The Experts below are selected from a list of 111 Experts worldwide ranked by ideXlab platform
Andre Choo - One of the best experts on this subject based on the ideXlab platform.
-
effect of linker flexibility and length on the functionality of a Cytotoxic engineered Antibody fragment
Journal of Biotechnology, 2015Co-Authors: Andre Choo, Maximilian Klement, Chengcheng Liu, Bernard Loo, Dongyup LeeAbstract:Abstract Engineered Antibody fragments often contain natural or synthetic linkers joining the antigen-binding domain and multimerization regions, and the roles of these linkers have largely been overlooked. To investigate linker effects on structural properties and functionality, six bivalent Cytotoxic Antibody fragments with of linkers of varying flexibility and length were constructed: (1) 10-AA mouse IgG3 upper hinge region, (2) 20-AA mouse IgG3 upper hinge region repeat, (3) 10-AA glycine and serine linker, (4) 20-AA glycine and serine linker repeat, (5) 21-AA artificial linker, and (6) no-linker control. Interestingly, a higher Cytotoxicity was observed for fragments bearing the rigid short linkers compared to the flexible longer linkers. More importantly, amino acid composition related to the rigidity/flexibility was found to be of greater importance upon Cytotoxicity than linker length alone. To further study the structure–function relationship, molecular modelling and dynamics simulation were exploited. Resultantly, the rigid mouse IgG3 upper hinge region was predicted to enhance structural stability of the protein during the equilibrium state, indicating the improved Cytotoxicity over other combinations of fragments. This prediction was validated by measuring the thermal stability of the mouse IgG3 upper hinge as compared to the artificial linker, and shown to have a higher melting temperature which coincides with a higher structural stability. Our findings clearly suggest that appropriate linker design is required for enhancing the structural stability and functionality of engineered Antibody fragments.
-
Selective Removal of Undifferentiated Human Embryonic Stem Cells Using Magnetic Activated Cell Sorting Followed by a Cytotoxic Antibody
Tissue Engineering Part A, 2012Co-Authors: Kornelia Schriebl, Gernalia Satianegara, Wey Jia Fong, Henry He Yang, Alois Jungbauer, Austin Hwang, Andre ChooAbstract:One of the most pertinent concerns of using differentiated cells derived from human embryonic stem cells (hESC) is the presence of residual undifferentiated hESC, because they carry a risk of teratoma formation. A new cell-cell separation approach that eliminates teratoma-forming hESC in order to ensure safer cell therapy was developed. By combining antibodies (IgMs or IgGs) for the selective removal of undifferentiated hESC using magnetic activated cell sorting (MACS) followed by selective killing of residual hESC with the unique Cytotoxic Antibody mAb 84, the required purity of differentiated hESC can be achieved. The applicability and robustness of this separation strategy is shown here in a case study using pools of undifferentiated hESC and human fibroblast cells at different ratios (5%–50% hESC) to reflect the different scenario of contaminating hESC in a differentiated cell population. Notably, 97.2%–99.7% of the hESC were removed after the MACS step and 99.1%–100%, after the mAb 84 treatment step,...
-
Selective removal of undifferentiated human embryonic stem cells using magnetic activated cell sorting followed by a Cytotoxic Antibody.
Tissue engineering. Part A, 2012Co-Authors: Kornelia Schriebl, Gernalia Satianegara, Wey Jia Fong, Henry He Yang, Alois Jungbauer, Austin Hwang, Heng Liang Tan, Andre ChooAbstract:One of the most pertinent concerns of using differentiated cells derived from human embryonic stem cells (hESC) is the presence of residual undifferentiated hESC, because they carry a risk of teratoma formation. A new cell-cell separation approach that eliminates teratoma-forming hESC in order to ensure safer cell therapy was developed. By combining antibodies (IgMs or IgGs) for the selective removal of undifferentiated hESC using magnetic activated cell sorting (MACS) followed by selective killing of residual hESC with the unique Cytotoxic Antibody mAb 84, the required purity of differentiated hESC can be achieved. The applicability and robustness of this separation strategy is shown here in a case study using pools of undifferentiated hESC and human fibroblast cells at different ratios (5%-50% hESC) to reflect the different scenario of contaminating hESC in a differentiated cell population. Notably, 97.2%-99.7% of the hESC were removed after the MACS step and 99.1%-100%, after the mAb 84 treatment step, which was confirmed by double-staining flow cytometry and RT-qPCR analysis. These in vitro findings were further validated in an in vivo severe combined immunodeficiency (SCID) mouse model. Importantly, we observed the absence of teratoma formation in eight out of nine SCID mice 28 weeks postinjection of cells after the MACS step, whereas teratomas were observed in all of the controls. Thus, the combination of MACS with the unique Cytotoxic Antibody mAb 84 constitutes an indispensible tool for successful and safe cell therapy.
