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PubMed1985

Development of a mouse antiperoxidase secreting hybridoma for use in the production of a mouse PAP complex for immunocytochemistry and as a parent cell line in the development of hybrid hybridomas.

Mouse antibodies are increasingly used as primary antibodies for immunocytochemistry as more mouse monoclonal antibodies are being produced. The localisation of these antibodies by the PAP technique requires mouse antiperoxidase antibody. A monoclonal antiperoxidase would obviate the limitations of production of a polyclonal mouse antiperoxidase. This paper describes the development of a mouse hybridoma producing such an antibody (MAP A6-2) and the use of this antibody to localise a number of mouse primary antibodies by the PAP technique for both light and electron microscopy. The antibodies localised include monoclonal antienkephalin and antityrosine hydroxylase. MAP A6-2 had a higher affinity in immuno-diffusion experiments and gives slightly better staining with an horse radish peroxidase of a different type from that used for immunisation. Staining was optimum with horse radish peroxidase type X whereas horse radish peroxidase type VI was used for immunisation. Also described is the production of a HAT sensitive variant cell line allowing the possibility of using this hybridoma as a parent cell line for the production of hybrid hybridomas secreting bi-specific antibodies.

Animals↗
PubMed1985

Generation of human hybridomas producing migration inhibitory factor (MIF) and of murine hybridomas secreting monoclonal antibodies to human MIF.

Human T-cell hybridomas were established by hybridization of concanavalin A (Con A)-stimulated human peripheral blood T lymphocytes with cells from a 6-thioguanine-resistant, aminopterin-sensitive mutant line designated CEM-WH4, derived from the continuously growing human T cell line, CEM. High levels of MIF activity were demonstrated in the supernatants of two hybridoma lines, T-CEMA and T-CEMB but not of CEM-WH4 when stimulated with phorbol myristate acetate and phytohemagglutinin. In comparison, MIF derived from Con A-stimulated peripheral blood mononuclear cells showed 100 times less activity. Upon isoelectrofocusing, MIF activity of T-CEMB was found exclusively between pH 4.6 and 5.3 whereas MIF derived from T-CEMA showed heterogeneity with a major peak of MIF recovered at pH 4.6-5.3 and a minor peak at pH 2.4-3.3. These molecules, however, were all found to have an apparent MW of 68,000 and were resistant to trypsin. Most of these characteristics are in accordance with second day pH 3- and pH 5-MIF derived from peripheral blood mononuclear cells. When spleen cells from BALB/c mice immunized with T-CEMB-MIF were used to fuse with NS-1 mouse myeloma cells, nine hybridomas secreting antibodies to human MIF were obtained. Clone D112 which demonstrated the highest MIF-neutralizing activity was found to neutralize MIF derived from T-CEMA, peripheral blood mononuclear cells, and a T cell line, Mo.

Animals↗
PubMed1981

Estimation of the number of monoclonal hybridomas in a cell fusion experiment. Effect of post-fusion cell dilution on hybridoma survival.

A practical method is described for the estimation of the number of monoclonal hybridomas in a cell fusion experiment as a function of the percent of culture dishes showing hybridoma growth. Our method is based on the Poisson probability model. A justification for the method is included. The application of this model to our experimental results indicates that the probability of hybridoma survival decreases with post-fusion cell dilution even in the presence of a constant number of feeder cells.

Animals↗
PubMed1984

Hybridoma preservation at -70 degrees C: a simple and economical procedure for the short-term storage and individual recovery of hybridomas.

A simple, economical, and time-saving procedure for freezing freshly prepared hybridomas at--70 degrees C is described. The procedure allows for supernatant fluid collection and storage of hybridomas in 96-well flexible polyvinyl microtiter plates. As the plates are sealed, and the plate is relatively soft, individual wells can be removed and cells recovered at the investigator's discretion. Consequently, this procedure allows significant latitude with regard to the time-consuming steps usually associated with hybridoma screening, expansion, and sub-cloning techniques.

