Identification of potential biomarkers of P-glycoprotein substrate neurotoxicity in transgenic mice expressing the mutated canine ABCB1 gene.

Objective: To identify biomarkers of P-glycoprotein (P-gp) substrate neurotoxicity in transgenic mice expressing the mutant canine ABCB1 gene (ABCB1-1Δ).

Animals: 8 ABCB1 knock-in and knock-out transgenic mice expressing the ABCB1-1Δ gene and 8 control mice expressing the wild-type canine ABCB1 gene (ABCB1-WT).

Procedures: Groups including 2 ABCB1-1Δ mutant mice and 2 ABCB1-WT mice were administered the P-gp substrates ivermectin (10 mg/kg, SC), doramectin (10 mg/kg, SC), moxidectin (10 mg/kg, PO), or digoxin (1.

Neurotoxic effects of ivermectin administration in genetically engineered mice with targeted insertion of the mutated canine ABCB1 gene.

Objective: To develop in genetically engineered mice an alternative screening method for evaluation of P-glycoprotein substrate toxicosis in ivermectin-sensitive Collies.

Animals: 14 wild-type C57BL/6J mice (controls) and 21 genetically engineered mice in which the abcb1a and abcb1b genes were disrupted and the mutated canine ABCB1 gene was inserted.

Procedures: Mice were allocated to receive 10 mg of ivermectin/kg via SC injection (n = 30) or a vehicle-only formulation of propylene glycol and glycerol formal (5).

Immunodiagnostic reagents using llama single domain antibody-alkaline phosphatase fusion proteins.

Naive libraries of single domain antibodies (sdAbs) enable rapid isolation of binders to nearly any target. These binders, however, lack the benefits bestowed by in vivo affinity maturation and typically have low affinity toward their targets. We expressed five low-affinity toxin binding sdAbs, previously selected from a naive library derived from variable regions of llama heavy chain-only antibodies, as fusions with a hyperactive mutant Escherichia coli alkaline phosphatase (AP) and examined the impact on apparent affinity and utility.

Binding kinetics of antiricin single domain antibodies and improved detection using a B chain specific binder.

Single domain antibodies are the recombinantly expressed binding fragments derived from heavy chain antibodies found in camels and llamas. These unique binding elements offer many desirable properties such as their small size ( approximately 15 kDa) and thermal stability, which makes them attractive alternatives to conventional monoclonal antibodies. We created a phage display library from llamas immunized with ricin toxoid and selected a number of single domain antibodies.

Llama-derived single-domain antibodies for the detection of botulinum A neurotoxin.

Single-domain antibodies (sdAb) specific for botulinum neurotoxin serotype A (BoNT A) were selected from an immune llama phage display library derived from a llama that was immunized with BoNT A toxoid. The constructed phage library was panned using two methods: panning on plates coated with BoNT A toxoid (BoNT A Td) and BoNT A complex toxoid (BoNT Ac Td) and panning on microspheres coupled to BoNT A Td and BoNT A toxin (BoNT A Tx). Both panning methods selected for binders that had identical sequences, suggesting that panning on toxoided material may be as effective as panning on bead-immobilized toxin for isolating specific binders.

Amplification of immunoassays using phage-displayed single domain antibodies.

Single domain antibodies (sdAb) are recombinantly produced variable domains derived from the heavy-chain only antibodies found in camelids. Previously, we selected sdAb that were specific for both ricin and botulinum A (BoNT A) toxin complex from phage display libraries of sdAb and evaluated the solubly expressed protein. Here, phage-displayed sdAb were used as reporter reagents and compared to soluble, unfused sdAb.

Ricin detection using phage displayed single domain antibodies.

Phage-displayed single domain antibodies (sdAb) were compared to monomeric solubly expressed sdAb and llama polyclonal antibodies for the detection of ricin. SdAb are comprised of the variable domain derived from camelid heavy chain only antibodies (HcAb). Although HcAb lack variable light chains, they as well as their derivative sdAb are able to bind antigens with high affinity.

Development of antiricin single domain antibodies toward detection and therapeutic reagents.

Single domain antibodies (sdAb) that bind ricin with high affinity and specificity were selected from a phage display library derived from the mRNA of heavy chain antibodies obtained from lymphocytes of immunized llamas. The sdAb were found to recognize three distinct epitopes on ricin. Representative sdAb were demonstrated to function as both capture and tracer elements in fluid array immunoassays, a limit of detection of 1.

Unimolecular, soluble semiconductor nanoparticle-based biosensors for thrombin using charge/electron transfer.

Duplex DNA was attached to semiconductor nanoparticles providing selective detection of thrombin. Using the method reported here, semiconductor nanoparticles can have selective sensory functions for a host of additional analytes in the future. The system uses one DNA strand that selectively binds an analyte (thrombin), while the complementary DNA strand contains a redox-active metal complex.

Geometric preferences of crosslinked protein-derived cofactors reveal a high propensity for near-sequence pairs.

Protein-derived cofactors that are composed of covalently crosslinked amino acid side chains are of increasing importance in protein science. These crosslinked protein-derived cofactors (CPDC) are formed either through direct oxidation by metal/O(2)-derived intermediates or through outer sphere oxidation by highly oxidizing cofactors. CPDCs that are formed by outer sphere oxidation do not require side-chain precursors to be coordinated by a metal center, and therefore are more difficult to identify than those formed by direct oxidation.