[go: up one dir, main page]

WO1990000625A1 - Pre-criblage servant a l'appauvrissement et a l'enrichissement de lymphocytes b specifiques - Google Patents

Pre-criblage servant a l'appauvrissement et a l'enrichissement de lymphocytes b specifiques Download PDF

Info

Publication number
WO1990000625A1
WO1990000625A1 PCT/US1988/002407 US8802407W WO9000625A1 WO 1990000625 A1 WO1990000625 A1 WO 1990000625A1 US 8802407 W US8802407 W US 8802407W WO 9000625 A1 WO9000625 A1 WO 9000625A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
antigen
protein
screening
specific epitope
Prior art date
Application number
PCT/US1988/002407
Other languages
English (en)
Inventor
Richard E. Warrington
Original Assignee
Monoclonetics International, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Monoclonetics International, Inc. filed Critical Monoclonetics International, Inc.
Priority to PCT/US1988/002407 priority Critical patent/WO1990000625A1/fr
Publication of WO1990000625A1 publication Critical patent/WO1990000625A1/fr

Links

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
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • 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
    • 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
    • C07K16/4208—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/16—Animal cells
    • C12N5/163—Animal cells one of the fusion partners being a B or a T lymphocyte
    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood cells
    • 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
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6878—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids in epitope analysis

