ammonium sulfate fractionation.
- the representative hybridoma cell line designated 2G10 was deposited with the American Type Culture Collection (ATCC), Rockville, Md., U.S.A., on April 23, 1990 and assigned Deposit Accession No. HB 10436.
- ATCC American Type Culture Collection
- the deposits are available pursuant to the patent laws and regulations of the United States and of those countries foreign to the United States in which counterparts of this application are filed. The availability of the deposit does not constitute a license to practice the invention of this application in derogation of any patent issued thereon or on any division or continuation of this application.
- 2G10 culture supernatant (or ascites fluid from nude mice) was made 45% saturated in ammonium sulfate content (salting out) by the slow addition of an equal volume of 90% saturated ammonium sulfate solution.
- the sample was stirred for 30 minutes at 4°C and then centrifuged at 20,000 x g for 30 minutes. The pellet was resuspended in a 40% saturated ammonium sulfate solution, stirred 30 minutes and repelleted by centrifugation as described above.
- the pellet was
- IgM antibodies were purified by ammonium sulfate precipitation and gel filtration on a 2.6 x 40 cm column of chromatographic resin containing agarose, dextran and/or acrylamide eluting with PBS/0.01% sodium azide at room temperature at a flow rate of 1 ml/min.
- the subclass of 2G10 rat monoclonal antibody was determined by the method of Ouchterlony (Ouchterlony and Nilsson (1958) in Handbook of Exp. Immun. Weir, ed.,
- the subclass of antibody 2G10 is important in the subclass of antibody 2G10.
- immunoglobin sub-types was added to each of the satellite wells.
- a known standard or unknown sample was added and allowed to diffuse into the semisolid media.
- a precipitation band at the site of the specific antisera indicates the subtype.
- the positive control samples containing all rat sub-type were added to each of the satellite wells.
- a known standard or unknown sample was added and allowed to diffuse into the semisolid media.
- a precipitation band at the site of the specific antisera indicates the subtype.
- Figure 6 the positive control samples containing all rat sub-type
- antibody 2G10 is an IgG 2a antibody.
- rat monoclonal antibody LO-MM-9 from Serotec, cat#MCA 199
- LO-MM-9 rat monoclonal antibody against murine IgM
- One hundred nanograms of mu-k, gamma-k, or gamma-1 was added to each well of a 96 well plate.
- Various ammounts of LO-MM-9 rat antibody were then added and an ELISA assay for rat antibody was performed as described previously. As shown in Table 1, there was no binding of this rat antibody to murine IgM coating the wells.
- this antibody was not deemed useful for further study.
- this antibody preparation contained at least three major protein bands and at least five minor protein bands as assessed by SDS PAGE.
- hybridoma cells are first incubated with rat anti-mouse IgM antibody 2G10, washed, then stained with the fluorescein F(ab) 2 fragment goat anti-mouse immunoglobulin IgM and sorted by flow cytometry.
- antibody 2G10 bound specifically to IgM present on the surface of IgM secreting murine hybridoma cells and not to IgG secreting cells.
- the claimed antibodies were conjugated to ricin toxin A chain (RTA) treated with SPDP as described by Carlsson, J., et al, Biochem J (1978) 173: 723-737 or with iminothiolane (IT).
- RTA ricin toxin A chain
- SPDP ricin toxin A chain
- I iminothiolane
- 3-(2-pyridylditho) proprionate) (6mg/ml) in dry DMF was prepared.
- SPDP was slowly added to a 5-fold molar excess (approx. 10 ul of stock solution). The mixture was vortexed every 5 minutes for 30 minutes at room temperature.
- Antibody eluted at the void volume (fractions 14-20) and these fractions were pooled and kept at 4°C.
- TEA/HCl buffer to a final concentration of 60 mM TEA/HCl and adjusted to pH 8.0.
- the solution was made 1 mM EDTA.
- 2-iminothiolane stock solution 0.5M in 0.5M TEA/HC1 pH 8.0 was added to a final concentration of 1 mM and the sample was incubated for 90 minutes at 4°C under nitrogen gas.
