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V T Oi

Publications and source records attributed to V T Oi.

36 records · Page 2Linked to original sources
PubMed1983

Genetic characterization of mouse immunoglobulin allotypic determinants (allotopes) defined by monoclonal antibodies.

We have generated a new series of monoclonal antibodies recognizing allotypic determinants on mouse IgG1, IgG2a, and IgG2b. In this communication we describe their reactivities with immunoglobulins of the inbred mouse strains. Comparison with serology charts indicates that many of these monoclonal antibodies detect allotypic specificities previously defined by conventional antisera; others define previously undescribed specificities. Strain and isotype distribution allows us to assign five new allotypic specificities to Igh-1 and three new specificities to Igh-3. In addition, on the basis of reactivity with the monoclonal antibodies, we have defined a new Igh haplotype in SWR/J mice, Ighp.

Alleles↗
PubMed1983

Structural characterization of mouse immunoglobulin allotypic determinants (allotopes) defined by monoclonal antibodies.

We have generated a new series of monoclonal antibodies recognizing allotypic determinants on mouse IgG1, IgG2a, and IgG2b. In this communication we describe their reactivity at the molecular level. A number of genetic specificities (as defined by reactivity with sera from inbred strains) were divided into subspecificities (allotopes) by these analyses. With the exception of one allotope located in the hinge region of Igh-1b, all other 23 allotopes examined were preserved upon reduction and alkylation of immunoglobulin antigens. To further analyze the role of immunoglobulin conformation in presenting the allotopes, we assayed their presence on mixed Igh-1a/Igh-4a heavy chain molecules. The Igh-1a determinants were maintained, but the Igh-4a determinants were lost. Taken together, our results indicate that genetic polymorphisms at the Igh loci generate an enormous antigenic complexity, much of which relies on tertiary and quaternary protein structure for expression.

Alleles↗
PubMed1983

A tissue-specific transcription enhancer element is located in the major intron of a rearranged immunoglobulin heavy chain gene.

We have studied the DNA sequences required for high level expression of a cloned heavy chain immunoglobulin gene stably introduced into mouse myeloma cells by DNA transfection. We found that DNA sequences derived from the germ line JH-C mu region are required for accurate and efficient transcription from a functionally rearranged VH promoter. Similar to viral transcriptional enhancer elements, these cellular sequences stimulate transcription from either the homologous VH gene segment promoter or a heterologous SV40 promoter. They are active when placed on the 5' or 3' side of the rearranged VH gene segment and they function when their orientation is reversed. However, unlike viral enhancers, the Ig gene enhancer appears to act in a tissue-specific manner, since it is active in mouse B cells but not in mouse fibroblasts. The nucleotide sequence of the Ig enhancer region contains repeating elements that closely resemble sequence the possible role of tissue-specific transcription in cell differentiation and malignant transformation.

Animals↗
PubMed1983

Immunoglobulin gene expression in transformed lymphoid cells.

Myeloma, hybridoma, and thymoma cell lines have been successfully transfected for the Escherichia coli xanthine-guanine phosphoribosyltransferase gene (gpt) by using the plasmid vector pSV2-gpt. The transformed cells synthesize the bacterial enzyme 5-phospho-alpha-D-ribose-1-diphosphate:xanthine phosphoribosyltransferase (XGPRT; EC 2.4.2.22) and have been maintained in selective medium for over 4 months. Lymphoid cell lines expressing a K immunoglobulin light chain were obtained by transfecting cells with pSV2-gpt containing a rearranged K light chain genomic segment from the S107 myeloma cell line. The S107 light chain is synthesized in gpt-transformed J558L myeloma cells and is identical to the light chain synthesized by the S107 myeloma cell line, as judged by immunoprecipitation and two-dimensional gel electrophoresis. Furthermore, this light chain is synthesized and secreted as part of an intact antibody molecule by transformed hybridoma cells that normally secrete an IgGl (gamma, K) antibody molecule. No light chain synthesis was detected in a similarly transformed rat myeloma or a mouse thymoma line.

Animals↗
PubMed1983

Localization of murine Igh-1a allotypic determinants by using a panel of mouse myeloma variant immunoglobulins.

A number of monoclonal antibodies are available that are reactive with distinct mouse immunoglobulin allotypic determinants. By determining which ones are present on a panel of hybrid IgG2b-IgG2a immunoglobulins, we have localized some of the allotypic determinants present on the IgG2a heavy chain of the "a" allotype (Igh-1a proteins). In particular, one group of determinants--Ig(1a)9.8 (20.6B8), 17.2 (20.19.2), and 14.4 (21.74.4)--has been placed in the CH2 domain. A second group--Ig(1a)8.3 (20.8.3), 21.2 (20.11.2), and 15.3 (21.66.3)--is located in a segment spanning the C terminal 8 residues of the CH2 domain and the complete CH3 domain.

Animals↗
PubMed1982

Rapid isolation of cloned isotype switch variants using fluorescence activated cell sorting.

