技术资料

PURPOSE: The PRL-3 mRNA is consistently elevated in metastatic samples derived from colorectal cancers. We sought to generate a specific PRL-3 monoclonal antibody (mAb) that might serve as a potential diagnostic marker for colorectal cancer metastasis. EXPERIMENTAL DESIGN: PRL-3 is one of three members (PRL-1,PRL-2,and PRL-3) in a unique protein-tyrosine phosphatase family. Because the three PRLs are 76% to 87% identical in their amino acid sequences,it poses a great challenge to obtain mAbs that are specific for respective phosphatase of regenerating liver (PRL) but not for the other two in the family. We screened over 1,400 hybridoma clones to generate mAbs specific to each PRL member. RESULTS: We obtained two hybridoma clones specifically against PRL-3 and another two clones specifically against PRL-1. These antibodies had been evaluated by several critical tests to show their own specificities and applications. Most importantly,the PRL-3 mAbs were assessed on 282 human colorectal tissue samples (121 normal,17 adenomas,and 144 adenocarcinomas). PRL-3 protein was detected in 11% of adenocarcinoma samples. The PRL-3- and PRL-1-specific mAbs were further examined on 204 human multiple cancer tissues. The differential expressions of PRL-3 and PRL-1 confirmed the mAbs' specificity. CONCLUSIONS: Using several approaches,we show that PRL-3- or PRL-1-specific mAbs react only to their respective antigen. The expression of PRL-3 in textgreater10% of primary colorectal cancer samples indicates that PRL-3 may prime the metastatic process. These mAbs will be useful as markers in clinical diagnosis for assessing tumor aggressiveness. View Publication

Current methods for producing immunoglobulin G (IgG) antibodies in engineered cells often require refolding steps or secretion across one or more biological membranes. Here,we describe a robust expression platform for biosynthesis of full-length IgG antibodies in the Escherichia coli cytoplasm. Synthetic heavy and light chains,both lacking canonical export signals,are expressed in specially engineered E. coli strains that permit formation of stable disulfide bonds within the cytoplasm. IgGs with clinically relevant antigen- and effector-binding activities are readily produced in the E. coli cytoplasm by grafting antigen-specific variable heavy and light domains into a cytoplasmically stable framework and remodelling the fragment crystallizable domain with amino-acid substitutions that promote binding to Fcγ receptors. The resulting cytoplasmic IgGs-named 'cyclonals'-effectively bypass the potentially rate-limiting steps of membrane translocation and glycosylation. View Publication

To investigate retinal involvement in chronic Chagas' disease,we performed electroretinography and retinal fluorescein angiography studies in chagasic patients. Our results demonstrated a dissociated electrophysiological response characterized by both an abnormal reduction of the electroretinographic b-wave amplitude and a delayed latency,under the dark-adaptated condition. These alterations are compatible with a selective dysfunction of the rods. Antibodies raised against Trypanosoma cruzi that also interact with beta1-adrenergic receptor blocked light stimulation of cGMP-phosphodiesterase in bovine rod membranes. The specificity from the antibody-rhodopsin interaction was confirmed by Western blot analysis and antigenic competition experiments. Our results suggest an immunomediated rhodopsin blockade. T. cruzi infection probably induces an autoimmune response against rhodopsin in the chronic phase of Chagas' disease through a molecular mimicry mechanism similar to that described previously on cardiac human beta1-adrenergic and M2-cholinergic receptors,all related to the same subfamily of G-protein-coupled receptors. View Publication