- reaction mixture was applied to a Sephadex S-300 column (1.6 x 31 cm) previously
- the coupling mixture containing free 2G10 antibody, 2G10 gelonin and free gelonin was first purified by gel filtration on an S-300 column. As shown in Figure 8, a high molecular weight peak was detected (fractions 25-42 ) as well as a lower molecular weight peak (fractions 55-67).
- the 2G10 gelonin conjugate was added to wells at various concentrations. A standard ELISA assay was then performed to detect rat antibody. As shown in Figure 10, the 2G10 gelonin conjugate bound readily to IgM and only to a small extent to IgG coated plates. Only at the highest concentration tested (1000 ng/well) did the 2G10 gelonin conjugate bind significantly to the IgG-coated wells. In contrast, at a similar concentration the 2G10 gelonin conjugate bound extensively to the IgM coated wells. Thus, the immunoreactivity of the 2G10 gelonin conjugate towards IgM was preserved.
- Protocols employed currently for the production of murine monoclonal antibodies result in the generation of hybridomas which secrete antibodies of the IgM subclass.
- two techniques were employed for generation of monoclonal antibodies against a rat protein ornithine decarboxylase (ODC).
- the first technique involves immunization of murine spleen cells in vitro, i.e., in culture dishes, with ODC protein.
- ODC protein was incubated with mouse cells for
- thymocyte conditioned medium a source of immunoglobulin secreting cell type specific growth factors.
- the spleen cells were then fused with MPC myeloma cells using polyethylene glycol as a fusogen.
- the resultant hybrids cells were tested for the secretion of antibody reactive with ODC protein. The results are summarized on Table 3.
- a second technique employed for the production of monoclonal antibodies was the immunization of a mouse by injection with purified ODC protein.
- Mouse spleen cells were isolated after immunization, fused with P3 X 63 Ag 8.653 myeloma cells (utilizing PEG as described above).
- Fluorescein conjugated affinity purified F(ab) 2 fragment goat anti-mouse immunoglobulin IgM (Cappel) is diluted 1:100 in PBS (lx) and 20-40 ul is added to 20 ul cell suspension.
- hybridoma cells are first incubated with rat anti-mouse IgM antibody 2G10, washed, then stained with the fluorescein F(ab) 2 fragment goat anti-mouse immunoglobulin IgM and sorted by flow cytometry.
- Mouse hybridoma cells 238-57 ADR are washed three times in Iscove's medium containing 1% fetal bovine serum and 0.1% gentamicin. The cells are centrifuged at 500 x g for three minutes at room temperature and then counted. The pellet is resuspended in 0.5 ml medium and incubated for 60 minutes on ice with purified rat anti-mouse IgM antibody (2G10) at a
- Magnetic beads are washed 3 times in serum-free medium using a magnetic board.
- the cell pellet is mixed with the bead pellet at a ratio of 20 beads/cell.
- the total volume should not exceed 0.4 ml.
- the cell/bead mixture is incubated for 1/2 hour on ice, agitating every 10 minutes.
- the cell/bead mixture is resuspended in at least 2 ml of medium and separate magnetically perpendicular to gravity. Once separation is complete, the supernatant is removed without disturbing the magnetic pellet.
