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R B Bankert

Publications and source records attributed to R B Bankert.

At least 73 records · Page 4Linked to original sources
PubMed1985

Monoclonal antibodies specific for the mouse IgG1 allotypic determinants: reactivity with inbred and outbred mice.

C57BL/6J mice were immunized with an affinity purified monoclonal antibody (IgG1) derived from BALB/c mice and their spleen cells were fused with the mouse myeloma X63.Ag8.653. Two monoclonal antibodies (3A9, gamma 1,kappa and 1C10, gamma 1,kappa) derived from separate fusions were originally found to react with BALB/c monoclonal antibodies expressing a gamma 1 heavy chain isotype but not with other heavy chain isotypes. The results from strain distribution reactivity patterns indicate that these antibodies recognize determinants coded by the allotype locus designated Igh-4a. In a survey of 22 different inbred or congenic strains of mice, no additional polymorphism associated with this locus has been detected. However, allelic polymorphism of the gamma 1 heavy chain allotype does exist in the wild mouse population. The 3A9 and 1C10 antibodies recognize the same determinants (i.e., specificity 1) associated with Igh-4a allotype.

Animals↗
PubMed1985

Failure of PFC inhibition assays to distinguish idiotypically between clonotypes that are readily distinguishable by RIA analysis.

Two different hemolytic plaque assay protocols that are commonly used to quantitate idiotype-positive antibody-secreting cells have been compared to a standard radioimmunoassay (RIA) to test for their ability to discriminate between related, but idiotypically distinct, clonotypes. The idiotype proband used in this analysis is the individual specific idiotype associated with the dextran-binding myeloma protein M104E (M104E IdI). Antibodies specific for this private idiotype (anti-M104E IdI) were purified by a combination of adsorption and affinity chromatography of the immunoglobulin fraction isolated from the sera of rabbits repeatedly immunized with M104E. The same affinity-purified anti-M104E IdI antibodies were used in the hemolytic plaque assays and in the RIA. In one of the plaque assays, the putative idiotype-positive antibody-forming cells were scored by lysis of target erythrocytes to which the anti-idiotype had been covalently coupled. In the other plaque assay, the idiotype-positive cells were determined indirectly by anti-idiotype inhibition of PFC produced on dextran-coupled target erythrocytes. The fidelity of these two assays to quantitate the M104E private idiotype expression in individual BALB/c mice after a single immunization with dextran B-1355S was determined by comparing the plaque assay data to the data generated by a double-antibody, post-precipitation RIA of either the antibodies in the serum or of monoclonal antibodies produced by hybridomas. Our data indicate that both plaque assay protocols reflect an overestimate of the actual expression of M104E private idiotype. By using a library of dextran-specific hybridomas (that have been characterized in an RIA with respect to their M104E IdI cross-reactivity), we have shown that the PFC overestimate of the M104E expression observed in dextran-immune mice is due to the inability of both plaque assay protocols to distinguish between dextran-specific clonotypes that express idiotypes cross-reactive with, but not identical to, the 104E IdI. We conclude that the plaque assay should be used only in conjunction with an RIA to estimate the idiotype expression. This is especially true in situations where closely related cross-reactive idiotype families exist.

Animals↗
PubMed1985

Antibody response of BALB/c mice to dextran B1355S: alterations in the expression of an idiotype associated with the depletion of idiotype-binding cells.

In this study, we established that BALB/c mice recognize and respond to the idiotype (M104E IdI) of a major dextran-specific clonotype within the BALB/c mouse repertoire. This idiotype recognition is established by demonstrating the presence of idiotype-binding cells and by the production of antibodies specific for the private M104E idiotype. To determine whether or not the idiotype-recognizing cells play a regulatory role during an immune response to dextran, the idiotype-binding cells were selectively removed either by panning or by radiation-induced killing. Two significant effects are observed when the depleted spleen cells are immunized with dextran. First, there is a substantial increase in the proportion of anti-dextran antibodies that are M104E IdI+. The second effect of the idiotype-specific cell depletion is the production of significant amounts of M104E IdI+ immunoglobulin molecules which do not bind dextran. The depletion experiments produced no alteration in the concentration of anti-dextran antibodies found in the serum or in the number of dextran-specific PFC in the spleen. The data indicate that idiotype-reactive cells can play a role in regulating the level of individual clonotype expression (i.e., the M104E clonotype), but that an alternative mechanism must exist for regulating the absolute amount of anti-dextran antibody produced after immunization.

Animals↗
PubMed1985

Cross-reactive idiotype family observed in the phthalate-specific B cell repertoire of adult BALB/c mice: diversity of IgM compared with IgG monoclonal anti-phthalate antibodies.