Animals↗
PubMed1985

The circular dichroism of phosphocholine-specific mouse hybridoma and myeloma proteins: unusual properties of the hybridoma protein 101.6G6.

The circular dichroism (CD) spectra of five myeloma and six hybridoma proteins specific for phosphocholine were measured in the 250-310-nm range. The effect on the CD spectra of adding phosphocholine was also examined. The five myeloma proteins all had distinctive native spectra and, except for M603 and W3207, unique changes occurred on ligand binding. The hybridomas were chosen as pairs from each of the three known families of phosphocholine-specific immunoglobulins. Those from the T15 or M603 families resembled the appropriate prototype. However, the proteins from the M167 family were all distinctively different in their CD properties. In particular, the hybridoma protein 101.6G6 showed large CD changes on hapten binding and values for the association constant for phosphocholine of 1.1 X 10(5) M-1 and of 5.8 X 10(2) M-1 for acetylcholine were obtained by CD spectrophotometric titration. The CD properties of the proteins are interpreted in the light of the sequence data so far available, including the possible role of the D-segment.

Acetylcholine↗
PubMed1992

Enhanced transfection of a bacterial plasmid into hybridoma cells by electroporation: application for the selection of hybrid hybridoma (quadroma) cell lines.

A procedure was investigated to transduce a bacterial plasmid containing a specific drug resistance marker (pSV2-neo), into a hybridoma cell line using electroporation. The effect of several buffers and the form of plasmid DNA (circular or linearized) on the stable transfection frequency were examined. When complete cell culture medium (DMEM) was used as electroporation buffer, we observed a two-fold increase in post-pulse viability and a ten- to thirty-fold increase in the transfection frequency of pSV2-neo, as compared with HEPES buffered 0.15 M sodium chloride. Supplementing DMEM with fetal bovine serum (DMEM + FBS) had some beneficial effect on post-pulse viability of the cells after electroporation, but did not markedly increase stable transfection frequency as compared with DMEM alone. Furthermore, with DMEM + FBS, the intact plasmid was transfected as effectively as linearized PSV2-neo. However, when using HEPES buffered saline, the transfection frequency of pSV2-neo increased two-fold after linearization as compared with intact plasmid. The drug resistance was used successfully as a marker for the selection of hybrid hybridoma (quadroma) cell lines after fusing two different hybridoma cell lines, producing anti-fibrin and anti-plasminogen activator antibodies respectively. The quadroma cells produced bispecific antibodies that are capable of accumulating plasminogen activator on a fibrin surface.

Antibodies, Monoclonal↗
PubMed1987

Studies on immunity in hybridoma-bearing mice. A. Immune response to antigens. II. inhibition of production of anti-dinitrophenyl antibodies in mice which have rejected the B 53 anti-dinitrophenyl-producing IgE hybridoma.

When BALB/c mice, which had rejected the anti-dinitrophenyl (DNP) IgE-producing hybridoma B 53, were immunized with DNP proteins, they produced much less anti-DNP antibodies than control (normal) mice. The anti-DNP plaque-forming cell (PFC) number was much less when spleen cells from mice immunized with DNP proteins were treated with sera of mice which had rejected the hybridoma B 53 than the PFC number from the same spleen cells not treated by the sera. The sera of mice which had rejected the hybridoma B 53 contained an inhibitor which was adsorbed and eluted from an anti-mouse immunoglobulin column and also a mouse anti-DNP IgG2a column. The inhibition of PFC was hapten-reversible. In Western blotting the eluates from the anti-DNP IgG2a column reacted as well with the blotted anti-DNP IgE B 53 as an anti-idiotypic antibody to anti-DNP IgE B 53. These criteria establish that the inhibitor in the sera of the mice which had rejected the B 53 tumor was an anti-idiotypic antibody of the type which mimics the epitope (DNP) of the immunizing antigen.

Animals↗
PubMed1986

Studies on immunity in hybridoma-bearing mice. B. Immunity against the hybridoma. II. The phenotype and specificity of the immune cell.