- the beads are resuspended in 1-2 ml medium and observed miscroscopically.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Pharmacology & Pharmacy (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
Description
Claims
Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP3509144A JPH06504424A (en) | 1990-04-27 | 1991-04-24 | Monoclonal anti-IgM antibodies, their production and use, and hybridomas for producing them |
AU78664/91A AU650659C (en) | 1990-04-27 | 1991-04-24 | Monoclonal anti-IgM antibodies, their production and use, and hybridomas for producing the same |
RU92016351A RU2105062C1 (en) | 1990-04-27 | 1991-04-24 | ANTI-iGM-ANTIBODY-BASE CONJUGATE (VARIANTS) AND METHOD OF DECREASE OF IGM-ANTIBODY SECRETION BY LYMPHOCYTES (VARIANTS) |
FI924851A FI924851A0 (en) | 1990-04-27 | 1992-10-26 | MONOCLONAL ANTI-IGM ANTIKROPPAR, DERAS FRAMSTAELLNING OCH ANVAENDNING SAMT HYBRIDOMER FOER FRAMSTAELLNING AV DEM |
NO92924137A NO924137L (en) | 1990-04-27 | 1992-10-26 | MONOCLONAL ANTI-IGM ANTIBODIES, THEIR PREPARATION AND USE, AND HYBRIDOMAS FOR THE PREPARATION OF THE SAME |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US51597490A | 1990-04-27 | 1990-04-27 | |
US515,974 | 1990-04-27 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1991017257A1 true WO1991017257A1 (en) | 1991-11-14 |
Family
ID=24053580
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1991/002822 WO1991017257A1 (en) | 1990-04-27 | 1991-04-24 | MONOCLONAL ANTI-IgM ANTIBODIES, THEIR PRODUCTION AND USE, AND HYBRIDOMAS FOR PRODUCING THE SAME |
Country Status (12)
Country | Link |
---|---|
EP (1) | EP0538267A4 (en) |
JP (1) | JPH06504424A (en) |
CN (1) | CN1057072A (en) |
CA (1) | CA2079901A1 (en) |
IE (1) | IE911172A1 (en) |
IL (1) | IL97827A0 (en) |
NZ (1) | NZ237784A (en) |
PT (1) | PT97482B (en) |
RU (1) | RU2105062C1 (en) |
TW (1) | TW349995B (en) |
WO (1) | WO1991017257A1 (en) |
ZA (1) | ZA912695B (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN116143931A (en) * | 2021-11-20 | 2023-05-23 | 东莞市朋志生物科技有限公司 | Anti-human IgM antibody and preparation method and application thereof |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
MY171554A (en) | 2008-09-05 | 2019-10-18 | Sekisui Medical Co Ltd | Monoclonal antibody and immunoassay using the same |
CN108384761B (en) * | 2018-03-30 | 2020-08-11 | 四川迈克生物新材料技术有限公司 | Anti-human IgM monoclonal antibody, hybridoma cell strain and application thereof |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4792447A (en) * | 1981-07-23 | 1988-12-20 | Board Of Regents, The University Of Texas System | Anti-immunoglobulin toxin conjugates useful in the treatment of B cell tumors |
AU9016591A (en) * | 1990-10-25 | 1992-05-26 | Tanox Biosystems, Inc. | Glycoproteins associated with membrane-bound immunoglobulins as antibody targets on B cells |
-
1991
- 1991-04-08 IE IE117291A patent/IE911172A1/en unknown
- 1991-04-10 NZ NZ237784A patent/NZ237784A/en unknown
- 1991-04-11 ZA ZA912695A patent/ZA912695B/en unknown
- 1991-04-11 IL IL97827A patent/IL97827A0/en unknown
- 1991-04-17 TW TW080103007A patent/TW349995B/en active
- 1991-04-24 WO PCT/US1991/002822 patent/WO1991017257A1/en not_active Application Discontinuation
- 1991-04-24 JP JP3509144A patent/JPH06504424A/en active Pending
- 1991-04-24 EP EP19910909436 patent/EP0538267A4/en not_active Withdrawn
- 1991-04-24 RU RU92016351A patent/RU2105062C1/en active
- 1991-04-24 CA CA002079901A patent/CA2079901A1/en not_active Abandoned
- 1991-04-26 PT PT97482A patent/PT97482B/en not_active IP Right Cessation
- 1991-04-27 CN CN91102913A patent/CN1057072A/en active Pending
Non-Patent Citations (4)
Title |
---|
European Journal of Immunology, Volume 14, Number 8, issued August 1984, JULIUS et al., "Induction of resting B cells to DNA Synthesis by soluble Monoclonal Anti-Immunoglobulin", pages 753-757, see entire article. * |
See also references of EP0538267A4 * |
The Journal of Biological Chemistry, Volume 260, Number 22, issued 05 October 1985, LAMBERT et al., "Purified Immunotoxins that are reactive with human Lymphoid cells", pages 12035-12041, see page 12035, first paragraph of text and page 12038, all of methods. * |
