Development of Rabbit Monoclonal Antibodies for Quantitation of Therapeutic Human Antibodies in Preclinical Non-Human Primate Studies.

During preclinical studies, there is a great need to develop monoclonal antibodies (mAbs) that are specific to human immunoglobulin (IgG), without binding to monkey IgG, to detect therapeutic human mAb in non-human primates. We took advantage of the latest rabbit B cell cloning technology to develop six unique rabbit anti-human IgG mAb clones for this purpose. These clones are capable of binding to both human IgG and Fab with high affinity without nonspecific binding to cynomolgus monkey IgG.

Capture-stabilize approach for membrane protein SPR assays.

Measuring the binding kinetics of antibodies to intact membrane proteins by surface plasmon resonance has been challenging largely because of the inherent difficulties in capturing membrane proteins on chip surfaces while retaining their native conformation. Here we describe a method in which His-tagged CXCR5, a GPCR, was purified and captured on a Biacore chip surface via the affinity tag. The captured receptor protein was then stabilized on the chip surface by limited cross-linking.

Protein profiling of mouse livers with peroxisome proliferator-activated receptor alpha activation.

Peroxisome proliferator-activated receptor alpha (PPARalpha) is important in the induction of cell-specific pleiotropic responses, including the development of liver tumors, when it is chronically activated by structurally diverse synthetic ligands such as Wy-14,643 or by unmetabolized endogenous ligands resulting from the disruption of the gene encoding acyl coenzyme A (CoA) oxidase (AOX). Alterations in gene expression patterns in livers with PPARalpha activation were delineated by using a proteomic approach to analyze liver proteins of Wy-14,643-treated and AOX(-/-) mice. We identified 46 differentially expressed proteins in mouse livers with PPARalpha activation.

Profiling of acyl-CoA oxidase-deficient and peroxisome proliferator Wy14,643-treated mouse liver protein by surface-enhanced laser desorption/ionization ProteinChip Biology System.

Peroxisome proliferators induce hepatic peroxisome proliferation and hepatocellular carcinomas in rodents. These chemicals increase the expression of the peroxisomal beta-oxidation pathway and the cytochrome P-450 4A family, which metabolizes lipids, including fatty acids. Mice lacking fatty acyl-CoA oxidase (AOX-/-), the first enzyme of the peroxisomal beta-oxidation system, exhibit extensive microvesicular steatohepatitis, leading to hepatocellular regeneration and massive peroxisome proliferation.