Definitions

  • the invention relates to the field of immunology, and more particularly to a pre-screening procedure for selecting B-cells producing antibodies to specific epitopes carried on antigens.
  • the selected B-cells can be used for production of monoclonal antibodies.
  • B-cell lymphocytes are the cells of an animal which produce antibody when the animal is exposed to an antigen. When an antigen enters the body it causes a complex series of responses in the immune system. Certain B-cells are stimulated to produce antibody which can bind to the epitopes of that antigen. Some of the antibody produced is excreted by the B-cell, while some is carried on the cell surface.
  • B-cells have become of particular interest to the scientific community in recent years because they are essential in the production of monoclonal antibodies.
  • Monoclonal antibodies have a wide variety of applications. Their primary use is in detecting the presence of particular proteins or other antigens. Detection of such molecules can be valuable for diagnosis of disease states or diagnosis of other conditions, such as pregnancy.
  • Monoclonal antibodies are also useful in research, in collecting and purifying specific antigens, and in the development of vaccines.
  • Monoclonal antibodies bind specifically to a particular epitope of an antigen. They have this property because they are produced by cells, known as hybridomas, which are all cloned from a single cell. All the clones have the same genetic pattern as the parent. Accordingly, all clones produce identical antibodies which have identical characteristics.
  • a method of making monoclonal antibodies was first described by Koehler and Milstein. See Milstein et al., Nature, 256, 495-97 (1975); Koehler et al., Eur. J. Immunol., 6, 511-19 (1976).
  • a host animal usually a mouse
  • the tissue which is rich in B-cells usually the spleen or lymphoid tissue
  • the ground tissue is- fused with myeloma (cancer) cells to form hybridomas.
  • the hybridomas which produce antibody to the epitopes of the immunizing antigen are then isolated from the others, many of which produce antibodies to other antigens.
  • the selected hybridomas are then reproduced to manufacture monoclonal antibodies.
  • hybridomas desired i.e., those which produce antibody to the epitopes of the immunizing antigen.
  • Hybridomas which produce undesired antibody, or no antibody at all are formed when B-cells which respectively produce undesired antibody, or no antibody, are fused with myeloma cells.
  • B-cells which produce undesired antibody are present because every animal is exposed to numerous antigens during its lifetime, and each antigenic exposure stimulates B-cells to produce reactive antibody.
  • B-cells which produce no antibody are those which are never stimulated by antigens during the animal's lifetime.
  • the desired hybridomas are typically isolated by sufficiently diluting the hybridoma cell suspension which results from the fusion to ensure the presence of only one clone in a given volume of suspension.
  • the volume of the suspension which contains the single clone is then placed into a well.
  • the antibody produced by the hybridoma which is excreted by the clone and which therefore becomes suspended in the well, is then bound to the walls of the well. Thereafter, radioactively labeled immunizing antigen is placed into each well. If the antibody in the well is specific to the labeled antigen, the labeled antigen will be bound.
  • the labeled antigen will remain suspended.
  • the wells are then washed to remove any unbound labeled antigen. Clones from those wells which display the highest level of radioactivity, and thus the highest production of the desired antibody, are multiplied to produce desired monoclonal antibodies.
  • B-cells which produce antibody to the immunizing antigen could be separated from those which produce no antibody, this would shorten the isolation process which is used to locate desired hybridomas as described above. If a pre-screen selection of B-cells were performed, non-producing B-cells would not be used in the fusion, and fewer of the hybridomas would be undesired. A larger proportion of wells would contain desired hybridomas. Isolation of desired hybridomas would consequently be faster and easier.
  • a B-cell pre-screening procedure can be utilized in many specific applications. One application is when the objective is to produce monoclonal antibodies specific to only one of the multiple epitopes that are present on an antigen.
  • the aforementioned isolation process relies on selective binding of radioactively labeled antigen as a means for identifying appropriate antibodies.
  • Antibodies which are to any of the many epitopes of the labeled antigen - - whether it is the designated epitope or not - - will bind the labeled antigen.
  • wells which contain antibodies to the undesired epitopes on the antigen will display a high radioactivity level.
  • wells containing antibodies to undesired epitopes will bind labeled antigen to the same extent and display as high a radioactivity count as wells which contain antibody to the designated epitope.
  • the selection of hybridomas producing monoclonal antibodies which are specific to one of the epitopes on an antigen is done by breaking the antigen into subunits, if the particular antigen can be broken down.
  • the monoclonals are then tested for reactivity with the various subunits and with the whole antigen.
  • the monoclonals which react only with the whole antigen, and not with the subunits, are then isolated.
  • Another application of a B-cell pre-screening procedure is where different immunogeni ⁇ entities carry some of the same epitopes, and the objective is to produce monoclonal antibodies to the epitopes which are not common to all entities.
  • An example of such immunogenic entities is a normal cell and a cancerous cell. Both will have a large number of surface epitopes in common. Yet the cancerous cell should carry some epitopes which represent the alteration in its structure.
  • a B-cell pre-screening process can also be used to isolate antibodies to uncommon epitopes on other types of cells. For example, a B-cell pre-screening process could reduce the time involved with isolating antibodies which differentiate diseased cells or virus transformed cells from normal cells. In addition, a pre-screening process could reduce the time involved with differentiating antibodies to young ceils from those to quiescent cells or senescent cells, or differentiating antibodies to starved cancer cells from those to non-starved cancer cells, or differentiating antibodies to transformed cells causing arteriosclerosis from those to normal epithelial cells which line the blood vessels.
  • a B-cell pre-screening process would also be useful for reducing the time needed to produce antibodies to certain biological molecules which are closely related, or even nearly identical, to other biological molecules. For example, the time involved with producing antibodies to different strains of viruses, and to fully folded, slightly folded, and unfolded proteins could be reduced.
  • a B-cell pre-screening process could be useful in isolating B-cells producing antibodies which are specific to certain trace proteins, such as those associated with disease states. If the protein is truly a trace protein, by definition present only in minute quantities, a correspondingly small number of B-cells will produce antibody to it. If hybridomas are produced from the family of B-cells, then the few hybridomas which result from the fusion of these few B-celis will be very difficult to isolate from those surrounding them. However, if the B-cells were pre-screened to select those producing antibody to the trace protein, a greater proportion of the hybridomas in the wells would produce monoclonals to the trace proteins. Therefore, isolation of the desired hybridomas would be easier.