The Journal of Immunology, Volume 127, Number 3, issued September 1981, KUNG et al., "A mouse IgM Allotypic determinant (Igh-6.5) recognized by a Monoclonal rat Antibody", pages 873-876, see entire article. * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN116143931A (en) * | 2021-11-20 | 2023-05-23 | 东莞市朋志生物科技有限公司 | Anti-human IgM antibody and preparation method and application thereof |
CN116143931B (en) * | 2021-11-20 | 2023-10-31 | 东莞市朋志生物科技有限公司 | Anti-human IgM antibody and preparation method and application thereof |
Also Published As
Publication number | Publication date |
---|---|
EP0538267A1 (en) | 1993-04-28 |
JPH06504424A (en) | 1994-05-26 |
CN1057072A (en) | 1991-12-18 |
PT97482A (en) | 1992-01-31 |
ZA912695B (en) | 1993-02-24 |
CA2079901A1 (en) | 1991-10-28 |
EP0538267A4 (en) | 1994-06-01 |
IL97827A0 (en) | 1992-06-21 |
RU2105062C1 (en) | 1998-02-20 |
NZ237784A (en) | 1992-09-25 |
AU7866491A (en) | 1991-11-27 |
PT97482B (en) | 1998-08-31 |
AU650659B2 (en) | 1994-06-30 |
IE911172A1 (en) | 1991-11-06 |
TW349995B (en) | 1999-01-11 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP0153114B1 (en) | Monoclonal anti-human breast cancer antibodies, their production and use, and hybridomas for producing the same and their preparation | |
US5629197A (en) | Monoclonal anti-human breast cancer antibodies | |
EP0450479B1 (en) | Bispecific monoclonal anti-bodies, their production and use | |
Mason et al. | Preparation of peroxidase: antiperoxidase (PAP) complexes for immunohistological labeling of monoclonal antibodies. | |
NO174719B (en) | Procedure for Preparation of Monoclonal Antibodies | |
Ball et al. | Isolation and characterization of monoclonal antibodies to (Na++ K+)-ATPase | |
US6306626B1 (en) | Anti-IgM monoclonal antibodies and methods of their use | |
CA1297048C (en) | Monoclonal antibodies reactive with normal and oncogenic forms of the ras p21 protein | |
WO1991017257A1 (en) | MONOCLONAL ANTI-IgM ANTIBODIES, THEIR PRODUCTION AND USE, AND HYBRIDOMAS FOR PRODUCING THE SAME | |
AU650659C (en) | Monoclonal anti-IgM antibodies, their production and use, and hybridomas for producing the same | |
US5053492A (en) | Immunopurification using monoclonal antibodies to Mojave toxin | |
JPH02238894A (en) | Antibody against endoserine and use thereof | |
US5032521A (en) | Monoclonal antibody specific for a mammary tumor cell surface antigen | |
CA2050941C (en) | Antibody to smooth muscle myosin heavy chains | |
METTLER et al. | Monoclonal sperm antibodies: their potential for investigation of sperms as target of immunological contraception | |
CA1253090A (en) | Monoclonal anti-human breast cancer antibodies | |
AU609382B2 (en) | Monoclonal antibodies to terminal deoxynucleotidyl transferase | |
US5141865A (en) | Monoclonal antibodies which bind thromboxane A2 receptor antagonists and diagnostic methods based thereon | |
JP3167024B2 (en) | Monoclonal antibodies against endothelin-3 or endothelin-3 precursor and uses thereof | |
JPH05168494A (en) | Hybrid monoclonal antibody and medicine containing the same antibody | |
EP0492530A1 (en) | Monoclonal antibodies which bind mannose binding protein | |
CA2132632A1 (en) | Anti-zona pellucida antibodies for delivery of therapeutic agents to the ovary | |
EP0439117A1 (en) | Monoclonal antibodies which bind (E)-5-(2-bromovinyl)-arabinofuranosyluracil and diagnostic methods based thereon | |
NAIEM | Preparation of Peroxidase: Antiperoxidase (PAP) Complexes for Immunohistological Labeling of | |
PT101652B (en) | ANTI-IGM MONOCLONAL ANTIBODIES AND METHODS FOR THEIR USE |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AU CA FI JP KR NO SU |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): AT BE CH DE DK ES FR GB GR IT LU NL SE |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2079901 Country of ref document: CA |
|
WWE | Wipo information: entry into national phase |
Ref document number: 1991909436 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 924851 Country of ref document: FI |
|
WWP | Wipo information: published in national office |
Ref document number: 1991909436 Country of ref document: EP |
|
WWW | Wipo information: withdrawn in national office |
Ref document number: 1991909436 Country of ref document: EP |