Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1994-07-13
1998-11-03
Jones, W. Gary
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435911, 435912, 435914, 4351723, 536243, 536 2431, 536 2432, 536 2433, C12Q 168, C12P 1934, C07H 2102
Patent
active
058306633
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
This invention concerns the treatment of cell populations and relates to methods of treating cell populations, e.g. for analysing the linkage of genes within individual cells of a heterogeneous population, novel protein and nucleic acid compositions, and kits for performing said methods.
BACKGROUND OF THE INVENTION
This invention concerns techniques for the treatment of populations of cells, typically heterogeneous populations of cells, and has particular, but not exclusive, application to the treatment of lymphocytes such as those producing antibodies.
Antibodies are present in serum and bind to and help eliminate diverse pathogens such as toxins, bacteria and viruses. They consist of a Y-shaped protein built from two heavy chains and two light chains. Each chain has a modular construction: each light chain consists of two domains, and each heavy chain has at least four domains. The antigen binding site is fashioned by one domain from the heavy chain (VH domain) and one domain from the light chain (VL domain). Indeed small antigen binding fragments can be prepared which consist only of these two domains, either associated non-covalently, or via disulphide bonds or via a peptide linker. The antigen binding domains are more variable in amino acid sequence than the other domains of the antibody, and are therefore termed variable (V) domains in contrast to the constant (C) domains. The constant domains of the antibody are responsible for triggering antibody effector mechanisms such as complement lysis and cell mediated killing.
Antibodies are made by B-lymphocytes in a process of gene rearrangement. During the development of these cells, the genes encoding the variable domains are assembled from genetic elements. In the case of the VH domains there are three elements, the unrearranged VH gene, the D segment and JH-segment. In the case of the VL domains there are two elements, the unrearranged VL (V lambda or V Kappa) gene and the JL (J lambda or J Kappa) segment. Random combination of these gene segments and random combination of the rearranged VH and VL domains generates a large repertoire of diverse antibodies, capable of binding to diverse antigens. The potential diversity is large (primary repertoire in mouse is greater than 10.sup.10), although the repertoire expressed at any time must be less than the total number of lymphocytes (in mouse less than 10.sup.8)
In the first stage of the immune response, antigen is recognized by lymphocytes and they proliferate and secrete antibody which is usually of low affinity (less than 10.sup.6 M').
On further encounter with antigen (as in hyperimmunisation), the V-genes of these cells are subjected to mutation and lymphocytes displaying antibody with enhanced affinity are selected. This process, in which high affinity antibodies are made in two stages from a primary repertoire and subsequently from a hyperimmune repertoire is highly efficient. It has advantages over selecting antibodies in a single step, as this would require a much larger primary repertoire (the potential hyperimmune repertoire is perhaps greater than 10.sup.30)
Traditionally cell lines making high affinity antibodies have been prepared by fusing B-lymphocytes from a hyperimmunised animal with a myeloma cell line. This immortalises the B-lymphocytes and allows the cloning and screening of cells for production of monospecific antibodies binding to antigen. Such monoclonal antibodies have found a wide variety of uses in therapy and diagnostics. However the plasma cells (last stage of differentiation of B-lymphocytes) which are essentially factories for secretion of antibodies, do not fuse. Hence the repertoire of antibody specificities contributed by plasma cells is not accessible by hybridoma technology. This may explain why polyclonal antisera from animals can have higher average affinities than monoclonal antibodies derived from the same animal. There are other disadvantages of hybridoma technology, including the low efficiency of cell fusion, the time consuming steps req
REFERENCES:
Dialog Information Services, file 154, Medline 66-91/May, accession No. 07690409, Medline acession No. 91209490, Marks J.D. et al: "Oligonucleotide primers for polymerase chain reaction amplification of human immunoglobulin variable genes and design of family-specific oligonucleotide probes", Eur J Immunol Apr. 1991, 21 (4) pp. 985-991.
Haase et al: "Amplification and detection of lentiviral DNA inside cells", Proc.Natl. Acad. Sci.USA, vol. 87, Jul. 1990, see pp. 4971-4975.
Orlandi et al: "Cloning immunoglobulin variable domains for expression by the polymerase chain reaction", Proc.Natl.Acad.Sci. USA, vol. 86, May 1989, see pp. 3833-3837.
Clackson et al: "Sticky feet-directed mutagenesis and its application to swapping antibody domainssss", Nucleic Acids Research, vol. 17, No. 24, 1989, see pp. 10163-10170.
Ward et al: "Binding activities of a repertorire of single immunoglobulin variable domains secreted from Escherichia coli", Nature, vol. 341, Oct. 1989, see pp. 544-546.
Haase et al, "Amplification and detection of lentiviral DNA inside cells", Proc. Natl. Acad. Sci. 87:4971-4975, Jul. 1990.
Kawasaki, E., "Amplification of RNA" in PCR Protocols: A Guide to Methods and Applications, (Ed. Innis) Academic Press, pp. 21-27, 1990.
Higuchi, R., "Recombinant PCR" in PCR Protocols: A Guide to Methods and Applications, (Ed. Innis) Academic Press, pp. 177-183, 1990.
Chehab et al, "Detection of specific DNA sequences by fluorescence amplification: A color complementation assay", Proc. Natl. Acad. Sci. 86:9178-9182, Dec. 1989.
Tecott et al, "In situ transcription: Specific synthesis of complementary DNA in fixed tissue sections", Science 240:1661-1664, Jun. 1988.
Mogensen et al, "Nonradioactive, sequence detection of RNA in situ by primed in situ labeling", Exp. Cell Res. 196:92-98, 1991.
Spann et al, "In situ amplification of single copy gene segments in individual cells by the polymerase chain reaction", Infection 19(4):242-244, Jul. 1991.
Embleton Michael J.
Gorochov Guy
Jones Peter T.
Winter Gregory P.
Fredman Jeffrey
Jones W. Gary
Medical Research Council
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