  • a B-cell pre-screening process could also be useful in isolating anti-idiotype antibodies.
  • All antibodies carry "idiotypes," regions near their antigen-recognition sites that are themselves capable of acting as antigens and stimulating antibody production. See J.L. Marx, Science, 228 "Making Antibodies Without Antigens," 162-65 (1986).
  • Antibodies which are specific to the idiotype sites are known as anti-idiotype antibodies.
  • Anti-idiotype antibodies have the same molecular structure, at least in part, as does the antigen which stimulated antibody production.
  • Anti-idiotype antibodies can be made by immunizing an animal with antibody. In order to make the antibody, a particular antigen is first selected, and an animal is immunized with it. If a pathogenic antigen is selected, the anti-idiotype antibodies eventually produced will be suitable for use as vaccines. The anti-idiotype will be so suited because they will carry the same molecular structure, at lease in part, as the antigen. Thus, immunization of a healthy individual with anti-idiotype antibodies will cause the same immune response as does immunization with the pathogenic antigen. The anti-idiotype antibody will, therefore, immunize the individual to the pathogen. However, the anti-idiotypes are themselves totally non-pathogenic.
  • a B-cell pre-screening procedure is done by binding an antigen, or cell, to a solid support, and passing a family of B-cells over the antigen.
  • the B-cells producing antibody specific to the bound antigen carry that antibody on the cell surface. As a result these B-cells bind to the antigen.
  • pre-screening is done completely in suspension. This type of pre-screening is particularly useful when attempting to isolate anti- idiotype antibodies. B-cells producing anti-idiotype antibody and carrying it on their surface, will tend to aggregate with B-cells which produce and carry the idiotype (antibody). Accordingly, in this type of pre- screening one locates aggregates in suspension and then examines the aggregates for the presence of anti- idiotypes.
  • a B-cell pre-screening in suspension could be used to isolate specific B-cells.
  • B-cells to a specific epitope on a first antigen are selected in suspension by eliminating the B-cells to the other epitopes on that antigen. This is done by aggregating the B-cells carrying antibody to the other epitopes. The supernatant, which will contain a greater proportion of desired B-cells, is then collected.
  • a pre-screening process where an antigen is used to selectively bind certain B-cells, used as part of a monoclonal antibody production procedure, has never been done.
  • solid support selection processes have been used in the past, these involved antibodies, not antigen, bound to the solid support. Such processes are disclosed in the following articles: M.G.
  • the invention relates to a B-cell pre-screening process where antigens are used to select certain B-cells from a family of B-cells.
  • antigen includes cells, proteins or any other antigens.
  • a family of B-cells which have been taken from an immunized animal is exposed to antigen bound to a solid support.
  • the antigen chosen is one which selectively binds certain of the B-cells.
  • the selected B-cells are fused to produce hybridomas, which in turn yield monoclonal antibodies.
  • the antigen which is attached to the support is selected to bind the B-cells which produce desired antibody.
  • This procedure is known as an enrichment procedure.
  • the attached antigen can be one which binds undesired antibody.
  • the B-cells in the supernatant and not the bound cells that carry the desired antibody.
  • enrichment it is the bound B-cells which are separated from the plate and then fused with a myeloma cell to produce hybridomas.
  • the B-cells in the supernatant are fused to form hybridomas. Irrespective of whether the pre-screening is by depletion or enrichment, the number of B-cells which yield undesired antibody is reduced. As a result, a greater proportion of the hybridomas will produce the desired monoclonal antibodies.
  • the pre-screening is done completely in suspension.
  • undesired antigen carrying some epitopes in common with the desired antigen is added to a suspension of B-cells. Those carrying antibody to this antigen will form aggregates.
  • the supernatant, which contains a greater percentage of the desired B-cells, is used in the fusion to form hybridomas.
  • the attempt is to isolate anti-idiotype antibodies in a suspension of cells. This is done by locating aggregates of cells, and then separating these from the suspension. The aggregates should contain B-cells which produce anti-idiotype antibodies.
  • the pre-screening procedure of the invention allows B-cells which produce antibody to selected antigens to be isolated prior to fusion.
  • a greater proportion of the hybridomas will produce desired monoclonal antibodies.
  • the presence of a greater proportion of desired hybridomas makes it easier to locate desired hybridomas and reduces the time normally needed to do so.
  • the B-cell pre-screening procedure significantly reduces the time needed to locate hybridomas which are specific to an epitope which exists only on a combination of protein subunits. Such an epitope is termed a combinatorial epitope.
  • hybridomas unique to combinatorial epitopes are normally isolated by producing the monoclonal antibodies and then separately testing their reactivity with the subunits and the combinatorial epitope. This is a time-consuming procedure.
  • Pre-screening the B-cells to obtain a greater proportion of those which produce the antibody to the combinatorial epitope, and then using the pre- screened B-cells in the fusion produces a greater proportion of desired hybridomas. Consequently, the desired hybridomas can be isolated more easily and more quickly.
  • ovalbumin-hemocyanin conjugate was prepared by using a one to one molecular ratio of each protein, and adding 25% glutaraldehyde as the cross linker. A more complete description of the cross-linking process is in S . Avrameas, "Coupling of Enzymes to Proteins with Glutaraldehyde,” Immunochemistry Vol. 6, 43 (1969), which is incorporated by reference.
  • conjugate After preparation of the conjugate it was used to immunize ten (10) female Balb/c mice.
  • the conjugate was diluted in normal saline (0.9%) and then emulsified in Freunds complete adjuvant by drawing it in and out of a syringe.
  • the final solution contained 100 micrograms of conjugate per 100 microliters. 100 microliters were injected intradermally.
  • mice were, given booster injections, either subcutaneously or intra-peritoneally, every thirty days.
  • the conjugate was emulsified in incomplete Freunds adjuvant. At least two booster injections were given to each mouse.
  • mice Four to six weeks after the last booster was given, the mice were again immunized with the same amount and concentration of conjugate (mixed in normal saline) by intraspleenic injection. Three to four days later the mice were sacrificed and their spleens were removed in preparation for the pre-screening procedure.
  • B-cells producing antibody to the ovalbumin attached to the antigen which was bound to the petri dish. Thereafter, the unattached B-cells
  • Single cell suspensions of the recovered cells were prepared.
  • the recovered cells were diluted in Dulbeccos Modification of Eagles Basal Medium and spun in a centrifuge at 1000 g for ten minutes. The supernatant was withdrawn, and Sp2/0-M5 myeloma cells (available from the American Tissue Culture Collection in Rockville, Maryland) were added to the cell pellet and resuspended.
  • the resultant suspension was then spun at 1000 g for ten minutes, and the supernatant was withdrawn. To complete the fusion, 2 ml of polyethylene glycol 4000 was added gradually over ten minutes, together with 20 ml of Dulbeccos Medium.