-
mab 84 a Cytotoxic Antibody that kills undifferentiated human embryonic stem cells via oncosis
Stem Cells, 2009Co-Authors: Heng Liang Tan, Wey Jia Fong, Eng Hin Lee, Miranda Yap, Andre ChooAbstract:The monoclonal Antibody mAb 84, which binds to podocalyxin-like protein-1 (PODXL) on human embryonic stem cells (hESCs), was previously reported to bind and kill undifferentiated cells in in vitro and in vivo assays. In this study, we investigate the mechanism responsible for mAb 84-induced hESCs Cytotoxicity. Apoptosis was likely not the cause of mAb 84-mediated cell death because no elevation of caspase activities or increased DNA fragmentation was observed in hESCs following incubation with mAb 84. Instead, it was preceded by cell aggregation and damage to cell membranes, resulting in the uptake of propidium iodide, and the leakage of intracellular sodium ions. Furthermore, examination of the cell surface by scanning electron microscopy revealed the presence of pores on the cell surface of mAb 84-treated cells, which was absent from the isotype control. This mechanism of cell death resembles that described for oncosis, a form of cell death resulting from membrane damage. Additional data suggest that the binding of mAb 84 to hESCs initiates a sequence of events prior to membrane damage, consistent with oncosis. Degradation of actin-associated proteins, namely, alpha-actinin, paxillin, and talin, was observed. The perturbation of these actin-associated proteins consequently permits the aggregation of PODXL, thus leading to the formation of pores. To our knowledge, this is the first report of oncotic cell death with hESCs as a model.
-
selection against undifferentiated human embryonic stem cells by a Cytotoxic Antibody recognizing podocalyxin like protein 1
Stem Cells, 2008Co-Authors: Andre Choo, Wey Jia Fong, Angela Chin, Jennifer Chi Yi Lo, Lu Zheng, Hannes Hentze, Robin Philp, Steve OhAbstract:Future therapeutic applications of differentiated human embryonic stem cells (hESC) carry a risk of teratoma formation by contaminating undifferentiated hESC. We generated 10 monoclonal antibodies (mAbs) against surface antigens of undifferentiated hESC, showing strong reactivity against undifferentiated, but not differentiated hESC. The mAbs did not cross react with mouse fibroblasts and showed weak to no reactivity against human embryonal carcinoma cells. Notably, one Antibody (mAb 84) is Cytotoxic to undifferentiated hESC and NCCIT cells in a concentration-dependent, complementindependent manner. mAb 84 induced cell death of undifferentiated, but not differentiated hESC within 30 minutes of incubation, and immunoprecipitation of the mAb-antigen complex revealed that the antigen is podocalyxin-like protein-1. Importantly, we observed absence of tumor formation when hESC and NCCIT cells were treated with mAb 84 prior to transplantation into severe combined immunodeficiency mice. Our data indicate that mAb 84 may be useful in eliminating residual hESC from differentiated cells populations for clinical applications. STEM CELLS 2008;26:1454–1463 Disclosure of potential conflicts of interest is found at the end of this article.
Wey Jia Fong - One of the best experts on this subject based on the ideXlab platform.
-
Selective Removal of Undifferentiated Human Embryonic Stem Cells Using Magnetic Activated Cell Sorting Followed by a Cytotoxic Antibody
Tissue Engineering Part A, 2012Co-Authors: Kornelia Schriebl, Gernalia Satianegara, Wey Jia Fong, Henry He Yang, Alois Jungbauer, Austin Hwang, Andre ChooAbstract:One of the most pertinent concerns of using differentiated cells derived from human embryonic stem cells (hESC) is the presence of residual undifferentiated hESC, because they carry a risk of teratoma formation. A new cell-cell separation approach that eliminates teratoma-forming hESC in order to ensure safer cell therapy was developed. By combining antibodies (IgMs or IgGs) for the selective removal of undifferentiated hESC using magnetic activated cell sorting (MACS) followed by selective killing of residual hESC with the unique Cytotoxic Antibody mAb 84, the required purity of differentiated hESC can be achieved. The applicability and robustness of this separation strategy is shown here in a case study using pools of undifferentiated hESC and human fibroblast cells at different ratios (5%–50% hESC) to reflect the different scenario of contaminating hESC in a differentiated cell population. Notably, 97.2%–99.7% of the hESC were removed after the MACS step and 99.1%–100%, after the mAb 84 treatment step,...
