Continuous cultures of fused cells secreting antibody of predefined specificity
TL;DR: The derivation of a number of tissue culture cell lines which secrete anti-sheep red blood cell (SRBC) antibodies is described here, made by fusion of a mouse myeloma and mouse spleen cells from an immunised donor.
Abstract: THE manufacture of predefined specific antibodies by means of permanent tissue culture cell lines is of general interest. There are at present a considerable number of permanent cultures of myeloma cells1,2 and screening procedures have been used to reveal antibody activity in some of them. This, however, is not a satisfactory source of monoclonal antibodies of predefined specificity. We describe here the derivation of a number of tissue culture cell lines which secrete anti-sheep red blood cell (SRBC) antibodies. The cell lines are made by fusion of a mouse myeloma and mouse spleen cells from an immunised donor. To understand the expression and interactions of the Ig chains from the parental lines, fusion experiments between two known mouse myeloma lines were carried out.
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TL;DR: This work speculates on the reasons behind this large discrepancy between the expectations arising from proteomics and the realities of clinical diagnostics and suggests approaches by which protein-disease associations may be more effectively translated into diagnostic tools in the future.
4,062 citations
Cites background from "Continuous cultures of fused cells ..."
...The requirement for an isolated antigen was circumvented, however, following the introduction of monoclonal mouse antibodies by Kohler and Milstein (11) in 1975....
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TL;DR: A human IgGI antibody has been reshaped for serotherapy in humans by introducing the six hypervariable regions from the heavy- and light-chain variable domains of a rat antibody directed against human lymphocytes.
Abstract: A human IgGI antibody has been reshaped for serotherapy in humans by introducing the six hypervariable regions from the heavy- and light-chain variable domains of a rat antibody directed against human lymphocytes. The reshaped human antibody is as effective as the rat antibody in complement and is more effective in cell-mediated lysis of human lymphocytes.
3,167 citations
TL;DR: It is proposed that one of the major long-term consequences of inadequate early nutrition is impaired development of the endocrine pancreas and a greatly increased susceptibility to the development of Type 2 diabetes.
Abstract: In this contribution we put forward a novel hypothesis concerning the aetiology of Type 2 (non-insulin dependent) diabetes mellitus. The concept underlying our hypothesis is that poor foetal and early post-natal nutrition imposes mechanisms of nutritional thrift upon the growing individual. We propose that one of the major long-term consequences of inadequate early nutrition is impaired development of the endocrine pancreas and a greatly increased susceptibility to the development of Type 2 diabetes. In the first section we outline our research which has led to this hypothesis. We will then review the relevant literature. Finally we show that the hypothesis suggests a reinterpretation of some findings and an explanation of others which are at present not easy to understand.
3,107 citations
Cites methods from "Continuous cultures of fused cells ..."
...Further work, this time exploiting the monoclonal antibody technique [18], was required to devise assays with adequate specificity to resolve the complex mixture of insulin-like molecules present in plasma [19]....
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Patent•
10 Jul 1991TL;DR: In this paper, a member of a specific binding pair (sbp) is identified by expressing DNA encoding a genetically diverse population of such sbp members in recombinant host cells in which the sbps members are displayed in functional form at the surface of a secreted recombinant genetic display package (rgdp) containing DNA encoding the sbp member or a polypeptide component thereof.
Abstract: A member of a specific binding pair (sbp) is identified by expressing DNA encoding a genetically diverse population of such sbp members in recombinant host cells in which the sbp members are displayed in functional form at the surface of a secreted recombinant genetic display package (rgdp) containing DNA encoding the sbp member or a polypeptide component thereof, by virtue of the sbp member or a polypeptide component thereof being expressed as a fusion with a capsid component of the rgdp. The displayed sbps may be selected by affinity with a complementary sbp member, and the DNA recovered from selected rgdps for expression of the selected sbp members. Antibody sbp members may be thus obtained, with the different chains thereof expressed, one fused to the capsid component and the other in free form for association with the fusion partner polypeptide. A phagemid may be used as an expression vector, with said capsid fusion helping to package the phagemid DNA. Using this method libraries of DNA encoding respective chains of such multimeric sbp members may be combined, thereby obtaining a much greater genetic diversity in the sbp members than could easily be obtained by conventional methods.
2,740 citations
TL;DR: Monoclonal antibodies specific for 5-bromodeoxyuridine have been produced and applied in detecting low levels of DNA replication on a cell-by-cell basis in vitro and do not cross-react with thymidine.