  • The. fused cells were spun down, the supernatant was withdrawn, and the cells were resuspended in 100 ml of Dulbeccos Medium and added in aliquots of 100 microliters to each of ninety- six wells of a linbro plate. Ten of the ninety-six well plates were so prepared, giving a total of nine hundred and sixty wells available for growth. The fusion products were grown in the plates for two weeks.
  • a similar hybridoma production procedure is described in K. Flurkey, M.B. Bolger & D.S. Linthicum, "Preparation and Characteristics of Antisera and Monoclonal Antibodies to Serotonergic and Dopaminergic Ligands, " 8 J. Neuroimmunol. at 115-27 (1985), which is incorporated by reference.
  • the pre-screening was to be done by the enrichment method, essentially the same procedure described above would be followed. The only difference would be that the conjugate, not the ovalbumin, would be bound to the petri dishes. The B-cells would be passed over the petri dish, as described above. However, B-cells which are specific to the conjugate, not the ovalbumin, would bind to the plate. These bound B-cells would then be separated from the plate and fused with myeloma cells to form hybridomas.
  • Example 1(A) A fundamentally identical procedure to that described in Example 1(A) was performed, i.e., the same immunization and fusion process was done. The only difference was that no pre-screening was performed.
  • the hybridomas were limit diluted so that there was one clone per well.
  • the monoclonal antibodies in the wells were checked for reactivity with ovalbumin, hemocyanin, and the ovalbumin-hemocyanin conjugate. The results appear in Table I below.
  • a B-cell pre-screening procedure would be useful for producing monoclonals to other types of combinatorial epitopes. For example, it could be used in producing monoclonals to the dimer CK-MB.
  • CK-MB is known to be found in body fluids following myocardial infarction.
  • a monoclonal antibody specific to CK-MB could be useful in developing a test for the presence of myocardial infarction.
  • the problem with developing a monoclonal to CK-MB is that the proteins CK-MM and CK-BB, both of which are normally present in body fluids, also carry the CK subunit. Therefore, immunization with CK-MB will yield antibodies which react with CK-MM, CK-BB and with CK-MB.
  • a B-cell pre-screening procedure could be used in the manner set forth below to select B-cells which produce antibody specific only to CK-MB, which will not bind with CK-MM or CK-BB.
  • Example I Essentially the same procedure described in Example I is used to produce monoclonal antibodies to the heart muscle enzyme CK-MB. The major difference is that the immunization is done with CK-MB instead of the ovalbumin-hemocyanin conjugate. The other main difference is that the pre-screening procedure is done with CK-MM and CK-BB bound to the solid support. As a result, B-cells which produce antibody to the CK subunit bind to the support. The supernatant, which contains B-cells specific to CK-MB, is then used in the fusion process. A high percentage of the hybridomas should produce monoclonal antibodies specific to CK-MB.
  • Pre-screening also reduces production time where different immunogenic entities carry most of the same epitopes, and the objective is to produce monoclonal antibodies to the epitopes which are not common to all entities.
  • An example of this is where antibodies are desired which can differentiate a cancerous from a normal cell.
  • the monoclonal antibodies produced could be used in the diagnosis of the disease conditions.
  • An example of using a pre-screening procedure in the selection of monoclonals to cancerous cells is set forth below.
  • Example I Using the immunization technique of Example I, host animals are immunized with cancerous cells. The resultant family of B-cells are then pre-screened with normal healthy cells, which have been bound to a solid support. The B-cells which produce antibody to epitopes on the normal cells will bind. The supernatant, which contains B-cells producing antibody specific to cancer cell epitopes, is then collected. A greater proportion of the monoclonals produced from the fusion of the supernatant B-cells should be specific to the cancerous cells.
  • Pre-screening the B-cells is also valuable where one is attempting to produce monoclonal antibodies to trace proteins, for example, those proteins which are present only in a diseased individual. Monoclonals to these proteins could be used in diagnosis of disease states.
  • mice are immunized with whole serum taken from a healthy individual.
  • Other animals are immunized with whole serum from a diseased individual. All serum proteins from the healthy individual are then attached to a petri dish.
  • B-cells from animals immunized with unhealthy serum are passed over the petri dish. Those B-cells that do not attach to the bound proteins, i.e., those which carry antibody to the diseased proteins, are fused to myeloma cells to produce hybridomas.
  • the resulting monoclonal antibodies are likely to be specific and bind only to disease-induced, or related, proteins.
  • This method may also be used in the opposite mode, in which monoclonals are produced to proteins that are only present in healthy individuals and absent in diseased individuals.
  • B-cell pre-screening would also be useful in producing monoclonal antibodies to trace proteins, for example, those which are present on different strains of viruses.
  • the monoclonal antibodies produced can be used to isolate quantities of the trace proteins for study and analysis.
  • a related technique to that described directly above can be used to produce monoclonals which differentiate between other closely-related proteins.
  • the bound antigen used in the pre-screening would be a protein which is closely related to the immunizing protein. By choosing appropriate immunizing and bound antigens, antibodies can be produced which differentiate among fully folded, slightly folded and unfolded proteins.
  • a host animal is immunized with CK-MB, and its spleen is removed.
  • the spleen is ground and suspended.
  • CK-MM is added to the suspension.
  • the aggregates formed by the B-cells and CK-MM are allowed to settle, and the supernatant is withdrawn.
  • CK-BB is then added to the supernatant, the aggregates are again allowed to settle, and the supernatant is withdrawn.
  • the final supernatant should contain a higher proportion of B-cells which produce antibody specific to CK-MB.
  • Pre-screening, in suspension is also useful in isolating anti-idiotype antibodies.
  • B-cells which produce anti-idiotype antibody will carry it on their surface and will tend to aggregate with B-cells which produce the idiotype. The occurrence of aggregates in a suspension of B-cells suggests the presence of anti-idiotype antibodies.
  • B-cell aggregates are relatively easy to isolate because they are heavier than the other components in the suspension and tend to settle. Settled aggregates can be easily collected and examined for the presence of anti-idiotypes. If the aggregates do not settle freely, the suspension can be centrifuged and the aggregates collected. The B-cells which produce the anti-idiotype antibody can then be separated from the other B-cells in the aggregate, and used to produce monoclonals. It should be understood that the examples herein are intended to be exemplary only and not limiting, and that the scope of protection is defined in the claims which follow, and includes all equivalents of the subject matter of the claims.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Analytical Chemistry (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • General Engineering & Computer Science (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