We have used highly specific, directly fluorescein-conjugated heterologous (conventional) and monoclonal antibodies directed against mouse immunoglobulin isotypes in conjunction with the fluorescence activated cell sorter (FACS) to enrich and clone hybridoma cells producing new immunoglobulin heavy chain constant regions. Each variant retains the parental heavy chain variable region and the parental immunoglobulin light chain; thereby each variant binds the same dansyl (DNS) hapten. These isotype switch variants occur at frequencies of approximately 10-5 to 10-6. We were able to isolate the variants by first sorting for an approximate 1000-fold enrichment of the desired immunoglobulin-producing cells, growing these cells for five to nine days, followed by a second 1000-fold enrichment and direct cell cloning into 96 well culture trays. Clones were screened only 3-5 weeks after the original selection for secretion of dansyl-binding immunoglobulin of the selected isotype. Judicious combination of existing methods permits improved analytical techniques using the cell sorter. These include: first, "red" fluorescence staining of dead cells with ethidium bromide or propidium iodide and using the red fluorescence measurement to exclude dead cells from the green fluorescence selection; and second, the use logarithmic amplification of fluorescence signals, allowing for more succinct selection of fluorescence parameters for sorting.

Animals↗
PubMed1982

Fluorescent phycobiliprotein conjugates for analyses of cells and molecules.

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(-15) mole) quantities of phycoerythrin conjugates can be detected because of the high extinction coefficient (epsilon M = 2.4 x 10(6) cm-1 M-1 for 2.4 x 10(5) 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 cytofluorimetric 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 two-color 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.

Antigens, Surface↗
PubMed1980

Lymphocyte membrane IgG and secreted IgG are structurally and allotypically distinct.

We have demonstrated that there are structurally distinct membrane and secreted IgG2a immunoglobulin molecules. The membrane heavy chain is both larger and more acidic than the secreted molecule. This difference is not a result of different N-glycosidic-linked oligosaccharide chains. The membrane heavy chain also is antigenically different from its secreted homologue. This is based on the fact that secreted IgG2a molecules express an allotypic determinant absent on membrane molecules. We discussed the genetic control and gene organization of membrane and secreted immunoglobulin heavy chain sequences and suggest mechanisms controlling the expression of the simian virus 40 genome as models for alternate gene expression of membrane and secreted heavy chain polypeptide chains from the same DNA sequence. The possible biological significance of the membrane immunoglobulin acting as a recognition site for regulatory T cells also is discussed. The difference between membrane and secreted immunoglobulin is proposed as a possible explanation for the manner in which T cells interact with IgG on memory B cells in the presence of a large excess of IgG present in body fluids.

Animals↗
PubMed1979

Antigen-specific identification and cloning of hybridomas with a fluorescence-activated cell sorter.

Myeloma-spleen cell hybrids (hybridomas) producing antibody to mouse immunoglobulin allotypes have been labeled with fluorescent microspheres coupled with myeloma protein antigens. The ratio of specific to nonspecific microsphere binding by viable hybridoma cells was about 100:1. By using a modified fluorescence-activated cell sorter (FACS), selected hybridoma cells in a mixture have been sorted individually into media in microculture wells, where, with thymocyte feeder cells, they developed into clones producing a desired monoclonal antibody. Viable cells were selected by measurement of their light scattering and autofluorescence properties. Rare antibody-producing clones were obtained without laborious screening and repeated subculturing. This technique should expand the range of monoclonal antibodies readily obtained from hybridomas and greatly facilitate the process of obtaining desired hybridomas.

Animals↗
PubMedDate not supplied

Correlation between segmental flexibility and effector function of antibodies.

Mouse monoclonal anti-dansyl antibodies with the same antigen-binding sites but different heavy chain constant regions were generated. The extent of segmental flexibility in times of nanoseconds and the capacity to fix complement were greatest for IgG2b, intermediate for IgG2a, and least for IgG1 and IgE. Hence, the effector functions of immunoglobulin isotypes may be controlled in part by the freedom of movement of their Fab arms.

Animals↗
PubMedDate not supplied

Expression of a VHC kappa chimaeric protein in mouse myeloma cells.

The heavy (H) and light (L) chains of antibodies consist of variable (V) and constant (C) regions. The V regions of the heavy and light chains form the antibody combining site. To determine whether a V region could be functional when joined to a polypeptide other than its own C region, we constructed a chimaeric gene encoding the V region of a mouse heavy chain and the C region of a mouse kappa light chain ( VHC kappa). The heavy-chain gene is derived from an A/J mouse hybridoma cell line 36-65 whose antibody product (gamma 1, kappa) is specific for the hapten azophenylarsonate. We report here that, when introduced into a mouse myeloma cell line, the chimaeric gene is expressed and a protein of the expected molecular weight is secreted into the medium. As light chains tend to dimerize we expected that the VHC kappa protein might associate with light chain from the cell line 36-65 to form an antibody-binding molecule. Affinity binding experiments and Ka determination indicate that this is the case. Dimers of this type offer a novel and interesting alternative to existing antibody-binding molecules.

Animals↗