A highly conserved clonotype has been identified within the repertoire of B cells specific for the negatively charged hapten phthalate. The prototype of this phthalate-specific clonotype is a primary-response hybridoma (2E9) that produces a mu,kappa anti-phthalate antibody. The 2E9 monoclonal antibody was found to share idiotypic determinants with several other independently-derived mu,kappa and gamma 1,kappa anti-phthalate monoclonal antibodies and with a significant proportion of conventional anti-phthalate antibodies derived from all of the BALB/c mice immunized with phthalate-keyhole limpet hemocyanin. Competitive RIA analysis of the 2E9 idiotypic relatedness between primary and secondary response antibodies was consistent with the hypothesis that the primary response mu,kappa antibodies represent a conserved germ-line product, whereas the secondary response to gamma 1,kappa antibodies reflect somatic variants of the 2E9 clonotype. Further analysis with a site-specific anti-idiotype reagent suggests that the idiotypic differences between mu,kappa and gamma 1,kappa monoclonal antibodies occur at positions outside of the combining site. Fine specificity analysis of the monoclonal antibodies expressing the 2E9 cross-reactive idiotype (CRI) also supports this hypothesis. Seven to 35% of the anti-phthalate antibodies after a single immunization with phthalate-KLH and 1 to 10% of the antibodies after a second immunization express the 2E9 CRI. The 2E9 CRI was also found in several other strains of mice, and its expression was associated exclusively with anti-phthalate antibodies.

Aging↗
PubMed1984

Spectrin immunofluorescence distinguishes a population of naturally capped lymphocytes in situ.

Immunofluorescence analysis of mammalian lymphocytes using antiserum directed against chicken erythrocyte alpha-spectrin revealed a lymphocyte population in which spectrin antigen was arranged in the form of a discrete cap (hereafter referred to as capped lymphocytes). This subset could be easily distinguished from other lymphocytes in which the spectrin antigen was diffusely distributed near the plasma membrane (noncapped lymphocytes). The subset of capped lymphocytes could be visualized in situ and in isolated cells in the absence of added ligand. Using frozen sections of lymphoid organs that were fixed in formaldehyde prior to the immunofluorescence procedure, capped lymphocytes were found in characteristic locations depending on the tissue examined. In the thymus, the major population of medullary lymphocytes were capped whereas cortical lymphocytes were mostly noncapped. In Peyer's patches, capped lymphocytes were interspersed with noncapped lymphocytes throughout the tissue. In the spleen, capped lymphocytes were concentrated in the periarterial lymphoid sheath of the white pulp and in lymph nodes they were found predominantly in the paracortical and cortical regions. Capped lymphocytes were not visible in the thymus until just before birth and did not appear in the spleen until 3 d after birth. When lymphocytes were isolated from lymphoid organs, fixed in formaldehyde and prepared for immunofluorescence, capped and noncapped lymphocytes were still identifiable and present in the same relative proportions as seen in situ. Results identical to those described above are obtained using antisera directed against guinea pig fodrin. Natural capping of proteins previously shown to co-migrate with a variety of cell surface macromolecules after cross-linking may be a new means of identifying various stages of lymphocyte activation or differentiation.

Animals↗
PubMed1984

Monoclonal antibodies specific for two different histological types of human lung carcinoma.

Hybridomas secreting monoclonal antibodies specific for human lung cancer were produced by fusing immunized mouse spleen cells with mouse myeloma line X63-Ag8.653. Prior to fusion, BALB/c mice were immunized with two different histological types of human lung cancer (Squamous cell carcinoma and adenocarcinoma) obtained from surgery. An immunocytoadherence test was used to select hybridomas secreting antibodies that bound the patient's lung tumor, but did not bind to a B-lymphoblastoid cell line derived from the same patient. Five stable antibody-producing hybrids have been established and cloned. The antibodies produced by these clones have been characterized according to their light and heavy chain isotypes and for their specificity. In addition to binding to the tumor used for immunization, the antibodies bound to other lung tumors of the same histological type (i.e., squamous cell or adenocarcinoma). This reactivity was observed with both established lung tumor cell lines and with fresh tumors obtained from biopsy of patients in our clinic. Some significant reactivity was also observed with large cell carcinoma but the antibodies did not react with small cell carcinomas of the lung, bronchiolo-alveolar cell carcinoma, cancer of the esophagus and stomach, melanomas, several types of leukemias, normal human lung tissue, fibroblasts, or erythrocytes of type A, B, or O. Two of the five antibodies, 5C7 and 5E8 cross-reacted with one breast cancer obtained from surgery, and 5C7 also cross-reacted with one melanoma biopsy specimen. These results suggest that we have generated monoclonal antibodies that recognize a set of antigenic determinants that are commonly expressed on a portion of human lung tumors that are not detectable on a variety of other human tumors or normal human tissue.