Dans le procédé de pré-criblage décrit, des antigènes sont utilisés pour sélectionner certains lymphocytes B qui se trouvent dans une famille de lymphocytes B. Dans un premier mode de réalisation, des lymphocytes B réactifs sont liés à un antigène et les lymphocytes B non réactifs contenus dans la couche surnageante sont ensuite recueillis (c'est ce qu'on appelle l'appauvrissement), ou alors les lymphocytes B qu'on désire sélectionner peuvent être liés directement (c'est ce qu'on appelle l'enrichissement). Dans un deuxième mode de réalisation, des lymphocytes B liés à un premier antigène comportant un épitope spécifique sont sélectionnés dans une suspension, ce qu'on effectue en ajoutant un second antigène comportant au moins un épitope en commun avec le premier antigène à une suspension de lymphocytes B et en formant des aggrégats de lymphocytes B qui produisent un anticorps contre le second antigène. La couche surnageante, qui contient alors des lymphocytes B produisant un anticorps contre l'épitope spécifique, est ensuite recueillie. Dans un troisième mode de réalisation, des lymphocytes B portant un anticorps d'anti-idiotype sont sélectionnés par incorporation d'aggrégats dans une suspension de lymphocytes B. Les aggrégats sont constitués de lymphocytes B portant l'anti-idiotype et d'autres lymphocytes B portant l'anticorps. Les lymphocytes B sélectionnés par n'importe lequel des procédés mentionnés ci-dessus peuvent être agglomérés à des cellules de myélome et utilisés pour produire des anticorps monoclonaux.
PCT/US1988/002407 1988-07-14 1988-07-14 Pre-criblage servant a l'appauvrissement et a l'enrichissement de lymphocytes b specifiques WO1990000625A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/US1988/002407 WO1990000625A1 (fr) 1988-07-14 1988-07-14 Pre-criblage servant a l'appauvrissement et a l'enrichissement de lymphocytes b specifiques

