We have created mouse-human antibody molecules of defined antigen-binding specificity by taking the variable region genes of a mouse antibody-producing myeloma cell line with known antigen-binding specificity and joining them to human immunoglobulin constant region genes using recombinant DNA techniques. Chimeric genes were constructed that utilized the rearranged and expressed antigen-binding variable region exons from the myeloma cell line S107, which produces an IgA (K) anti-phosphocholine antibody. The heavy chain variable region exon was joined to human IgG1 or IgG2 heavy chain constant region genes, and the light chain variable region exon from the same myeloma was joined to the human ic light chain gene. These genes were transfected into mouse myeloma cell lines, generating transformed cells that produce chimeric mouse-human IgG (K) or IgG (K) anti-phosphocholine antibodies. The transformed cell lines remained tumorigenic in mice and the chimeric molecules were present in the ascitic fluids and sera of tumor-bearing mice.The capability to transfer immunoglobulin genes into lymphoid cells where they produce protein in quantities sufficient for structural studies (1-3) provides us with the opportunity to generate and characterize novel immunoglobulin molecules. Cloned variable (V) region genes from mouse or rat hybridoma cell lines can be ligated to human constant (C) region genes and we would expect that these chimeric genes can be transfected into mouse myeloma cells, which then will produce novel human antibody molecules. We would thus produce antibodies that are largely human but which have antigen-binding specificities generated in mice. The additional potential for in vitro manipulation and alteration of both the antigen-binding site and the structures correlated with biological effector functions of these antibody molecules using recombinant DNA techniques would introduce a powerful approach for further understanding antibody structure, function, and immunogenetics.As we show here, both chimeric mouse heavy chain V region exon (VH)-human heavy chain C region genes and chimeric mouse light chain V region exon (VK)-human K light chain gene constructs are expressed when transfected into mouse myeloma cell lines. When both chimeric heavy and light chain genes are transfected into the same myeloma cell, an intact tetrameric (H2L2) chimeric antibody is produced.In this study we used VH and VK exons from the mouse phosphocholine (PCho)-binding antibody-producing S107 myeloma cell line (4, 5). Chimeric mouse-human anti-PCho antibodies were produced in culture by appropriate transfected cell lines or by "transfectomas" obtained when such cell lines are injected into mice.
The synthesis of a novel class of reagents for fluorescence analyses of molecules and cells is reported . These compounds consist of a highly fluorescent phycobiliprotein conjugated to a molecule having biological specificity . Phycoerythrin-immunoglobulin, phycoerythrin-protein A, and phycoerythrin-avidin conjugates were prepared . These conjugates bind specifically to beads containing a covalently attached target molecule and render them highly fluorescent. Femtomole (10-'5 mole) quantities of phycoerythrin conjugates can be detected because of the high extinction coefficient (e m = 2.4 x 106 cm -' M-' for 2.4 X 105 daltons) and high fluorescence quantum yield (Q = 0.8) of the phycobiliprotein moiety . An important feature of these conjugates is that they emit in the orange-red spectral region, where background fluorescence is less than at shorter wavelengths. Phycoerythrin conjugates are well-suited for two-color flow cytofIuorimetric analyses employing a single excitation line . The distributions of Leu antigens (also called OKT antigens) on the surface of T-lymphocytes were analyzed using fluoresceinated antibody as the green-fluorescent stain and biotinylated antibody counter-stained with phycoerythrin-avidin as the red one. This one-laser twocolor analysis showed that cells express Leu-3a and Leu-3b or neither antigen. In contrast, the distributions of Leu-2a (a marker of suppressor and cytotoxic T-cells) and Leu-3a (a marker of helper and inducer T-cells) are mutually exclusive . These studies show that phycobiliprotein conjugates can be applied to fluorescence-activated cell sorting and analysis, fluorescence microscopy, and fluorescence immunoassay.Single-cell analysis and sorting by flow cytofluorimetry, fluorescence microscopy, and other fluorescence techniques are important methods in cell biology (1-4) . The scope and value of these experimental approaches are closely tied to the availability of suitable fluorescent probes. In particular, there is a need for orange and red-fluorescent probes to complement fluorescein, a green emitter, in analyzing for multiple markers on cells and macromolecules. Rhodamine and other currently available longer wavelength emitters are not optimal in this regard. For example, fluorescein and rhodamine cannot both be excited with high efficiency by a single laser line . It seemed to us that phycobiliproteins, a naturally occurring family of highly fluorescent macromolecules, could serve as the fluorescent moieties of a broad range of specific reagents for analyses of cells and molecules . Phycobiliproteins are constituents of the light-harvesting apparatus of blue-green bacteria, red algae, and cryptomonads (5, 6) . The light energy absorbed by these proteins is funneled by dipole-dipole energy transfer to chlorophyll and then used in photosynthesis . Some characteristics of phycobiliproteins that make them well-suited for fluorescence analyses are : (a) they contain multiple bilin chromo-THE JOURNAL OF CELL BIOLOGY " VOLUME 93 JUNE 1982 981-986 © The Rockefel...
We generated a family of chimeric immunoglobulin G (IgG) molecules having identical antigen‐combining sites for the dansyl (DNS) hapten, in conjunction with nine heavy chain constant (CH) regions. This family of antibody molecules allows comparison of CH dependent properties independent of possible variable region contributions to IgG function. The segmental flexibility and complement fixation activity were measured of six genetically engineered molecules (the four human IgG isotypes, mouse IgG3 and rabbit IgG) and the remaining three mouse IgG isotypes, (IgG1, IgG2a and IgG2b), isolated previously by somatic cell genetic techniques. These properties of antibody molecules each correlate with the length of the immunoglobulin hinge region which separate the first and second CH (CH1 and CH2) domains. These results attribute a structural basis for two critical properties of antibody molecules.
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