Adenocarcinoma↗
PubMed1984

Generation of heavy chain-loss mutants in a B cell hybrid mediated by syngeneic idiotype-specific spleen cells.

Idiotype-specific spleen cells from appropriately primed BALB/c mice cause a marked and irreversible suppression of the membrane and secreted forms of idiotype-positive immunoglobulin (Ig) of an antigen-specific B cell hybrid clone (2C3E1). The suppression of this BALB/c B cell line has been observed in vitro and in vivo, and appears to require intimate contact between effector spleen cells and target 2C3E1 cells. The observed suppression in the 2C3E1 cell line is due to an induced mutation or a selection of pre-existing mutants within the 2C3E1 cell population, because the resultant light and heavy chain-loss variants are phenotypically stable in vitro and in vivo in the absence of any further active suppression. Biochemical analysis of the 2C3E1 cells after this suppression indicates that all of the variants are negative for the production of idiotype-positive Ig. Heavy chain synthesis by the variants is almost totally eliminated, and light chain synthesis is decreased by 10 to 90%. Spleen cells from identically primed nude mice do not induce any alteration in the 2C3E1 cell line, suggesting that induction or selection of the heavy and light chain-loss mutants requires the presence of mature T lymphocytes. The generation of idiotype-negative 2C3E1 variants during the period of tumor growth in the spleen (but not elsewhere) may represent one mechanism by which this tumor escapes the host's immune recognition.

Animals↗
PubMed1983

A filtration double antibody radioimmunoassay that simplifies and semi-automates the isolation of immune precipitates.

A semi-automation of fluid phase double antibody radioimmunoassay has been developed. The immune precipitate that was formed in 96-well microtitration plates was harvested and washed on microfibre filters using a Titertek cell harvester. A disc transfer system originally designed for use with the harvester was used as a quick and easy method of transferring the filter discs containing immune precipitate into vials for counting. The results of radioimmunoassay using the microtitration plate-filtration and conventional tube-centrifugation method are essentially identical. The microtitration plate-filtration radioimmunoassay has the following advantages over the conventional tube-centrifugation method: (1) there is no centrifugation required; (2) handling of microtitration plate is easier than the tubes in racks; and (3) it requires much less time to perform the assay.

Animals↗
PubMed1982

Monoclonal antibodies and a heterobifunctional reagent: a novel approach to the vectorial labeling of selected membrane proteins.

Many applications exist and others are envisioned for the chemical coupling of macromolecules to membrane proteins on the surface of mammalian cells. The ability to use antibody as a means to label and subsequently to follow the distribution of cell surface proteins is reported here. A new procedure is outlined for covalently coupling monoclonal antibodies to thiol-containing membrane proteins. The key reagent in the coupling reaction is the commercially available heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP). The coupling proceeds in a simple two-step reaction in aqueous medium under very mild conditions. This results in a very efficient and stable attachment of anti-hapten antibodies to a selected set of cell surface proteins without any loss in cell viability and without denaturing the antibody molecule. The hapten-binding activity of the antibody is exploited to monitor the re-distribution of the antibody-labeled cell surface proteins periodically after the coupling reaction. The hapten binding activity can also be utilized to isolate membrane macromolecules via affinity chromatography.

Animals↗
PubMed1982

Hapten-specific B cell repertoire probed by hybridoma technology: selection and characterization of representative clonotypes from the antibody-forming cell pool.

In the course of this study, more than 3000 phthalate-specific antibody-forming cell hybrids were identified using the hybridoma technology. With the aid of a rapid screening assay and an extensive library of phthalate analogs, it was possible to assign selected hapten-specific clones to one of 11 distinct fine-specificity sets. This compartmentalization of the phthalate-specific hybridomas has made it possible to focus attention upon a single manageable portion of the phthalate-specific repertoire. Fourteen clones from a single fine-specificity set were selected for further immunochemical characterization. Five of these clones were found to secrete an antibody that was indistinguishable in isoelectric focusing. Affinity-purified, high-resolution anti-idiotype antibodies were prepared with specificity for the antibodies produced by one of these clones (i.e., 4C7). A major portion of the serologically defined private idiotype (4C7 IdI) was shown to be associated with the ligand-combining site. Our results indicate that the five clones that share a common spectrotype also express the 4C7 IdI. Two other independently derived clones from two distinct fusions also share this idiotype. The 4C7 IdI was also identified in affinity-purified anti-phthalate antibodies derived from a pool of phthalate-immune serum (conventional antibody) and from affinity-purified antibodies derived from a pool of serum from unimmunized BALB/c mice (natural antibody). The 4C7 IdI is thus considered to represent a repeating clonotype in the phthalate-specific repertoire of BALB/c mice, and will serve as one of several useful clonal markers that are being developed for studies of the mechanism regulating idiotype expression.