-
Selective removal of undifferentiated human embryonic stem cells using magnetic activated cell sorting followed by a Cytotoxic Antibody.
Tissue engineering. Part A, 2012Co-Authors: Kornelia Schriebl, Gernalia Satianegara, Wey Jia Fong, Henry He Yang, Alois Jungbauer, Austin Hwang, Heng Liang Tan, Andre ChooAbstract:One of the most pertinent concerns of using differentiated cells derived from human embryonic stem cells (hESC) is the presence of residual undifferentiated hESC, because they carry a risk of teratoma formation. A new cell-cell separation approach that eliminates teratoma-forming hESC in order to ensure safer cell therapy was developed. By combining antibodies (IgMs or IgGs) for the selective removal of undifferentiated hESC using magnetic activated cell sorting (MACS) followed by selective killing of residual hESC with the unique Cytotoxic Antibody mAb 84, the required purity of differentiated hESC can be achieved. The applicability and robustness of this separation strategy is shown here in a case study using pools of undifferentiated hESC and human fibroblast cells at different ratios (5%-50% hESC) to reflect the different scenario of contaminating hESC in a differentiated cell population. Notably, 97.2%-99.7% of the hESC were removed after the MACS step and 99.1%-100%, after the mAb 84 treatment step, which was confirmed by double-staining flow cytometry and RT-qPCR analysis. These in vitro findings were further validated in an in vivo severe combined immunodeficiency (SCID) mouse model. Importantly, we observed the absence of teratoma formation in eight out of nine SCID mice 28 weeks postinjection of cells after the MACS step, whereas teratomas were observed in all of the controls. Thus, the combination of MACS with the unique Cytotoxic Antibody mAb 84 constitutes an indispensible tool for successful and safe cell therapy.
-
mab 84 a Cytotoxic Antibody that kills undifferentiated human embryonic stem cells via oncosis
Stem Cells, 2009Co-Authors: Heng Liang Tan, Wey Jia Fong, Eng Hin Lee, Miranda Yap, Andre ChooAbstract:The monoclonal Antibody mAb 84, which binds to podocalyxin-like protein-1 (PODXL) on human embryonic stem cells (hESCs), was previously reported to bind and kill undifferentiated cells in in vitro and in vivo assays. In this study, we investigate the mechanism responsible for mAb 84-induced hESCs Cytotoxicity. Apoptosis was likely not the cause of mAb 84-mediated cell death because no elevation of caspase activities or increased DNA fragmentation was observed in hESCs following incubation with mAb 84. Instead, it was preceded by cell aggregation and damage to cell membranes, resulting in the uptake of propidium iodide, and the leakage of intracellular sodium ions. Furthermore, examination of the cell surface by scanning electron microscopy revealed the presence of pores on the cell surface of mAb 84-treated cells, which was absent from the isotype control. This mechanism of cell death resembles that described for oncosis, a form of cell death resulting from membrane damage. Additional data suggest that the binding of mAb 84 to hESCs initiates a sequence of events prior to membrane damage, consistent with oncosis. Degradation of actin-associated proteins, namely, alpha-actinin, paxillin, and talin, was observed. The perturbation of these actin-associated proteins consequently permits the aggregation of PODXL, thus leading to the formation of pores. To our knowledge, this is the first report of oncotic cell death with hESCs as a model.
-
selection against undifferentiated human embryonic stem cells by a Cytotoxic Antibody recognizing podocalyxin like protein 1
Stem Cells, 2008Co-Authors: Andre Choo, Wey Jia Fong, Angela Chin, Jennifer Chi Yi Lo, Lu Zheng, Hannes Hentze, Robin Philp, Steve OhAbstract:Future therapeutic applications of differentiated human embryonic stem cells (hESC) carry a risk of teratoma formation by contaminating undifferentiated hESC. We generated 10 monoclonal antibodies (mAbs) against surface antigens of undifferentiated hESC, showing strong reactivity against undifferentiated, but not differentiated hESC. The mAbs did not cross react with mouse fibroblasts and showed weak to no reactivity against human embryonal carcinoma cells. Notably, one Antibody (mAb 84) is Cytotoxic to undifferentiated hESC and NCCIT cells in a concentration-dependent, complementindependent manner. mAb 84 induced cell death of undifferentiated, but not differentiated hESC within 30 minutes of incubation, and immunoprecipitation of the mAb-antigen complex revealed that the antigen is podocalyxin-like protein-1. Importantly, we observed absence of tumor formation when hESC and NCCIT cells were treated with mAb 84 prior to transplantation into severe combined immunodeficiency mice. Our data indicate that mAb 84 may be useful in eliminating residual hESC from differentiated cells populations for clinical applications. STEM CELLS 2008;26:1454–1463 Disclosure of potential conflicts of interest is found at the end of this article.
