A simple procedure for large-scale density separation of bone marrow cells for transplantation.
Explore the source record for details and available documents.
Adolescent↗
Biomedical subjects
T E Thomas
Publications and source records attributed to T E Thomas.
Single laser three color immunofluorescence staining procedures based on energy transfer between phycoerythrin and cyanine 5.
Monoclonal antibodies specific for phycoerythrin (PE) were covalently labeled with the fluorescent dye cyanine 5 (Cy5). Excitation at 488 nm of immune complexes obtained by mixing Cy5-anti-PE with PE resulted in a 4-fold reduction of PE fluorescence measured at 565 nm and an increase of fluorescence measured at 655 nm. The observed energy transfer between PE and Cy5-anti-PE was used to develop three color immunofluorescence staining procedures for flow cytometers equipped with an Argon laser tuned at 488 nm. Mouse IgG1 monoclonal antibodies specific for cell surface antigens were cross-linked with either unlabeled or Cy5 labeled mouse IgG1 anti-PE using F(ab')2 fragments of monoclonal rat anti-mouse IgG1. PE was added to these immune complexes in sufficient amounts to saturate all PE binding sites. Cells were incubated with PE-labeled and PE/Cy5-labeled tetrameric antibody complexes together with FITC labeled antibodies and analyzed by flow cytometry. The emission from FITC, PE and PE/Cy5 could be readily separated and bright three color immunofluorescence staining of mononuclear cells from human peripheral blood and bone marrow was observed. The results of these experiments demonstrate that useful probes for single laser three color staining of cell surface antigens can be readily obtained by mixing of selected reagents. Compared to standard procedures for the covalent labeling of PE (tandem) molecules to antibodies, the non-covalent procedures described in this report provide significant advantages in terms of the amount of reagents, time and equipment required to obtain suitable reagents for three color immunofluorescence staining.
Animals↗
Purification and analysis of bispecific tetrameric antibody complexes.
In order to study the type and yield of immune complexes obtained by the mixing of purified F(ab')2 fragments of rat monoclonal antibodies specific for mouse IgG1 with equimolar amounts of purified mouse IgG1 size exclusion HPLC of the reaction mixture was performed. Immune complexes eluted as a single peak at a position compatible with a tetrameric antibody complex configuration. The yield of tetramers could be increased by incubation of the antibody mixture for several hours at 37 degrees C, indicating a preference of the tetrameric composition over other immune complex compositions. Size exclusion HPLC also showed that greater than 80% of purified tetramers retained their original dimensions after storage for 1 year at 4 degrees C, thus indicating the long-term stability of tetrameric antibody complexes. When complexes were prepared with a mixture of two different mouse IgG1 antibodies, bispecific tetramers were obtained that could be separated from monospecific tetramers using DEAE-HPLC. Purified bispecific antibody complexes of mouse IgG1 anti-CD34 (My10) cross-linked to mouse IgG1 anti-desferal with F(ab')2 rat anti-mouse IgG1 were useful for the purification of cells expressing CD34 from human bone marrow. For this purpose cells were labelled with the antibody complexes, selectively adsorbed onto columns containing desferal coated glass beads and then selectively eluted by treatment with dithiothreitol resulting in reductive cleavage of the disulfide bonds of the F(ab')2 fragments. This relatively simple cell fractionation technique illustrates the unique cross-linking properties of bispecific tetrameric antibody complexes. The procedure appears useful for further studies of hemopoietic cells and bone marrow transplantation.
Antibodies↗
Specific binding and release of cells from beads using cleavable tetrameric antibody complexes.
A two-step separation procedure is described for the positive selection of cells based on their reactivity with mouse monoclonal antibodies. In the first step cells are specifically cross-linked to hapten-modified glass beads using tetrameric monoclonal antibody complexes. In the second step bound cells are selectively eluted by reductive cleavage of the tetrameric antibody complexes. The latter are comprised of two mouse IgG1 monoclonal antibodies (one recognizing a cell surface antigen on target cells and the other a hapten coupled to the glass beads) bound together by two F(ab')2 fragments of rat anti-mouse IgG1 monoclonal antibody. The complexes provide a specific cleavable cross-link between cell and bead because the disulfide bonds between the two Fab' arms of the F(ab')2 fragments can be broken under relatively mild conditions using dithiothreitol. This specific cleavage of the cross-linker allows elution of the specifically absorbed cells without co-elution of non-specifically bound cells. This is shown in the purification of CD3+ T cells from human peripheral blood, where the removed fractions were over 90% pure and approximately 50% of the positive cells were recovered. Separation of cells labelled with limiting amounts of tetrameric antibody complexes demonstrated that this separation technique was also effective for the purification of cells expressing low amounts of antigens. This was confirmed by the purification of CD34-positive cells from human bone marrow. With this approach, colony-forming cells were enriched 15-24-fold over density separated marrow.
Antibodies, Monoclonal↗
Immunoadsorption of T cells onto glass beads using tetramolecular complexes of monoclonal antibodies.
Tetramolecular monoclonal antibody complexes were used to selectively cross-link a subset of human peripheral blood T cells (CD8 positive) to glass beads coated with hapten (fluorescein) modified bovine serum albumin. Tetramolecular antibody complexes were prepared with anti-CD8 (Leu 2a), anti-FITC mouse IgG1 monoclonal antibodies and monoclonal rat anti-mouse IgG1. Optimum conditions for depletion of CD8 positive cells from peripheral blood mononuclear cell suspensions were determined with 1 ml columns. 90-99% of the CD8 positive cells could be removed with 0-17% non-specific adsorption of CD8 negative cells. The weakest link in this system was the bond between hapten-modified albumin and the glass beads. These results indicate that tetramolecular antibody complexes are useful for the specific immunoadsorption of cells to a defined affinity matrix.
Antibodies, Anti-Idiotypic↗
Use of tetrameric antibody complexes to stain cells for flow cytometry.
Bifunctional tetrameric complexes of monoclonal antibodies were used to stain cells for flow cytometry. These complexes consist of two different mouse monoclonal IgG1 antibodies (one with specificity for a cell surface antigen, the other with specificity for a fluorochrome) cross-linked by two molecules of a monoclonal rat anti-mouse IgG1. The use of this immunological approach to cross-link fluorochromes to cell surface antigens was studied with tetrameric complexes containing Leu-3a or Leu-2a antibodies and monoclonal antibodies specific for the fluorochromes B- and R-phycoerythrin. The ability of such cyclic immune complexes to stain T-cell subset antigens on human peripheral blood lymphocytes was demonstrated in single and double-staining experiments. The results demonstrate that tetrameric antibody complexes provide a simple and efficient alternative to covalently labeled antibodies for the flow cytofluorimetric analysis of cell-surface antigens.
Animals↗
NMR in cancer: XVII. dewar for a 53-inch superconducting NMR magnet.
A giant nitrogen-jacketed liquid-helium metal dewar built in this laboratory is described for housing 53-inch superconducting magnet used in the human FONAR experiments. This dewar is 10 feet tall, 6 feet wide, 18 inches deep, and weighs 1 1/2 tons. It consists of the main magnet hoop connected through a demountable gooseneck to a liquid helium reservoir tank.
Electromagnetic Phenomena↗