Animals↗
PubMed1981

Coupling of protein antigens to erythrocytes through disulfide bond formation: preparation of stable and sensitive target cells for immune hemolysis.

An efficient technique has been developed for coupling protein antigens to erythrocyte membranes. The procedure involves three steps. First, 3-(2-pyridyldithio)propionyl residues are introduced into the protein by reaction with a heterobifunctional reagent, N-succinimidyl 3-(pyridyldithio) propionate. Second, the addition of disulfide groups to sheep erythrocytes (SRBC) is achieved by coupling dithiodiglycolic acid to SRBC with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The disulfide bonds of the dithiodiglycolyl-SRBC conjugate are then reduced with dithiothreitol. Finally, the 3-(2-pyridyldithio)propionyl-protein conjugate is covalently coupled to the thiolated SRBC through thiol/disulfide exchange to form the disulfide-linked antigen-SRBC conjugate. The procedure requires only 10-500 microgram of protein antigen for the preparation of 50 microliter of packed protein-coupled SRBC. Antibodies binding to antigen on the erythrocyte initiate a complement-dependent immune lysis of the target cells. Target cells prepared by this method are stable for at least 4 wk at 4 degrees C in phosphate buffer (pH 7.2) and are capable of detecting as little as 40 pg of antibody in a hemolytic assay without noticeable nonspecific lysis.

Antigen-Antibody Reactions↗
PubMed1981

Hybridomas producing hemolytic plaques used to study the relationship between monoclonal antibody affinity and the efficiency of plaque inhibition with increasing concentrations of antigen.

Hybridoma clones producing anti-hapten antibodies were established by fusing spleen cells from mice immunized with 4-azophthalate keyhole limpet hemocyanin to drug-resistant non-producing myelomas. The affinity and homogeneity of the immunochemically purified anti-phthalate antibodies were determined by equilibrium dialysis. We report here a broad distribution of the monoclonal antibody affinities ranging from a low of 4.0 X 10(4) to a high of 4.0 X 10(7) (L/M). The binding data for eleven anti-phthalate antibodies described a straight line in a Scatchard plot as would be expected for homogeneous antibodies. We determined that all hybridomas, except those secreting very low affinity antibody, produced hemolytic plaques. The hybridomas made it possible to test several assumptions regarding the association of plaque morphology and plaque inhibition with the affinity of the antibody secreted by the plaque-forming cells. Our studies indicate that the affinity of the antibody secreted by the hybridoma clones does not correlate with either the size of the plaque or with the efficiency with which the hybridoma-produced plaques are inhibited by free hapten. By comparing hybridoma-produced plaques to plaques produced by phthalate-immune spleen cells it has been demonstrated that the plaque size within each hybridoma clone was substantially less heterogeneous than that observed for the immune spleen cells. Our results do support the assumption that the range of free hapten concentration over which PFC are inhibited is a reflection of PFC heterogeneity. The analysis of the PFC inhibition of hybridomas produced an interesting and unexpected result which is reported here. It was observed that subinhibitory doses of free hapten caused an enhancement in the number of hybridoma-produced plaques. The relevance of this finding to the recent observation and interpretation of plaque enhancement (i.e., the displacement of anti-idiotype antibodies) observed for immune spleen cells is discussed.

Animals↗
PubMed1980

Screening and replica plating of anti-hapten hybridomas with a transfer template hemolytic spot assay.

A localized hemolysis in gel assay is described for screening microcultures of hybridomas for the production of anti-hapten antibody. The keys to the rapid screening assay reported here are a special fenestrated transfer template and an improved hapten conjugated target cell. The transfer template is a 96-well plate with a calibrated hole in the bottom center of each well. To assay for anti-hapten antibodies, the transfer template is positioned over a 96-well microculture plate containing the growing hybridomas. After making contact with the tissue culture supernatant each orifice of the transfer template retains approximately 2 microliter of tissue culture supernatant. The transfer template is then placed onto an assay slide containing a thin layer of hapten conjugated target erythrocytes incorporated into agarose. After incubation with an anti-immunoglobulin and complement, areas of localized hemolysis in the gel indicate hybridomas which are secreting anti-hapten antibodies. The assay detects as little as 10 pg of antibody. Since the transfer template can be used as a replica plate, one can repeatedly transfer samples to various slides which contain either different hapten target cells or different hapten analog inhibitors in the agarose layer. Therefore, in addition to rapidly screening microcultures for positive hybridomas this procedure permits the characterization of each monoclonal antibody's fine specificity.

Animals↗