When appropriate numbers of anti-dinitrophenyl (DNP) immunoglobulin (Ig) E-secreting hybridoma (B 53) cells were injected s.c. into normal BALB/c mice, some of the recipients rejected the tumors. These mice were shown to be immune to B 53 as they withstood, without any ill effect, the i.p. injection of lethal doses of B 53 cells. In previous studies, it was shown that the spleen cells of these mice protected against the growth of B 53 cells. In this study, the characteristics and specificity of the immune spleen cells were examined. The cells responsible for this immunity were shown to be T cells that express Ly-2 on their surface. These cells were shown in in vivo and in vitro assays to limit the growth of the immunizing hybridoma, as well as some but not all BALB/c plasmacytomas and hybridomas.

Animals↗
PubMed1982

Antigen presentation by Ia+ B cell hybridomas to H-2-restricted T cell hybridomas.

The Ia+, H-2d BALB/c lymphoma cell line L10.A 2J was fused to T cell-depleted spleen cells from mouse strains bearing other H-2 haplotypes. A portion of the selected hybrids expressed Ia antigens of the normal spleen cell partner in the fusion as evidenced by their presentation of various antigens to a set of antigen-specific I-region-restricted T cell hybridomas. Three cloned hybrids were studied in detail. Antigen presentation was shown to be inhibited specifically by monoclonal anti-Ia antibodies. Both I-A and I-E molecules were expressed, including in the one case examined hybrid I-A and I-E molecules between the H-2d and H-2b haplotypes. These Ia+ B cell hybridomas provide a useful set of tools for studying the role of I-region-encoded molecules in antigen presentation to T cells.

Antibody Formation↗
PubMed1984

Studies on immunity in hybridoma-bearing mice. A. Immune response to antigens. I. Role of subcutaneously injected IgE-producing hybridoma on antibody production of different isotypes against immunizing antigens.

When immunized with ovalbumin (OVA), mice bearing subcutaneously injected anti-dinitrophenyl (DNP) IgE-secreting hybridoma cells do not produce anti-OVA IgE antibodies as long as anti-DNP IgE is present in abundance in their sera. As soon as the titer of anti-DNP IgE antibody falls, anti-OVA IgE antibody appears. This inhibition of production of anti-OVA IgE is attributed to Fc epsilon-bearing T cells which have a suppressive action of limited duration. Other isotypes of anti-OVA antibodies are produced in similar amounts as in controls which were immunized with OVA but not injected with hybridoma cells.

Animals↗
PubMed1987

Role of the macrophage-derived hybridoma growth factor in the in vitro and in vivo proliferation of newly formed B cell hybridomas.

It has been shown recently that monocyte-macrophage cells produce a growth factor (HGF) active on newly formed B cell hybridomas. We have studied the effect of HGF on the proliferation of an HGF-sensitive clone of B cell hybridoma. Results obtained showed that the murine P388D1 cell-derived HGF has a m.w. of 29,000 and an isoelectric point (pI) of 6.2 whereas the human monocyte-derived HGF has a m.w. of 34,000 and a pI of 4.9. The HGF activity was not mediated by interleukin 1 because the two activities could be completely separated by gel filtration. Results obtained in in vivo experiments showed that HGF-sensitive cells are tumorigenic in mice. The finding that the HGF biochemical parameters (m.w. and pI) are similar to the ones of a recently described plasmacytoma growth factor suggests that HGF and the plasmacytoma growth factor are similar and that the HGF sensitivity of SP 2/O myeloma cells is reactivated after fusion with normal B lymphocytes.

Animals↗
PubMed1983

T cell hybridomas coexpressing Fc receptors (FcR) for different isotypes. II. IgA-induced formation of suppressive IgA binding factor(s) by a murine T hybridoma bearing Fc gamma R and Fc alpha R.