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US1988/002407 WO1990000625A1 (fr) 1988-07-14 1988-07-14 Pre-criblage servant a l'appauvrissement et a l'enrichissement de lymphocytes b specifiques

Publications (1)

Publication Number Publication Date
WO1990000625A1 true WO1990000625A1 (fr) 1990-01-25

Family

ID=22208798

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1988/002407 WO1990000625A1 (fr) 1988-07-14 1988-07-14 Pre-criblage servant a l'appauvrissement et a l'enrichissement de lymphocytes b specifiques

Country Status (1)

Country Link
WO (1) WO1990000625A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5700379A (en) * 1995-02-23 1997-12-23 Siemens Aktiengesellschaft Method for drying micromechanical components
WO2000042176A1 (fr) * 1999-01-11 2000-07-20 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Selection d'anticorps monoclonaux
EP1418808A4 (fr) * 2001-07-30 2004-09-15 Smithkline Beecham Corp Transgenes marqueurs d'imagerie
WO2014146074A3 (fr) * 2013-03-15 2014-11-06 Alder Biopharmaceuticals, Inc. Protocole d'identification et d'isolement de lymphocytes b spécifiques d'un antigène et et production d'anticorps dirigés contre des antigènes souhaités
CN107058235A (zh) * 2017-06-05 2017-08-18 深圳大学 一种b细胞筛选方法及其在单克隆抗体制备中的应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3932221A (en) * 1971-06-09 1976-01-13 Merck Patent Gesellschaft Mit Beschrankter Haftung Immunological isozyme determination method
US4746539A (en) * 1983-11-23 1988-05-24 The Ohio State University Research Foundation Purification of cancer-associated protein and preparation of antibody thereto

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3932221A (en) * 1971-06-09 1976-01-13 Merck Patent Gesellschaft Mit Beschrankter Haftung Immunological isozyme determination method
US4746539A (en) * 1983-11-23 1988-05-24 The Ohio State University Research Foundation Purification of cancer-associated protein and preparation of antibody thereto