-
selection against undifferentiated human embryonic stem cells by a Cytotoxic Antibody recognizing podocalyxin like protein 1
Stem Cells, 2008Co-Authors: Andre Choo, Wey Jia Fong, Heng Liang Tan, Angela Chin, Lu Zheng, Hannes Hentze, Robin Philp, Sheu Ngo Ang, Miranda YapAbstract:Future therapeutic applications of differentiated human embryonic stem cells (hESC) carry a risk of teratoma formation by contaminating undifferentiated hESC. We generated 10 monoclonal antibodies (mAbs) against surface antigens of undifferentiated hESC, showing strong reactivity against undifferentiated, but not differentiated hESC. The mAbs did not cross react with mouse fibroblasts and showed weak to no reactivity against human embryonal carcinoma cells. Notably, one Antibody (mAb 84) is Cytotoxic to undifferentiated hESC and NCCIT cells in a concentration-dependent, complement-independent manner. mAb 84 induced cell death of undifferentiated, but not differentiated hESC within 30 minutes of incubation, and immunoprecipitation of the mAb-antigen complex revealed that the antigen is podocalyxin-like protein-1. Importantly, we observed absence of tumor formation when hESC and NCCIT cells were treated with mAb 84 prior to transplantation into severe combined immunodeficiency mice. Our data indicate that mAb 84 may be useful in eliminating residual hESC from differentiated cells populations for clinical applications. Disclosure of potential conflicts of interest is found at the end of this article.
Andrew J. Weiland - One of the best experts on this subject based on the ideXlab platform.
-
Immunologic and ultrastructural changes during early rejection of vascularized bone allografts.
Plastic and Reconstructive Surgery, 1991Co-Authors: M. F. Gornet, Mark A. Randolph, B. H. Schofield, Michael J. Yaremchuk, Andrew J. WeilandAbstract:This investigation evaluated ultrastructural changes during the earliest phase of immunologic rejection of vascularized bone allografts in a genetically defined rat model. These results were correlated with the cell-mediated and humoral immunologic responses during this time period. Employing a model for heterotopic allograft transplantation, 33 rats divided into four categories were evaluated. Group I consisted of ungrafted (naive) Lewis and Brown Norway rats; group II consisted of Lewis-to-Lewis vascularized bone isografts; group III consisted of Lewis-to-Brown Norway vascularized bone allografts; and group IV consisted of Lewis-to-Brown Norway vascularized bone allografts in rats receiving cyclosporine (10 mg/kg/day). Experimental animals were sacrificed at 3, 5, and 7 days. Immunologic analysis was performed using a cell-mediated lymphoCytotoxicity assay and a complement-dependent Cytotoxic Antibody assay. The results of this study show that rejection of vascularized bone allografts appears as early as 3 days postoperatively, with osteocytes and vascular endothelium being the first elements affected. This early rejection is probably a manifestation of the humoral response. All changes secondary to rejection were arrested by cyclosporine.
-
Vascularized bone allografts: in vitro assessment of cell-mediated and humoral responses.