T2D4 murine T hybridoma cells have previously been shown to express Fc receptors (FcR) for IgG (Fc gamma R) and for IgA (Fc alpha R) and to produce an IgG binding factor (IgGBF) that suppresses IgG and IgM responses. In the present work we report on the behavior of IgA bound to T2D4 cells and on the production of IgA binding factor (IgABF) and its ability to suppress IgA antibody production. A dose-dependent binding of MOPC315 IgA with anti-TNP activity by T2D4 cells was demonstrated by rosette formation with trinitrophenylated ox red blood cells (TNP-ORBC) and fixation of iodinated DNP-BSA. IgA bound to the cells disappeared after a short-term culture of 3 hr at 37 degrees C, but not at 4 degrees C. Because this phenomenon was inhibited by 0.1% sodium azide and 100 microM dansylcadaverine, a transglutaminase inhibitor, Fc alpha R-IgA complexes seemed to be released by an active process involving receptor movement. In the culture supernatant of IgA-treated T2D4 cells, we detected a factor(s) that binds to IgA-Sepharose and competitively inhibits the binding of IgA to T2D4 cells. The factor (IgABF) failed to inhibit the rosette formation of Fc gamma R(+) cells with IgG-sensitized ORBC (EAox gamma), indicating that it binds specifically to IgA. IgABF was undetectable in the culture supernatants of untreated T2D4 cells of Fc alpha R(-) BW5147 T lymphoma cells used as parent cells for the establishment of the hybridoma. To study the effect of IgABF on antibody formation, culture filtrates of IgA-treated or untreated T2D4 cells were fractionated on IgA-Sepharose beads and were added to BALB/c spleen cells cultured with pokeweed mitogen. By use of a reverse plaque assay, it was shown that the IgA plaque-forming cell (PFC) response was suppressed by the acid eluate but not by the effluent of IgA-Sepharose beads incubated with the filtrates of IgA-treated T2D4 cell cultures. The suppression was IgA specific, because neither IgG nor IgM responses were suppressed by the eluate. As expected, there was no significant IgA suppressive activity in the acid eluates of the beads incubated with the culture filtrate of untreated T2D4 cells or IgA-treated BW5147 cells. IgA-specific suppressive activity proved to be due to IgA binding factor(s), because suppressive activity in the eluate was completely adsorbed by IgA-Sepharose but not by IgG- nor BSA-Sepharose.

Animals↗
PubMed1988

Suppressor factor secreted by T hybridoma established from peripheral blood lymphocytes of a bone marrow transplantation patient. I. Establishment of human T-cell hybridoma and partial characterization of suppressor factor.

A stable human T-cell hybridoma was established by cell fusion between activated human peripheral blood lymphocytes from an allogeneic bone marrow transplantation patient and the JD1-17 cell line, a subclone of the human T leukemia Jurkat cell line. This hybrid clone 1-8, which bore the surface phenotype of suppressor cells (CD8+HNK1+), spontaneously secreted a factor which, at high dilutions, suppressed the responses of T and B cells induced by mitogens and alloantigens. This suppressor factor was found to be heat-resistant (56 degrees C, 30 min), stable at alkaline but not acid pH, unaffected by 2-mercaptoethanol, and sensitive to trypsin. Preparative isoelectric focusing revealed an isoelectric point of 5.35. The suppressor activity was selectively absorbed by blast T cells. By gel filtration on Sephacryl S-200 and HPLC, the suppressor activity was found in two peaks corresponding to 40-45 kDa (monomer) and 90-95 kDa (dimer).

Absorption↗
PubMed1989

Regulation of antibody secretion by hybridoma cells. II. Mechanism of idiotype-induced suppression of antibody secretion by hybridoma cells.

We have previously reported that antibody secretion by B.22 hybridoma cells can be suppressed in an MHC-restricted manner, by idiotype-specific T cells. It was shown that T cells of both helper and suppressor phenotypes are involved, and that the suppression is mediated by soluble factors. In the present paper, we have characterized the effects of T-cell-mediated suppression at the level of B.22 antibody mRNA expression and stability. Nuclear run-on analysis comparing suppressed and control B.22 cells indicates no change in the transcription rates of heavy and light chains. Northern blot analysis demonstrates that steady-state levels of heavy and light chain mRNAs are also unchanged. Thus, the suppression of antibody secretion by B.22 cells probably occurs at the levels of translation or secretion.