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
B. ALBERTS et al., "Molecular Biology of the Cell", published 1983 by Garland Publishing Inc. (New York), see pages 928-929. *
D.P. STITES et al., "Basic and Clinical Immunology", published 1982 by Lange Medical Publishers (Los Altos, Calfornia), see pages 165 and 373. *
E.D. SEVIER et al., "Monoclonal Antibodies in Clinical Immunology", in CLINICAL CHEMISTRY, 1981, 27(11), 1797-1806. *
F.C. HAY, "Idiotype Vaccines", in IMMUNE INTERVENTION, published 1984 by Academic Press (London), see pages 117-138. *
H.N. EISEN, "Immunology", published 1980 by Harper and Row, Publishers (Philadelphia), see page 394. *
PHARMACIA FINE CHEMICALS TECHNICAL DISCLOSURE, printed August 1982 by Ljungforetagen AB (Sweden), "Affinity Chromatography Principles and Methods", pages 96-97. *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5700379A (en) * 1995-02-23 1997-12-23 Siemens Aktiengesellschaft Method for drying micromechanical components
WO2000042176A1 (fr) * 1999-01-11 2000-07-20 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Selection d'anticorps monoclonaux
EP1418808A4 (fr) * 2001-07-30 2004-09-15 Smithkline Beecham Corp Transgenes marqueurs d'imagerie
WO2014146074A3 (fr) * 2013-03-15 2014-11-06 Alder Biopharmaceuticals, Inc. Protocole d'identification et d'isolement de lymphocytes b spécifiques d'un antigène et et production d'anticorps dirigés contre des antigènes souhaités
CN107058235A (zh) * 2017-06-05 2017-08-18 深圳大学 一种b细胞筛选方法及其在单克隆抗体制备中的应用
CN107058235B (zh) * 2017-06-05 2020-08-11 深圳大学 一种b细胞筛选方法及其在单克隆抗体制备中的应用

Similar Documents

Publication Publication Date Title
CA1190873A (fr) Anticorps monoclonaux recombinants
US4714681A (en) Quadroma cells and trioma cells and methods for the production of same
KR900003923B1 (ko) 인체 암종양과 관련된 항원에 대한 단일클론성 항체
JP4414900B2 (ja) Ｌ鎖欠落免疫グロブリン
WO1982001461A1 (fr) Hybridomes humains, precurseurs et produits
Moshly-Rosen et al. Monoclonal antibodies against defined determinants of acetylcholine receptor
JPS595120A (ja) 単クロ−ン性抗ｃｅａ抗体
Eisenbarth Application of monoclonal antibody techniques to biochemical research
US7871621B2 (en) Anti-HBs monoclonal antibody
WO1987001131A1 (fr) Anticorps monoclonaux de primates non humains et procedes
JP3550410B2 (ja) 好塩基球に結合するモノクローナル抗体、好塩基球の分離方法、好塩基球からのケミカルメディエーターの遊離方法、及び好塩基球由来ケミカルメディエーター遊離試験法
French et al. The production of more useful monoclonal antibodies I. Modifications of the basic technology
DK172659B1 (da) Fremgangsmåde til diagnosticering af myasthenia gravis og substrater og sæt til anvendelse derved
EP0131424A2 (fr) Préparations d'anticorps
WO1990000625A1 (fr) Pre-criblage servant a l'appauvrissement et a l'enrichissement de lymphocytes b specifiques
JPH03198795A (ja) 種々のヒト白血病およびリンパ腫細胞上に広く存在する特有の抗原に反応するモノクローナル抗体、および診断および治療を目的とした該抗体の使用法
JP2726264B2 (ja) Ｎ‐ｍｙｃタンパク質のためのアッセイ及び抗体
JPH0683666B2 (ja) 腫瘍▲下−▼胎児に特異的なモノクロナ−ル抗体の調製方法および使用方法
US4668629A (en) Human hybridomas, precursors and products
Bankert et al. Screening and replica plating of anti-hapten hybridomas with a transfer template hemolytic spot assay
Fletcher et al. Monoclonal antibodies specific for blood groups A and B
Devaux et al. Cross-reactive idiotypes of monoclonal anti-Iak antibodies: characterization with xenogeneic anti-idiotypic reagents and expression in anti-H-2 humoral responses.
JPH03112485A (ja) Ｈｌａ―ｃ遺伝子およびｄｎａプローブ並びに形質転換細胞
JPH06153979A (ja) 魚類イリドウイルスに対するモノクローナル抗体並びに該抗体を製造するためのハイブリドーマ及び方法
CN112094345B (zh) 可应用于肿瘤细胞捕获的小鼠抗免疫球蛋白关联βCD79b的单克隆抗体

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE FR GB IT LU NL SE