Plastic and Reconstructive Surgery, 1991Co-Authors: Peter C. Innis, Mark A. Randolph, Michael J. Yaremchuk, James P. Paskert, James F. Burdick, Lawrence W. Clow, Andrew J. WeilandAbstract:The immunologic consequences of transplantation of vascularized bone allografts have not been previously characterized. In this study, knee allografts, both vascularized and nonvascularized, were transplanted from Lewis rats to Brown Norway rats across a strong histocompatibility barrier. A total of 66 transplants and 8 control animals were evaluated. The vascularized knee grafts consisted of 1 cm of proximal tibia and distal femur with a minimal muscular cuff isolated on the femoral vessels, and these were transplanted to a heterotopic, subcutaneous position on the abdominal wall of the recipient rat. Nonvascularized allografts (identical but without anastomoses) were transplanted for comparison. The cell-mediated response was measured by lymphoCytotoxicity assay, and the humoral response was measured by Cytotoxic Antibody assay, both employing 51Cr-labeled target cells. The timing and intensity of the immune response differed according to the type of graft. The vascularized bone allografts generated significant cell-mediated and humoral responses as early as 5 days posttransplant. A significant humoral response in nonvascularized bone allografts was not apparent until day 14, while cell-mediated response in these grafts was variable. These findings were correlated with the histologic appearance of the grafted tissue. Cyclosporine, which was administered to one group of vascularized bone allografts, resulted in the suppression of both types of immune responses. The histologic appearance of this group resembled that of isografts transplanted as controls. The clinical application of vascularized bone allografts may offer significant advantages over nonvascularized allografts in the reconstruction of massive bone defects. Complications such as nonunion, fracture, and collapse of articular segments seen in nonvascularized allograft transplantation may be avoided by preservation of the blood supply to the graft. Characterization of the immune response to vascularized bone allografts may subsequently allow the manipulation of the host and/or graft tissue and promote graft incorporation.
Heng Liang Tan - One of the best experts on this subject based on the ideXlab platform.
-
Selective removal of undifferentiated human embryonic stem cells using magnetic activated cell sorting followed by a Cytotoxic Antibody.
Tissue engineering. Part A, 2012Co-Authors: Kornelia Schriebl, Gernalia Satianegara, Wey Jia Fong, Henry He Yang, Alois Jungbauer, Austin Hwang, Heng Liang Tan, Andre ChooAbstract:One of the most pertinent concerns of using differentiated cells derived from human embryonic stem cells (hESC) is the presence of residual undifferentiated hESC, because they carry a risk of teratoma formation. A new cell-cell separation approach that eliminates teratoma-forming hESC in order to ensure safer cell therapy was developed. By combining antibodies (IgMs or IgGs) for the selective removal of undifferentiated hESC using magnetic activated cell sorting (MACS) followed by selective killing of residual hESC with the unique Cytotoxic Antibody mAb 84, the required purity of differentiated hESC can be achieved. The applicability and robustness of this separation strategy is shown here in a case study using pools of undifferentiated hESC and human fibroblast cells at different ratios (5%-50% hESC) to reflect the different scenario of contaminating hESC in a differentiated cell population. Notably, 97.2%-99.7% of the hESC were removed after the MACS step and 99.1%-100%, after the mAb 84 treatment step, which was confirmed by double-staining flow cytometry and RT-qPCR analysis. These in vitro findings were further validated in an in vivo severe combined immunodeficiency (SCID) mouse model. Importantly, we observed the absence of teratoma formation in eight out of nine SCID mice 28 weeks postinjection of cells after the MACS step, whereas teratomas were observed in all of the controls. Thus, the combination of MACS with the unique Cytotoxic Antibody mAb 84 constitutes an indispensible tool for successful and safe cell therapy.
-
mab 84 a Cytotoxic Antibody that kills undifferentiated human embryonic stem cells via oncosis
Stem Cells, 2009Co-Authors: Heng Liang Tan, Wey Jia Fong, Eng Hin Lee, Miranda Yap, Andre ChooAbstract:The monoclonal Antibody mAb 84, which binds to podocalyxin-like protein-1 (PODXL) on human embryonic stem cells (hESCs), was previously reported to bind and kill undifferentiated cells in in vitro and in vivo assays. In this study, we investigate the mechanism responsible for mAb 84-induced hESCs Cytotoxicity. Apoptosis was likely not the cause of mAb 84-mediated cell death because no elevation of caspase activities or increased DNA fragmentation was observed in hESCs following incubation with mAb 84. Instead, it was preceded by cell aggregation and damage to cell membranes, resulting in the uptake of propidium iodide, and the leakage of intracellular sodium ions. Furthermore, examination of the cell surface by scanning electron microscopy revealed the presence of pores on the cell surface of mAb 84-treated cells, which was absent from the isotype control. This mechanism of cell death resembles that described for oncosis, a form of cell death resulting from membrane damage. Additional data suggest that the binding of mAb 84 to hESCs initiates a sequence of events prior to membrane damage, consistent with oncosis. Degradation of actin-associated proteins, namely, alpha-actinin, paxillin, and talin, was observed. The perturbation of these actin-associated proteins consequently permits the aggregation of PODXL, thus leading to the formation of pores. To our knowledge, this is the first report of oncotic cell death with hESCs as a model.
