ABSTRACT
The purpose of this study was to prepare a fully human anti-VEGF (vascular endothelial growth factor) monoclonal antibody with anti-tumor activity from five-feature mice which express human immunoglobin loci. Four hybridomas secreting mAb stably were isolated successfully. Some characters such as isotypes, cross-reactivity, inhibition on the binding of hVEGF to VEGFR-2, dissociation constants and the idiotypic characteristic were determined. Proliferation of T24 and Ls-174-T cell line and nude mice bearing human colorectal cancer were used to evaluate therapeutic effects and safety of this mAb. Pharmacokinetics data shows the half life of this mAb was about 5 days after a single intravenous injection. These results suggest the fully human anti-VEGF mAb maybe safe and efficient for cancer treatment.
Subject(s)
Antibodies, Monoclonal/isolation & purification , Antineoplastic Agents/isolation & purification , Immunoglobulin M/metabolism , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Animals , Antibodies, Monoclonal/pharmacokinetics , Antibodies, Monoclonal/pharmacology , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Colonic Neoplasms , Humans , Male , Mice , Mice, Nude , Mice, Transgenic , Protein Binding , Rats , Rats, Sprague-Dawley , Recombinant Proteins/genetics , Vascular Endothelial Growth Factor A/geneticsABSTRACT
AIM: To prepare monoclonal antibody (mAb) against erythropoietin(EPO), characterize its biological properties and use it to purify the rhEPO from transgenic goat milk. METHODS: A crude rhEPO product was used as the antigen to immunize BALB/c mice for preparing mAbs against rhEPO. The mAbs were characterized by Western blot and indirect ELISA. Purified mAb 2E6 was coupled with pre-activated Sepharose 4B to prepare the immunoaffinity chromatography column for purifying the rhEPO from transgenic goat milk. RESULTS: Two hybridoma cell lines(1E7 and 2E6)were obtained. mAbs 1E7 and 2E6 were shown to be IgG1 and IgG2b respectively, and their light chains were both kappa. Western blot analysis confirmed that the two mAbs could bind to rhEPO. The immunoaffinity chromatography column could adsorb 70% of rhEPO in purifying the rhEPO from transgenic goat milk. CONCLUSION: Two hybridoma cell lines secreting anti-rhEPO mAbs were successfully established. The mAb-immunoaffinity chromatography column could be used to purify the rhEPO from transgenic goat milk.
Subject(s)
Antibodies, Monoclonal/immunology , Erythropoietin/immunology , Hybridomas/immunology , Immunoglobulin kappa-Chains/immunology , Recombinant Proteins/immunology , Animals , Antibodies, Monoclonal/isolation & purification , Chromatography, Affinity , Erythropoietin/genetics , Erythropoietin/isolation & purification , Goats , Immunoglobulin kappa-Chains/genetics , Immunoglobulin kappa-Chains/isolation & purification , Mice , Mice, Inbred BALB C , Milk/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purificationABSTRACT
UNLABELLED: Shaneng goat is a famous milking species. Boer goat is world famous goat breed for creophagism. In this study, we evaluated the development potential of adult Boer goat's somatic nuclei after nuclear tansfer (NT) into enucleated MII oocytes of the Shaneng goat. Somatic donor cells were obtained from two different sources: 1) adult granulosa cells (GCs) and 2) adult skin fibroblasts (FCs). The reconstructed embryos that developed to morula or blastocyst stage in vivo were transferred to 38 synchronized recipient. CONTROL: Somatic donor fibroblast cells were obtained from a fetal at 35 day. In the same way the reconstructed embryos were directly transferred to synchronized recipient of Shaneng goats. (1) Experimental group: NT embryos derived from GC and FC developed into morulas and blastocysts at a frequency of 46.8% and 31.4% respectively. Fifty-two NT morula and blastocyst stage embryos were transferred in to 38 recipients, Three of which were confirmed to be pregnant (7.9%). All pregnancies were not maintained to term. (2) CONTROL group: 136 NT embryos were transferred in to 14 recipients, Six of which were confirmed to be pregnant (42.9%). Four of those were maintained to term. Four recipients delivered four male kids (2.9% of embryos transferred). One male kid died at birth, the dead lamb shows as "large offspring syndrome". the others appeared health and normal. DNA analysis confirmed that those kids were genetically identical to their donor. These results demonstrated that Shaneng goat somatic cells could direct normal development and Shaneng goat oocyte cytoplasm supported development of preimplantation embryos produced by NT of somatic cell nuclei from Boer goat.