Animals↗
PubMedDate not supplied

[Allelic exclusion in hybridomas. II. Characteristics of the synthesis of 2 allelic variants of rat immunoglobulin kappa-chains in a cloned hybridoma line].

Production of Igk-1a and Igk-1b allelic variants of rat Ig kappa-chains in hybridoma clone 4C2 was studied. Gel-filtration of the culture fluid demonstrates that the cells secrete isolated L-chains in monomeric and dimeric form. Different L-chain allotypes fail to associate in dimers. Immunofluorescence studies prove that both allelic variants are produced in a single cell. Cells with double allotype production loose the ability to produce one or both allotypes at an essentially heightened rate as compared to those producing Igk-1a or Igk-1b alone. The results fit well into the regulated allelic exclusion model.

Alleles↗
PubMed1983

Idiotypes on monoclonal antibodies to bovine insulin. I. Two public idiotypes on anti-bovine insulin hybridomas define idiotypically distinct families of hybridomas.

We used isoelectric focusing (IEF) to show that individual mice responding to bovine insulin (BI) present complex spectrotypes that are not conserved between individuals. Competition of 125I-BI-reactive bands with cold BI or cold pork insulin (PI), showed that the majority of the antibody response is sensitive to the two amino acid difference between BI and PI. This difference resides in the three amino acid intrachain disulfide-bonded A-loop. To begin dissection of the antibody response to BI, we prepared a panel of 30 monoclonal antibodies against insulin. Anti-idiotypic sera were produced in guinea pigs against seven of these hybridoma proteins (HP), and two, anti-5. 10C6 and anti-5.2B8, were found to define public idiotypes. The level of the 5.10C6 idiotype is controlled by genes linked to the Igh locus. Both the G.P. anti-5.10C6 and G.P. anti-5.2B8 were found to define distinct groups of idiotypically related HP. The 5.10C6 idiotype group contains three closely related members, 5.10C6, 5.5F10, and 5.4C5, which are shown to bear the same L chain by IEF. The 5.2B8 idiotype group has six members that are less closely related, defined by inhibition solid phase RIA. The two most closely related members of this family, 5.2B8 and 5.3C6, possess co-focusing L chains. Within each idiotype group, however, members were found that bind to distinct determinants on insulin. Four of the members of the 5.2B8 idiotype group appear to bind overlapping determinants on insulin that define a topographic region that includes the three amino acid A-loop. Two other HP that have been shown to bind the A-loop do not bear determinants that cross-react with the G.P. anti-5.2B8 antiserum.

Animals↗
PubMed1982

Human T cell hybridomas producing lymphokines. I. Establishment and characterization of human T cell hybridomas producing lymphotoxin and migration inhibitory factor.

Stable human T cell hybridomas were produced by cell fusion between PHA-activated human peripheral blood lymphocytes and human acute lymphatic leukemia cells of a CEM cell line whose proliferation had been inhibited by treatment with an irreversible protein synthesis inhibitor (emetine) and an irreversible RNA synthesis inhibitor (actinomycin D). Two hybrid cell lines (E10, F8) thus produced expressed OKT3-reactive antigen, HLA-A1 and -B8 antigens derived from CEM cells, and HLA-A2 antigen derived from PBL, and they have 10 more chromosomes than CEM cells. Furthermore, these cell lines continuously secreted lymphotoxin over 3 mo. E10 was found to produce MIF in addition to lymphotoxin. Cloning of E10 and the relationship between the functions and surface phenotypes of E10 sublines were examined. Lymphotoxin-producing hybrids (E10-15 and E10-37) express more OKT8-reactive antigen than OKT4-reactive antigen, while lymphotoxin and MIF-producing hybrids (E10-25, E10-42, and E10-43) express more OKT4-reactive antigen than OKT8-reactive antigen.

Antibodies, Monoclonal↗