-
selection against undifferentiated human embryonic stem cells by a Cytotoxic Antibody recognizing podocalyxin like protein 1
Stem Cells, 2008Co-Authors: Andre Choo, Wey Jia Fong, Heng Liang Tan, Angela Chin, Lu Zheng, Hannes Hentze, Robin Philp, Sheu Ngo Ang, Miranda YapAbstract:Future therapeutic applications of differentiated human embryonic stem cells (hESC) carry a risk of teratoma formation by contaminating undifferentiated hESC. We generated 10 monoclonal antibodies (mAbs) against surface antigens of undifferentiated hESC, showing strong reactivity against undifferentiated, but not differentiated hESC. The mAbs did not cross react with mouse fibroblasts and showed weak to no reactivity against human embryonal carcinoma cells. Notably, one Antibody (mAb 84) is Cytotoxic to undifferentiated hESC and NCCIT cells in a concentration-dependent, complement-independent manner. mAb 84 induced cell death of undifferentiated, but not differentiated hESC within 30 minutes of incubation, and immunoprecipitation of the mAb-antigen complex revealed that the antigen is podocalyxin-like protein-1. Importantly, we observed absence of tumor formation when hESC and NCCIT cells were treated with mAb 84 prior to transplantation into severe combined immunodeficiency mice. Our data indicate that mAb 84 may be useful in eliminating residual hESC from differentiated cells populations for clinical applications. Disclosure of potential conflicts of interest is found at the end of this article.
A J Cumber - One of the best experts on this subject based on the ideXlab platform.
-
Preparation of Cytotoxic Antibody—Toxin Conjugates
Methods of Molecular Biology, 1992Co-Authors: A J Cumber, Edward J. WawrzynczakAbstract:: Conjugates of antibodies with plant toxins, such as ricin and abrin, are potent Cytotoxic agents that selectively eliminate target cells from mixed cell cultures in vitro, and have great promise as antitumor agents in cancer therapy (1). Ricin and abrin are protein toxins consisting of two different polypeptide subunits, the A and B chains, which are of similar size (between 30 and 34 kDa) and are joined by a single disulfide bond. The A chain is a ribosome-inactivating protein (RIP) that inactivates eukaryotic ribosomes by a specific irreversible covalent modification of the ribosomal RNA (2). The B chain binds to cell surface galactose-containing oligosaccharide residues. Following receptor-mediated endocytosis of toxin bound to the cell surface, the A chain gains access to the cytosol and destroys the ability of the cell to make protein (3).
-
Preparation of Cytotoxic Antibody-toxin conjugates.
Methods in molecular biology (Clifton N.J.), 1992Co-Authors: A J Cumber, Edward J. WawrzynczakAbstract:Conjugates of antibodies with plant toxins, such as ricin and abrin, are potent Cytotoxic agents that selectively eliminate target cells from mixed cell cultures in vitro, and have great promise as antitumor agents in cancer therapy (1). Ricin and abrin are protein toxins consisting of two different polypeptide subunits, the A and B chains, which are of similar size (between 30 and 34 kDa) and are joined by a single disulfide bond. The A chain is a ribosome-inactivating protein (RIP) that inactivates eukaryotic ribosomes by a specific irreversible covalent modification of the ribosomal RNA (2). The B chain binds to cell surface galactose-containing oligosaccharide residues. Following receptor-mediated endocytosis of toxin bound to the cell surface, the A chain gains access to the cytosol and destroys the ability of the cell to make protein (3).
-
Immunoaffinity purification and quantification of Antibody-toxin conjugates.
Methods in molecular biology (Clifton N.J.), 1992Co-Authors: Edward J. Wawrzynczak, A J CumberAbstract:Cytotoxic Antibody-toxin conjugates made using antibodies and ribs some-inactivating proteins (RIPs) are prepared using chemical crosslinking methods (1,2 and this vol., Chapter 31 ). Gel permeation chromatography is used as a first step to purify conjugate molecules from the reaction mixture. This procedure removes protein aggregates, the excess of RIP employed in the conjugation reaction, and low molecular weight byproducts. However, a significant fraction of the resulting conjugate preparation consists of unconjugated Antibody that cannot be completely separated from the conjugate on the basis of size discrimination alone (see chapter 31 ).
