Validation of Humanized Mouse Antibodies

Antibody therapy is being developed and tested as one of the most promising agents for treatment of various human diseases. As of March 2016, 350 antibody candidates are in clinical trials. Many of these antibodies have been taken from animals and “humanized” by genetic modification. Our experiment tests monoclonal antibodies that have been harvested from mouse hybridoma (spleen-derived) cells and cloned until the heavy and light chains of the antibody can be recognized by human cells. Because of this “humanization” procedure, basic antibody assays are needed to demonstrate that the binding, specificity and functional parameters of the antibodies are not lost during cloning. The purpose of this research is to perform this validation through assays. The antibodies are harvested from cell supernatants and purified using affinity chromatography. Then, the antibody fractions are tested for reactivity with human target protein PTP-Beta, via western blot and ELISA procedures. Cross-reactivity of the antibody is tested against human eta and cynomolgus beta proteins. The work presented in this poster describes results from one particular mouse antibody, R15, which has been humanized to functionally enhance endothelial survival. The goal is to generate a therapeutic antibody candidate that improves endothelium survival and stability

Religion

Pharmacists should be well-trained and sensitive to patient religious concerns and proactive in their research of any drug ingredients that may conflict with a patient\u27s beliefs. Pharmaceutical manufacturing companies should be comprehensive in their product labeling to accommodate religious sensitivities.https://digitalcommons.cedarville.edu/public_health_posters/1008/thumbnail.jp

Transgene Expression Is Associated with Copy Number and Cytomegalovirus Promoter Methylation in Transgenic Pigs

Transgenic animals have been used for years to study gene function, produce important proteins, and generate models for the study of human diseases. However, inheritance and expression instability of the transgene in transgenic animals is a major limitation. Copy number and promoter methylation are known to regulate gene expression, but no report has systematically examined their effect on transgene expression. In the study, we generated two transgenic pigs by somatic cell nuclear transfer (SCNT) that express green fluorescent protein (GFP) driven by cytomegalovirus (CMV). Absolute quantitative real-time PCR and bisulfite sequencing were performed to determine transgene copy number and promoter methylation level. The correlation of transgene expression with copy number and promoter methylation was analyzed in individual development, fibroblast cells, various tissues, and offspring of the transgenic pigs. Our results demonstrate that transgene expression is associated with copy number and CMV promoter methylation in transgenic pigs

The Adaptor Function of TRAPPC2 in Mammalian TRAPPs Explains TRAPPC2-Associated SEDT and TRAPPC9-Associated Congenital Intellectual Disability

Background: The TRAPP (Transport protein particle) complex is a conserved protein complex functioning at various steps in vesicle transport. Although yeast has three functionally and structurally distinct forms, TRAPPI, II and III, emerging evidence suggests that mammalian TRAPP complex may be different. Mutations in the TRAPP complex subunit 2 (TRAPPC2) cause X-linked spondyloepiphyseal dysplasia tarda, while mutations in the TRAPP complex subunit 9 (TRAPPC9) cause postnatal mental retardation with microcephaly. The structural interplay between these subunits found in mammalian equivalent of TRAPPI and those specific to TRAPPII and TRAPPIII remains largely unknown and we undertook the present study to examine the interaction between these subunits. Here, we reveal that the mammalian equivalent of the TRAPPII complex is structurally distinct from the yeast counterpart thus leading to insight into mechanism of disease. Principal Findings: We analyzed how TRAPPII- or TRAPPIII- specific subunits interact with the six-subunit core complex of TRAPP by co-immunoprecipitation in mammalian cells. TRAPPC2 binds to TRAPPII-specific subunit TRAPPC9, which in turn binds to TRAPPC10. Unexpectedly, TRAPPC2 can also bind to the putative TRAPPIII-specific subunit, TRAPPC8. Endogenous TRAPPC9-positive TRAPPII complex does not contain TRAPPC8, suggesting that TRAPPC2 binds to either TRAPPC9 or TRAPPC8 during the formation of the mammalian equivalents of TRAPPII or TRAPPIII, respectively. Therefore, TRAPPC2 serves as an adaptor for the formation of these complexes. A disease-causing mutation of TRAPPC2, D47Y, failed to interact with either TRAPPC9 or TRAPPC8, suggesting that aspartate 47 in TRAPPC2 is at or near the site of interaction with TRAPPC9 or TRAPPC8, mediating the formation of TRAPPII and/or TRAPPIII. Furthermore, disease-causing deletional mutants of TRAPPC9 all failed to interact with TRAPPC2 and TRAPPC10. Conclusions: TRAPPC2 serves as an adaptor for the formation of TRAPPII or TRAPPIII in mammalian cells. The mammalian equivalent of TRAPPII is likely different from the yeast TRAPPII structurally. © 2011 Zong et al.published_or_final_versio

Proceedings of the 38th International Symposium on Multiparticle Dynamics (ISMD08)

Proceedings of ISMD08Comment: Edited by: J. Bartels, K. Borras, G. Gustafson, H. Jung, K. Kutak, S. Levonian, and J. Mnic