Abstract: Monoclonal antibodies specific for 5-bromodeoxyuridine have been produced and applied in detecting low levels of DNA replication on a cell-by-cell basis in vitro. The immunoglobulin-producing hybridomas were derived from spleen cells of mice immunized with a conjugate of iodouridine and ovalbumin. The cells were fused with the plasmacytoma line SP2/0Ag14. The antibodies produced are highly specific for bromodeoxyuridine and iododeoxyuridine and do not cross-react with thymidine. DNA synthesis in cultured cells exposed to bromodeoxyuridine for as short a time as 6 minutes can be detected easily and rapidly by an immunofluorescent staining method and quantitated by flow cytometry.
2,722 citations
References
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TL;DR: In this form the technique has been successfully applied to studies on the heterogeneity of myoglobins, cytochrome c6, lactoperoxidase7 and a number of other proteins (see review by Haglund8).
Abstract: THE principle of isoelectric fractionation was applied to the separation of amino-acids as early as 1912 by Ikeda and Suzuki (Japanese patent quoted in ref. 8), but the technique did not become practical for the fractionation of macromolecular ampholytes such as proteins until the introduction of natural pH gradients1 and suitable carrier ampholytes2–4. Svensson described an apparatus for isoelectric focusing in shallow pH gradients formed by low molecular weight ampholytes and stabilized by sucrose density gradients3. In this form the technique has been successfully applied to studies on the heterogeneity of myoglobins4,5, cytochrome c6, lactoperoxidase7 and a number of other proteins (see review by Haglund8).
471 citations
469 citations
TL;DR: It is demonstrated that fusion between two immunoglobulin-secreting cell lines produces hybrid cells which secrete immunoglOBulin of both parental types.
Abstract: EACH immunoglobulin chain is the integrated expression of one of several V and C genes, coding respectively for their variable and constant sections1. The restricted expression of these genes leads to molecules of a single class and type with identical combining sites at both halves of the antibody molecule2–4. This symmetry is essential for the formation of the antigen-antibody lattice. Its importance is emphasized by allelic exclusion, whereby each cell expresses only one of two possible alleles4. To understand this problem further we have studied the expression of these genes in hybrid cells obtained by fusion of two cell lines producing different immunoglobulins. Successful fusion of this type has not yet been demonstrated but fusion between immunoglobulin-producing cells and non-immunoglobulin-producing cells has been reported. Thus myeloma cells have been fused to fibroblasts5–7 and also to a non-immunoglobulin-producing lymphoma line8. Substantial immunoglobulin production in the hybrids was observed only in the latter case. Here, we demonstrate that fusion between two immunoglobulin-secreting cell lines produces hybrid cells which secrete immunoglobulin of both parental types.
155 citations
TL;DR: A method has been developed for the screening of immunoglobulins produced by large numbers of individual clones of the mouse myeloma in tissue culture using autoradiography to detect variations between different clones.
Abstract: A method has been developed for the screening of immunoglobulins produced by large numbers of individual clones of the mouse myeloma (MOPC 21) in tissue culture. Clones grown in agar were incubated in the presence of radioactive amino acids and applied directly onto polyacrylamide plates for isoelectric focusing. A discrete number of well-defined immunoglobulin bands were then detected by autoradiography. The following variations between different clones were observed:
1. Variation in relative intensity of the different bands.
2. Total absence of immunoglobulin bands.
3. Variation in the position of the bands along the pH gradient.
When a mutant clone exhibiting this third type of variation was purified by subcloning, only the mutant pattern was observed. Thus, the cells appear to express only a single allele, although they are known to be aneuploid.
We consider the third type of variation to be relevant in the evaluation of the contribution of somatic mutation to the diversity of antibody structure.
151 citations
TL;DR: The complete amino acid sequence of the kappa-chain of the mouse myeloma protein MOPC 21 was established after it was reduced and alkylated with iodo[2-(14)C]acetic acid, and 21 tryptic peptides were isolated, mainly by paper electrophoresis and paper chromatography.
Abstract: The complete amino acid sequence of the kappa-chain of the mouse myeloma protein MOPC 21 was established. The protein was reduced and alkylated with iodo[2-(14)C]acetic acid, and 21 tryptic peptides were isolated, mainly by paper electrophoresis and paper chromatography. Three large tryptic peptides (of 35, 36 and 42 residues), which were difficult to isolate in this manner, were obtained pure and in excellent yields by a combination of Sephadex G-50 gel filtration in 1% (w/v) NH(4)HCO(3) and chromatography on a DEAE-cellulose column in ammonium acetate buffer, pH8.1. Peptides overlapping the tryptic peptides were isolated from a chymotryptic digest. The chain is 214 residues long. Microheterogeneity of two peptides was observed and is believed to be due to deamidation. It was not excluded that such deamidation could occur in serum from which the protein was isolated. The sequence is compared with the sequences of two other mouse kappa-chains, and with the human kappa-chain basic sequences.
110 citations